Publications by authors named "Tian-Yun Wang"

75 Publications

Serum-Free Medium for Recombinant Protein Expression in Chinese Hamster Ovary Cells.

Front Bioeng Biotechnol 2021 15;9:646363. Epub 2021 Mar 15.

Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Xinxiang, China.

At present, nearly 70% of recombinant therapeutic proteins (RTPs) are produced by Chinese hamster ovary (CHO) cells, and serum-free medium (SFM) is necessary for their culture to produce RTPs. In this review, the history and key components of SFM are first summarized, and its preparation and experimental design are described. Some small molecule compound additives can improve the yield and quality of RTP. The function and possible mechanisms of these additives are also reviewed here. Finally, the future perspectives of SFM use with CHO cells for RTP production are discussed.
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http://dx.doi.org/10.3389/fbioe.2021.646363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006267PMC
March 2021

Progress of cationic gene delivery reagents for non-viral vector.

Appl Microbiol Biotechnol 2021 Jan 4;105(2):525-538. Epub 2021 Jan 4.

International Joint Research Laboratory for Recombinant Pharmaceutical Protein Expression System of Henan, Xinxiang Medical University, Xinxiang, 453003, Henan, China.

Gene delivery systems play a vital role in gene therapy and recombinant protein production. The advantages of using gene delivery reagents for non-viral vector include the capacity to accommodate a large packaging load and their low or absent immunogenicity. Furthermore, they are easy to produce at a large scale and preserve. Gene delivery reagents for non-viral vector are commonly used for transfecting a variety of cells and tissues. It is mainly composed of liposomes and non-liposome cationic polymers. According to the different head structures used, the non-viral cationic transfection reagents include a quaternary ammonium salt, amine, amino acid or polypeptide, guanidine salt, and a heterocyclic ring. This article summarizes these approaches and developments of types and components of transfection reagents and optimization of gene delivery. The optimization of mammalian cell transient recombinant protein expression system and cationic reagents for clinical or clinical trials are also discussed.
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http://dx.doi.org/10.1007/s00253-020-11028-6DOI Listing
January 2021

Woodchuck hepatitis post-transcriptional regulatory element improves transgene expression and stability mediated by episomal vectors in CHO-K1 cells.

Acta Biochim Biophys Sin (Shanghai) 2020 Dec;52(11):1285-1288

Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Xinxiang 453003, China.

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http://dx.doi.org/10.1093/abbs/gmaa105DOI Listing
December 2020

The CAG promoter maintains high-level transgene expression in HEK293 cells.

FEBS Open Bio 2021 Jan 3;11(1):95-104. Epub 2020 Dec 3.

Department of Biochemistry and Molecular Biology, Xinxiang Medical University, China.

The vast majority of therapeutic recombinant proteins are produced in mammalian cell lines. However, proteins generated in nonhuman cell lines, such as Chinese hamster ovary (CHO) cells, are decorated with human-like glycan structures that differ from those of human cells, and these may induce immunogenic responses in human cells. Human embryonic kidney cells (HEK293F) are also extensively used as hosts for the expression of recombinant therapeutic proteins, but their utility is limited by the low expression of transgenes in these cells. Here, we investigated recombinant protein expression from eight frequently used promoters in transfected HEK293F cells. The expression levels and stability of the transgenes were evaluated by flow cytometry and qRT-PCR. The most efficient expression (in terms of both mRNA and protein yields) was achieved using a cytomegalovirus (CMV) major immediate-early enhancer combined with the chicken beta-actin promoter (CAG) promoter, as compared to all other tested promoters under both transient and stable transfection conditions. In addition, application of mild hypothermia (i.e., 33 °C) after transfection improved the positive effect of the CMV enhancer fused to the chicken beta-actin promoter (CAG promoter) on enhanced green fluorescent protein (eGFP) expression. Although the temperature sensitivity of the CMV promoter is greater than that of CAG promoter, recombinant protein levels were still highest when expression was driven by the CAG promoter. When eGFP was replaced with hepatitis B surface antigen, the CAG promoter still showed the highest transgene expression. In conclusion, our data show that the CAG promoter is a strong promoter for recombinant protein expression in HEK293F cells.
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http://dx.doi.org/10.1002/2211-5463.13029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7780116PMC
January 2021

MS-based metabolite analysis of two licorice chalcones in mice plasma, bile, feces, and urine after oral administration.

Biomed Chromatogr 2021 Mar 29;35(3):e4998. Epub 2020 Oct 29.

Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou, China.

Isoliquiritigenin (ILG) and isoliquiritin (ILQ), two kinds of major flavonoids in licorice, are biological active substances with antioxidant, anti-inflammatory, and tumor-suppressive effects. However, their in vivo metabolites, possible material basis of this two licorice chalcones for the treatment of diseases, have not been studied completely. To determine the metabolism of ILG and ILQ, after oral administration of 100 mg/kg/day of these compounds for consecutive 8 days, the metabolites of these two licorice chalcones in mice plasma, urine, feces, and bile were determined using liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry in this study. The structures of those metabolites were tentatively identified according to their fragment pathways, accurate masses, characteristic product ions, metabolism law, and reference standards-matching. As a result, a total of 25 and 29 metabolites of ILG and ILQ were identified, respectively. Seven main metabolic pathways, oxidation and reduction, deglycosylation and glycosylation, dehydroxylation and hydroxylation, demethoxylation and methoxylation, acetylation, glucuronidation, and sulfation, were summarized to tentatively explain how the metabolites were biologically transformed. These results provide the important information on the metabolism of ILG and ILQ, which may be helpful for the further research of their pharmacological mechanism.
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http://dx.doi.org/10.1002/bmc.4998DOI Listing
March 2021

Chromatin-modifying elements for recombinant protein production in mammalian cell systems.

Crit Rev Biotechnol 2020 Nov 10;40(7):1035-1043. Epub 2020 Aug 10.

Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Xinxiang, China.

Mammalian cells are the preferred choice system for the production of complex molecules, such as recombinant therapeutic proteins. Although the technology for increasing the yield of proteins has improved rapidly, the process of selecting, identifying as well as maintaining high-yield cell clones is still troublesome, time-consuming and usually uncertain. Optimization of expression vectors is one of the most effective methods for enhancing protein expression levels. Several commonly used chromatin-modifying elements, including the matrix attachment region, ubiquitous chromatin opening elements, insulators, stabilizing anti-repressor elements can be used to increase the expression level and stability of recombinant proteins. In this review, these chromatin-modifying elements used for the expression vector optimization in mammalian cells are summarized, and future strategies for the utilization of expression cassettes are also discussed.
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http://dx.doi.org/10.1080/07388551.2020.1805401DOI Listing
November 2020

Fusion with matrix attachment regions enhances expression of recombinant protein in human HT-1080 cells.

J Biosci Bioeng 2020 Nov 6;130(5):533-538. Epub 2020 Aug 6.

Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Xinxiang 453003, Henan, China; International Joint Research Laboratory for Recombinant Pharmaceutical Protein Expression System of Henan, Xinxiang Medical University, Xinxiang 453003, Henan, China. Electronic address:

Like endogenous proteins, recombinant foreign proteins produced in human cell lines also need post-translational modifications. However, high and long-term expression of a gene of interest (GOI) presents significant challenges for recombinant protein production in human cells. In this work, the effect of human matrix attachment region elements (MARs), including the β-globin MAR (gMAR), chicken lysozyme MAR (cMAR), and a combination of these two, on the stable expression of GOI was assessed in human HT-1080 cells. After transfection with vectors containing the MAR elements and eGFP, stably HT-1080 cell pools were obtained under selective pressure. eGFP protein expression was analyzed by flow cytometry, while transgene copy number and eGFP mRNA expression levels were determined with qPCR and qRT-PCR technology. We found that MARs could not enhance transfection efficiency, but gMAR could significantly increase eGFP expression in stable HT-1080 cell pools by approximately 2.69-fold. Moreover, gMAR could also increase eGFP expression stability during long-term culture. Lastly, we showed that the effect of the MARs on transgenes was related to the gene copy number. In summary, this study found that MARs could both enhance the transgene expression and stability in HT-1080 cells.
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http://dx.doi.org/10.1016/j.jbiosc.2020.07.007DOI Listing
November 2020

Construction of an expression vector mediated by the dual promoter for prokaryotic and mammalian cell expression system.

Mol Biol Rep 2020 Jul 20;47(7):5185-5190. Epub 2020 Jun 20.

Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Jinsui Road, Xinxiang, 453003, Henan, China.

The aim of this study was to construct an expression vector mediated by the dual promoter that can simultaneously drive the recombinant protein production in eukaryotic and prokaryotic cells. The prokaryotic T7 promoter and ribosome binding site (RBS) was cloned downstream of CMV promoter in the eukaryotic expression vector pIRES-neo, and T7 termination sequence was inserted upstream of neomycin phosphotransferase gene to generate the dual promoter vector. The enhanced green fluorescent protein (eGFP) gene was used as reporter gene. Then, the resultant vector was transfected into Chinese hamster ovary (CHO) cells and transformed into Escherichia coli (E. coli) BL21, and the eGFP expression levels were analyzed by fluorescence microscopy, flow cytometry and Western blot, respectively. Fluorescence microscopy revealed that the eGFP was expressed in both CHO cells and E. coli BL21. Flow cytometry showed that the eGFP expression level had no significant difference between the dual promoter vector and control vector in transfected CHO cells. Western blot analysis indicated the eGFP expressed in transformed E. coli. In conclusion, a prokaryotic-eukaryotic double expression vector was successfully constructed, which has potential applications in rapid cloning and expression of recombinant proteins in both prokaryotic and eukaryotic expression systems.
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http://dx.doi.org/10.1007/s11033-020-05593-2DOI Listing
July 2020

Corrigendum to "Metabolic profiling of mice plasma, bile, urine and feces after oral administration of two licorice flavonones" [J. Ethnopharmacol. 257 15 July 2020 112892].

J Ethnopharmacol 2020 07 20;257:112977. Epub 2020 May 20.

Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou, China; Department of Pharmaceutical Analysis, Xuzhou Medical University, Xuzhou, China. Electronic address:

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http://dx.doi.org/10.1016/j.jep.2020.112977DOI Listing
July 2020

Expression vector cassette engineering for recombinant therapeutic production in mammalian cell systems.

Appl Microbiol Biotechnol 2020 Jul 6;104(13):5673-5688. Epub 2020 May 6.

International Joint Research Laboratory for Recombinant Pharmaceutical Protein Expression System of Henan, Xinxiang Medical University, Xinxiang, 453003, Henan, China.

Human tissue plasminogen activator was the first recombinant therapy protein that successfully produced in Chinese hamster ovary cells in 1986 and approved for clinical use. Since then, more and more therapeutic proteins are being manufactured in mammalian cells, and the technologies for recombinant protein production in this expression system have developed rapidly, with the optimization of both upstream and downstream processes. One of the most promising strategies is expression vector cassette optimization based on the expression vector cassette. In this review paper, these approaches and developments are summarized, and the future strategy on the utilizing of expression cassettes for the production of recombinant therapeutic proteins in mammalian cells is discussed.
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http://dx.doi.org/10.1007/s00253-020-10640-wDOI Listing
July 2020

Metabolic profiling of mice plasma, bile, urine and feces after oral administration of two licorice flavonones.

J Ethnopharmacol 2020 Jul 19;257:112892. Epub 2020 Apr 19.

Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou, China; Department of Pharmaceutical Analysis, Xuzhou Medical University, Xuzhou, China. Electronic address:

Ethnopharmacological Relevance: Licorice is an ancient food and medicinal plant. Liquiritigenin and liquiritin, two kinds of major flavonoes in licorice, are effective substances used as antioxidant, anti-inflammatory and tumor-suppressive food, cosmetics or medicines. However, their in vivo metabolites have not been fully explored.

Aim Of Study: To clarify the metabolism of liquiritigenin and liquiritin in mice.

Materials And Methods: In this study, we developed a liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry approach to determine the metabolites in mice plasma, bile, urine and feces after oral administration of liquiritigenin or liquiritin. The structures of those metabolites were tentatively identified according to their fragment pathways, accurate masses, characteristic product ions, metabolism laws or reference standard matching.

Results: A total of 26 and 24 metabolites of liquiritigenin or liquiritin were respectively identified. The products related with apigenin, luteolin or quercetin were the major metabolites of liquiritigenin or liquiritin in mice. Seven main metabolic pathways including (de)hydrogenation, (de)hydroxylation, (de)glycosylation, (de)methoxylation, acetylation, glucuronidation and sulfation were summarized to tentatively explain their biotransformation.

Conclusion: This study not only can provide the evidence for in vivo metabolites and pharmacokinetic mechanism of liquiritigenin and liquiritin, but also may lay the foundation for further development and utilization of liquiritigenin, liquiritin and then licorice.
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http://dx.doi.org/10.1016/j.jep.2020.112892DOI Listing
July 2020

Distance effect characteristic of the matrix attachment region increases recombinant protein expression in Chinese hamster ovary cells.

Biotechnol Lett 2020 Feb 27;42(2):187-196. Epub 2019 Nov 27.

Department of Biochemistry and Molecular Biology, Xinxiang Medical University, No. 601 Jinsui Road, Xinxiang, 453003, Henan, China.

Objectives: Previously, we have found that the matrix attachment region (MAR) may confer a 'distance effect' on transgene expression. This work aims to systematically explore the increased transgene expression in transfected Chinese hamster ovary (CHO) cells due to the characteristics of MAR and its mechanism.

Results: Compared with the control vector, 500 and 1000 bp DNA distances between MAR and the cytomegalovirus promoter can increase transgene expression by 1.77- and 1.56-fold, respectively. Meanwhile, transgene expression was not affected when 2000 and 2500 bp spacer DNAs were inserted, but a declining trend was observed when a 1500 bp spacer DNA was inserted. The vector containing a 500 bp DNA distance significantly increased the expression of the enhanced green fluorescent protein, and this increase was not related to transgene copy numbers.

Conclusions: A short DNA distance-containing MAR confers high transgene expression level in transfected CHO cells, but a distance threshold does not exist in the vector system.
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http://dx.doi.org/10.1007/s10529-019-02775-2DOI Listing
February 2020

Effects of different 2A peptides on transgene expression mediated by tricistronic vectors in transfected CHO cells.

Mol Biol Rep 2020 Jan 28;47(1):469-475. Epub 2019 Oct 28.

Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Jinsui Road, Xinxiang, 453003, Henan, People's Republic of China.

Multicistronic vectors can increase transgene expression and decrease the imbalance of gene expression in the Chinese hamster ovary (CHO) cell expression system. Small, self-cleaving 2A peptides have a high cleavage efficiency and are essential for constructing high-expression multicistronic vectors. In this study, we investigated the effects of two different 2A peptides on transgene expression in CHO cells via their mediating action on tricistronic vectors. The enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) genes were linked by the porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) peptides in a multicistronic vector. We transfected CHO cells with these vectors and screened for the presence of blasticidin-resistant colonies. Flow cytometry and real-time quantitative PCR (qPCR) were used to detect the expression levels of eGFP and RFP and the copy numbers of stably transfected cells. The results showed that P2A could enhance eGFP and RFP expression by 1.48- and 1.47-fold, respectively, compared to T2A. The expression levels of the genes were not proportional to their copy numbers. In conclusion, we found that P2A can effectively drive transgene expression in CHO cells and a potent 2A peptide can be used for recombinant protein production in the CHO cell system.
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http://dx.doi.org/10.1007/s11033-019-05153-3DOI Listing
January 2020

Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells.

Mol Biol Cell 2019 10 11;30(22):2761-2770. Epub 2019 Sep 11.

Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Xinxiang 453003, Henan, China.

Matrix attachment regions (MARs) can mediate the replication of vector episomes in mammalian cells; however, the molecular mode of action remains unclear. Here, we assessed the characteristics of MARs and the mechanism that mediates episomal vector replication in mammalian cells. Five shortened subfragments of β-interferon MAR fragments were cloned and transferred into CHO cells, and transgene expression levels, presence of the gene, and the episomal maintenance mechanism were determined. Three shortened MAR derivatives (position 781-1320, 1201-1740, and 1621-2201) retained full MAR activity and mediated episomal vector replication. Moreover, the three shortened MARs showed higher transgene expression levels, greater efficiency in colony formation, and more persistent transgene expression compared with those of the original pEPI-1 plasmid, and three functional truncated MARs can bind to SAF-A MAR-binding protein. These results suggest that shortened MARs are sufficient for replication and maintenance of episomes in CHO cells.
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http://dx.doi.org/10.1091/mbc.E19-02-0108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789156PMC
October 2019

Novel short synthetic matrix attachment region for enhancing transgenic expression in recombinant Chinese hamster ovary cells.

J Cell Biochem 2019 10 6;120(10):18478-18486. Epub 2019 Jun 6.

International Joint Research Laboratory for Recombiant Pharmaceutical Protein Expression System of Henan, Xinxiang Medical University, Xinxiang, Henan, China.

Matrix attachment regions (MARs) are DNA fragments with specific motifs that enhance transgenic expression; however, the characteristics and functions of these elements remain unclear. In this study, we designed and synthesized three short chimeric MARs, namely, SM4, SM5, and SM6, with different numbers and orders of motifs on the basis of the features and motifs of previously reported MARs, namely, SM1, SM2, and SM3, respectively. Expression vectors with six synthetic MARs flanking the down or upstream of the expression cassette for enhanced green fluorescence protein (EGFP) were constructed and introduced into Chinese hamster ovary (CHO) cells. Results indicated that the EGFP expression of the CHO cells with transfection bySM4, SM5, or SM6-containing vectors was higher than that of those containing SM1, SM2, or SM3 regardless of the MAR insertion position. The improving effect of SM5 was particularly pronounced. Transgenic expression was further enhanced with the increasing SM5 copy number. Bioinformatics analysis indicated that several arrangements of the DNA-binding motifs for CEBP, FAST, Hox, glutathione, and NMP4 may help increase transgenic expression levels and the average population of highly expressed cells. Our findings on novel synthetic MARs will help establish stable expression systems in mammalian cells.
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http://dx.doi.org/10.1002/jcb.29165DOI Listing
October 2019

Enhancing expression level and stability of transgene mediated by episomal vector via buffering DNA methyltransferase in transfected CHO cells.

J Cell Biochem 2019 09 9;120(9):15661-15670. Epub 2019 May 9.

International Joint Research Laboratory for Recombiant Pharmaceutical Protein Expression System of Henan, Xinxiang Medical University, Xinxiang, Henan, China.

Nonviral episomal vectors present attractive alternative vehicles for gene therapy applications. Previously, we have established a new type of nonviral episomal vector-mediated by the characteristic motifs of matrix attachment regions (MARs), which is driven by the cytomegalovirus (CMV) promoter. However, the CMV promoter is intrinsically susceptible to silencing, resulting in declined productivity during long-term culture. In this study, Chinese hamster ovary (CHO) cells and DNA methyltransferase-deficient (Dnmt3a-deficient) CHO cells were transfected with plasmid-mediated by MAR, or CHO cells were treated with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine. Flow cytometry, plasmid rescue experiments, fluorescence in-situ hybridization (FISH), and bisulfite sequencing were performed to observe transgene expression, its state of existence, and the CpG methylation level of the CMV promoter. The results indicated that all DNA methylation inhibitor and methyltransferase deficient cells could increase transgene expression levels and stability in the presence or absence of selection pressure after a 60-generation culture. Plasmid rescue assay and FISH analysis showed that the vector still existed episomally after long-time culture. Moreover, a relatively lower CMV promoter methylation level was observed in Dnmt3a-deficient cell lines and CHO cells treated with 5-Aza-2'-deoxycytidine. In addition, Dnmt3a-deficient cells were superior to the DNA methylation inhibitor treatment regarding the transgene expression and long-term stability. Our study provides the first evidence that lower DNA methyltransferase can enhance expression level and stability of transgenes mediated by episomal vectors in transfected CHO cells.
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http://dx.doi.org/10.1002/jcb.28835DOI Listing
September 2019

Identification of consensus sequence from matrix attachment regions and functional analysis of its activity in stably transfected Chinese hamster ovary cells.

J Cell Biochem 2019 08 7;120(8):13985-13993. Epub 2019 Apr 7.

College of Forensic Medicine, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi, China.

Matrix attachment regions (MARs) can enhance transgene expression levels and maintain stability. However, the consensus sequence from MARs and its functional analysis remains to be examined. Here, we assessed a possible consensus sequence from MARs and assessed its activity in stably transfected Chinese hamster ovary (CHO) cells. First, we analyzed the effects of 10 MARs on transfected CHO cells and then analyzed the consensus motifs from these MARs using a bioinformatics method. The consensus sequence was synthesized and cloned upstream or downstream of the eukaryotic vector. The constructs were transfected into CHO cells and the expression levels and stability of enhanced green fluorescent protein were detected by flow cytometry. The results indicated that eight of the ten MARs increased transgene expression in transfected CHO cells. Three consensus motifs were found after bioinformatics analyses. The consensus sequence tandemly enhanced transgene expression when it was inserted into the eukaryotic expression vector; the effect of the addition upstream was stronger than that downstream. Thus, we found a MAR consensus sequence that may regulate the MAR-mediated increase in transgene expression.
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http://dx.doi.org/10.1002/jcb.28673DOI Listing
August 2019

Human genome-derived TOP1 matrix attachment region enhances transgene expression in the transfected CHO cells.

Biotechnol Lett 2019 Jul 5;41(6-7):701-709. Epub 2019 Apr 5.

International Joint Research Laboratory for Recombiant Pharmaceutical Protein Expression System of Henan, Xinxiang Medical University, Xinxiang, 453003, Henan, China.

Objectives: To investigate the effect of full-length fragment of DNA topoisomerase I gene (TOP1) matrix attachment regions (MARs) originating from the human genome on transgene expression in Chinese hamster ovary (CHO) cells and explore the underlying mechanisms.

Results: Results showed that TOP1 MAR cannot only enhance the transient and stable transgenic expression of enhanced green fluorescence protein (EGFP) but also increase long-term stability and ratio of positive colonies in transfected CHO cells with TOP1 MAR at the 5' or 3' ends of the EGFP expression cassette. Interestingly, the CHO cells were transfected with the 5',3' TOP1 MAR-containing vector featured the highest transient and stable expression, whereas those with the 3' TOP1 MAR-containing vector exhibited the most effective stability and ratio of positive colonies. We also observed that transgene copy numbers and mRNA of egfp gene were correlated with the expression levels of EGFP protein in polyclonal CHO cells. However, the heterogeneity of expression in monoclonal CHO cells was unaffected by transgene copy number.

Conclusions: The findings may aid in the potential application of TOP1 MAR in expression enhancement of recombinant proteins in mammalian cells.
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http://dx.doi.org/10.1007/s10529-019-02673-7DOI Listing
July 2019

Interaction of clopidogrel and fufang danshen dripping pills assay in coronary heart disease based on non-target metabolomics.

J Ethnopharmacol 2019 Apr 28;234:189-196. Epub 2019 Jan 28.

Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou, Jiangsu, China. Electronic address:

Ethnopharmacological Relevance: Clopidogrel is the recommended treatment by current clinical practice guidelines to prevent adverse cardiovascular events in patients with coronary heart disease (CHD), but this treatment regimen still fails and 5-40% patients display inadequate antiplatelet responses. Fufang Danshen Dripping Pill (FDDP), a Chinese patient drug, was used as the combination with clopidogrel to improve the therapeutic effect. However, the mechanism of the interaction between clopidogrel and FDDP has not been elucidated.

Materials And Methods: We have used non-targeted metabolism method based on GC-MS and LC-MS for the investigation of drug interactions between clopidogrel and FDDP. 63 patients were divided into four groups with different dosage regimen and the serum samples were collected for the analysis.

Results: We have found 5 and 55 differential metabolites between health volunteer group and CHD patients group, respectively. The contents of these differential metabolites had diverse changes in clopidogrel group, FDDP group, and drug combination group, indicating that the clopidogrel and FDDP combination can adjust the glycometabolism, lipid metabolism, and phospholipid metabolism, sequentially made the content of downstream related metabolites towards to the health volunteer group.

Conclusion: This work has explained the mechanism of the interaction between clopidogrel and FDDP from the point of view of metabolic product change, and revealed the potential metabolic pathways it affects, which provided the new ideas for clinical medication.
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http://dx.doi.org/10.1016/j.jep.2019.01.030DOI Listing
April 2019

Off-line two-dimensional liquid chromatography coupled with diode array detection and quadrupole-time of flight mass spectrometry for the biotransformation kinetics of Ginkgo biloba leaves extract by diabetic rat liver microsomes.

J Chromatogr B Analyt Technol Biomed Life Sci 2019 Mar 23;1109:1-9. Epub 2019 Jan 23.

Key Laboratory of New Drug Research and Clinical Pharmacy of Jiangsu Province, School of Pharmacy, Xuzhou Medical University, Xuzhou 221004, China; Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical University, Xuzhou 221004, China. Electronic address:

Ginkgo biloba leaves extract (GBE), one of the most widely used traditional Chinese medicines worldwide, can be used for the treatment of diabetes mellitus (DM). However, its biotransformation in liver is not fully known under the state of DM. In this study, an off-line hydrophilic interaction × reversed-phase two-dimensional liquid chromatography (HILIC × RP 2D-LC) system coupled with diode array detection (DAD) and quadrupole time-of-flight mass spectrometry (q/TOF-MS) was established for the qualification and quantification of the biotransformation of GBE in normal and diabetic rat liver microsomes (RLMs). 6 metabolites were tentatively identified according to the exact molecular weights and the characteristic fragment ions provided by q/TOF-MS data. The results of metabolic stability showed that the metabolic ratio of four target compounds including quercetin, genistein, kaempferol and isorhamnetin in diabetic RLMs were significantly enhanced when comparing with normal RLMs. The results of enzyme kinetics showed that compared with normal RLMs, the Michaelis-Menten constant (K) value of genistein was obvious increased while its maximal velocity (V) and intrinsic clearance (CL) values were significantly decreased by diabetic RLMs, and the V and CL values of kaempferol and isorhamnetin were notably enhanced while their K values were markedly reduced. For the half-time (t) values of four target compounds and the K, V and CL values of quercetin, there were not statistically significant changes between normal and diabetic RLMs. The results suggest that the developed off-line 2D LC-DAD-q/TOF-MS method is an easy and accurate approach for the study of GBE biotransformation in RLMs and may provide the essential data for further pharmacological and clinical studies of GBE.
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http://dx.doi.org/10.1016/j.jchromb.2019.01.015DOI Listing
March 2019

Two human MARs effectively increase transgene expression in transfected CHO cells.

J Cell Mol Med 2019 02 18;23(2):1613-1616. Epub 2018 Nov 18.

Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Henan, China.

Matrix attachment regions (MARs) can enhance the expression level of transgene in Chinese hamster ovaries (CHO) cell expression system. However, improvements in function and analyses of the mechanism remains unclear. In this study, we screened two new and more functional MAR elements from the human genome DNA. The human MAR-3 and MAR-7 element were cloned and inserted downstream of the polyA site in a eukaryotic vector. The constructs were transfected into CHO cells, and screened under G418 to produce the stably transfected cell pools. The expression levels and stability of enhanced green fluorescent protein (eGFP) were detected by flow cytometry. The transgene copy number and transgene expression at mRNA level were detected by quantitative real-time PCR. The results showed that the expression level of eGFP of cells transfected with MAR-containing vectors were all higher than those of the vectors without MARs under transient and stably transfection. The enhancing effect of MAR-7 was higher than that of MAR-3. Additionally, we found that MAR significantly increased eGFP copy numbers and eGFP gene mRNA expression level as compared with the vector without. In conclusion, MAR-3 and MAR-7 gene can promote the expression of transgene in transfected CHO cells, and its effect may be related to the increase of the number of copies.
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http://dx.doi.org/10.1111/jcmm.14018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349195PMC
February 2019

Chemical Profiling and Comparison of Sangju Ganmao Tablet and Its Component Herbs Using Two-Dimensional Liquid Chromatography to Explore Compatibility Mechanism of Herbs.

Front Pharmacol 2018 16;9:1167. Epub 2018 Oct 16.

Department of Pharmaceutical Analysis, Xuzhou Medical University, Xuzhou, China.

Sangju Ganmao tablet (SGT), a well-known Chinese patent medicine used to treat cold symptoms, is made from eight herbal medicines. In this study, an off-line hydrophilic interaction × reversed-phase two-dimensional liquid chromatography (HILIC × RP 2D-LC) method was developed to comprehensively separate the chemical constituents of SGT. Through optimization of the experimental conditions, a total of 465 peaks were finally detected in SGT, and the structures of 54 selected compounds were fully identified or tentatively characterized by quadrupole time-of-flight mass spectrometry (qTOF-MS) analysis. The established 2D-LC analysis showed high orthogonality (63.62%) and approximate 11-fold improvement in peak capacity (2399 and 1099, obtained by two calculation methods), in contrast to conventional one-dimensional RPLC separation. The eight component herbs of SGT were also respectively separated by using the 2D-LC system, and we found that a total of 12 peaks detected in SGT were not discovered in any component herbs. These newly generated chemical constituents would benefit better understanding of the compatibility mechanism of the component herbs. The strategy established in this study could be used for systematic chemical comparison of SGT and its component herbs, which contributes to exploration of herbal compatibility mechanism.
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http://dx.doi.org/10.3389/fphar.2018.01167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6198175PMC
October 2018

Burn Wound Bacteriological Profiles, Patient Outcomes, and Tangential Excision Timing: A Prospective, Observational Study.

Ostomy Wound Manage 2018 09;64(9):28-36

Research Center of Trauma and Orthopedic, Xinxiang Medical University, Xinxiang, Henan, China.

Purpose: Because infection can thwart burn healing, microorganisms, their susceptibility patterns, and the effect of tangential excision timing on outcomes of burn patients were examined.

Methods: A prospective, observational study was conducted that involved 318 patients with deep second-degree burns from a gas explosion treated in Xinxiang, Henan, China between January 2009 and December 2016. Patient demographic data, culture and antimicrobial susceptibility test results, and outcome variables (resuscitation fluid volume, signs of shock, body temperature, heart rate, and time to wound healing) were analyzed. Outcomes were compared among patients who had early (<24 hours), middle (2 to 7 days), and late (> 7 days) post burn excision.

Results: Bacterial culture and drug sensitivity data were available for 314 of the 318 persons with burns >10% of total body surface area (TBSA). Of the 486 bacterial isolates, 330 (67.9%) were gram-negative and 156 (32.1%) were gram-positive. The number of isolates and resistance to third-generation cephalosporins increased over time. Patients having early tangential excision had significantly lower heart rate (P <.05) and reduced time to healing (P <.01) than patients in the middle or late excision group.

Conclusion: Early tangential excision was found to be safe and to facilitate healing.
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September 2018

Construction strategies for developing expression vectors for recombinant monoclonal antibody production in CHO cells.

Mol Biol Rep 2018 Dec 6;45(6):2907-2912. Epub 2018 Sep 6.

Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Jinsui Road, Xinxiang, 453003, Henan, People's Republic of China.

Recent years have seen the use of recombinant proteins in the treatment of different diseases. Among them, monoclonal antibodies (mAbs) are currently the fastest growing class of bio-therapeutic recombinant proteins. Chinese hamster ovary (CHO) cells are the most commonly used host cells for production of these recombinant mAbs. Expression vectors determine the expression level and quality of recombinant mAbs. Currently, few construction strategies for recombinant mAbs expression vectors in CHO cells have been developed, including monocistronic vector, multiple-promoter expression vector, and tricistronic vector mediated by internal ribosome entry site (IRES) or Furin-2A element. Among them, Furin-2A-mediated vector is an effective approach due to advantages of high "self-cleavage" efficiency, and equal expression of light and heavy chains from a single open reading frame. Here, we have reviewed the progress in development of different strategies for constructing recombinant mAb expression vectors in CHO cells and its potential advantages and disadvantages.
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http://dx.doi.org/10.1007/s11033-018-4351-0DOI Listing
December 2018

Retraction Note: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells.

Sci Rep 2018 Sep 4;8(1):13482. Epub 2018 Sep 4.

Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Xinxiang, 453003, Henan, China.

This paper has been retracted at the request of the authors.
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http://dx.doi.org/10.1038/s41598-018-31224-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123454PMC
September 2018

Enhanced transgene expression using cis-acting elements combined with the EF1 promoter in a mammalian expression system.

Eur J Pharm Sci 2018 Oct 12;123:539-545. Epub 2018 Aug 12.

Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Xinxiang 453003, Henan, China; International Joint Research Laboratory for Recombinant Pharmaceutical Protein Expression System of Henan, Xinxiang Medical University, Xinxiang, Henan, China. Electronic address:

Transgene expression in eukaryotic cells suffers from epigenetic effects that result in low or unstable transgene expression and high clonal variability. The use of epigenetic regulators is a promising approach to alleviating such unwanted effects. we investigated the effect of the strong human EF1-α promoter combined with six cis-acting elements on transgene expression in transfected Chinese hamster ovary (CHO) cells. The six elements included the human cytomegalovirus immediate early core promoter element (hCPE), three synthetic enhancer element (SEE1, SEE2, Syn1), human cytomegalovirus immediate early enhancer (hCMV-IEE), and a regulatory element isolated from CHO-K1 genomic DNA (C77). The respective vectors were transfected into CHO cells, and stably transfected cell pools were screened and analyzed for transgene expression. The results showed that SEE1 increased transient expression most strongly. However, hCPE enhanced eGFP transgene expression most significantly in stably transfected CHO cells, by about 2.45-fold. Erythropoietin expression analysis showed that hCPE induced the highest EPO productivity, followed by hCMV-IEE. We found that the enhancing effect of hCPE and hCMV-IEE was related with transgene copy number. In conclusion, we found that hCPE and hCMV-IEE cis-acting elements combine with EF1-α can increase recombinant protein expression in CHO cells.
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http://dx.doi.org/10.1016/j.ejps.2018.08.016DOI Listing
October 2018

A Simple, Time-Saving Dye Staining of Proteins in Sodium Dodecyl Sulfate-Polyacrylamide Gel Using Coomassie Blue.

Methods Mol Biol 2018 ;1853:31-35

Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Henan, China.

Most traditional post-electrophoretic processes need several hours to several days to finish the whole staining process and traditional staining solutions all contain methanol, acetic acid, or phosphoric acid, which not only produce the unpleasant smell but also cause environmental pollution. Here a fixation-free, fast protein staining method in sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue is described. The protocol includes only staining and quick washing steps, can be completed in 0.5 h. It has a sensitivity of 10 ng. In addition, the dye stain does not contain any acid or methanol.
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http://dx.doi.org/10.1007/978-1-4939-8745-0_5DOI Listing
May 2019

CRISPR/Cas9-mediated gene knockout for DNA methyltransferase Dnmt3a in CHO cells displays enhanced transgenic expression and long-term stability.

J Cell Mol Med 2018 09 30;22(9):4106-4116. Epub 2018 May 30.

International Joint Research Laboratory for Recombiant Pharmaceutical Protein Expression System of Henan, Xinxiang Medical University, Xinxiang, Henan, China.

CHO cells are the preferred host for the production of complex pharmaceutical proteins in the biopharmaceutical industry, and genome engineering of CHO cells would benefit product yield and stability. Here, we demonstrated the efficacy of a Dnmt3a-deficient CHO cell line created by CRISPR/Cas9 genome editing technology through gene disruptions in Dnmt3a, which encode the proteins involved in DNA methyltransferases. The transgenes, which were driven by the 2 commonly used CMV and EF1α promoters, were evaluated for their expression level and stability. The methylation levels of CpG sites in the promoter regions and the global DNA were compared in the transfected cells. The Dnmt3a-deficent CHO cell line based on Dnmt3a KO displayed an enhanced long-term stability of transgene expression under the control of the CMV promoter in transfected cells in over 60 passages. Under the CMV promoter, the Dnmt3a-deficent cell line with a high transgene expression displayed a low methylation rate in the promoter region and global DNA. Under the EF1α promoter, the Dnmt3a-deficient and normal cell lines with low transgene expression exhibited high DNA methylation rates. These findings provide insight into cell line modification and design for improved recombinant protein production in CHO and other mammalian cells.
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http://dx.doi.org/10.1111/jcmm.13687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6111867PMC
September 2018

[Effects of Different Promoters and Combinations on Transgene Expression of Recombinant CHO Cells].

Sichuan Da Xue Xue Bao Yi Xue Ban 2018 Jan;49(1):18-23

Department of Biochemistry and Moiecular Biology,Xinxiang Medical University,Xinxiang 453003,China.

Objective: To analyze the effects of different promoters and matrix attachment region (MAR) on the expression of transgene in Chinese hamster ovary (CHO) cells.

Methods: The expression vector was constructed by the combination of beta globin MAR with the human cytomegalovirus immediate-early promoter and simian virus 40 promoter. These vectors were transfected into CHO cells,after 48 h,the transient expression of enhanced green fluorescent protein (eGFP) was observed; G418 was used to screen stably transformed cell lines,and the expression level of in CHO cells was analyzed by flow cytometry. The relative copy numbers of were analyzed by qPCR.

Results: Without expression vector,the expression of which was driven by promoter was stronger than that of promoter; could increase the expression level of driven by promoter,but did not show any enhancement in promoter. The expression level of which containing on both sides was stronger than that of on one side driven by promoter; After G418 screening,the expression level of containing driven by promoter wasunstable,the fluorescence gradually weakened,therefore,we only analyzed the expression vector stably expressing the gene driven by promoter by flow cytometry and qPCR. Compared with the expression vector without containing promoter,flow cytometry showed that the expression levels of on one and both sides with were increased by 9.85-fold and 12.94-fold,respectivley; The result of qPCR showed that the copy number of the gene without was set to 1,the copy number of the gene in the expression vector driven by with on one side and both sides were 3.68-fold and 9.25-fold,respectively.

Conclusion: The activity of promoter is stronger than that of promoter. can enhance the expression levels of transgene,which may be related to the increase of gene copy number.
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January 2018

Human rhinovirus internal ribosome entry site element enhances transgene expression in transfected CHO-S cells.

Sci Rep 2018 04 27;8(1):6661. Epub 2018 Apr 27.

Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Xinxiang, 453003, Henan, China.

Chinese hamster ovary (CHO) cells are mainly used for recombinant protein production. However, the unstable transgene expression and lower transgene copy numbers are the major issues need to be resolved. Here, eleven internal ribosome entry site (IRES) elements from viral and cellular IRES were evaluated for foreign gene expression in CHO-S cells. We constructed eleven fusing plasmids containing different IRES sequences downstream of the enhanced green fluorescent protein (EGFP) gene. EGFP expression was detected by flow cytometry and the transgene copy number was evaluated by quantitative PCR. The erythropoietin (EPO) protein was also used to assess the stronger IRES. The results showed that IRES from human rhinovirus (HRV) exhibited the highest EGFP expression level under transient and stable transfections. The EGFP expression level of vector with IRES from HRV was related to the gene copy number in stably transfected CHO-S cells. Moreover, IRES from HRV induced higher expression level of EPO compared with one mutant IRES from EMCV in transfected cells. In conclusion, IRES from HRV can function as a strong IRES element for stable expression in CHO-S cells, which could potentially guide more effective foreign gene expression in CHO-S cells.
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http://dx.doi.org/10.1038/s41598-018-25049-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923211PMC
April 2018