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Construction of Expression Vector Containing TβRⅡ-IgG1 Fc Genes and in Vitro Study on Its Anti-fibrotic Effects on Human Hepatocytes

Author: GaoWei
Tutor: ZhouYongXing；NieQingHe
School: Fourth Military Medical University
Course: Internal Medicine
Keywords: TβRII-IgG1 Fc gene liver fibrosis transforming growth factor β1 gene therapy retroviral vector tissue inhibitors of metalloproteinases hepatocyte apoptosis
CLC: R346
Type: PhD thesis
Year: 2005
Downloads: 136
Quote: 0
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Abstract


Objective: The aim of this study was to construct recombinant human retroviral vector, which carries soluble TGF β1 Type II receptor （TβRII） gene, and evaluate its anti-fibrotic effects on human hepatocytes in vitro. Methods: The T βRII -IgG1 Fc gene was amplified by RT-PCR and the purified PCR products were ligated into the sequencing vector pGEM-T-Easy. The positive clones were selected and the recombinant plasmid was purified, which was further identified by restriction endonucleases and was sequenced . T βRII -IgG1 Fc gene was subcloned into retroviral vector pLXSN by recombinant DNA technology. pL（T0 RII -IgGi Fc）SN was packaged with PA317 and screened by G418 to obtain the positive clones, which was able to produce steady retrovirus. The integration and expression of T P RII -IgGi Fc gene in PA317 cells were identified by RT-PCR. Then we examined the anti-fibrotic effects of the retro viral vector on human hepatocyte line HL-7702. The growth state of HL-7702 was estimated by MTT and flow cytometry, and the changes of TIMPs were studied by immunohischemistry and in situ hybridization methods. The apoptosis rate was determined by flow cytometry, TUNEL technology, electronic microscope. Results: We construct the retroviral vector pL（T 3 RII -IgGi Fc）SN successfully. PCR demonstrated that T 0 RII -IgGi Fc gene was integrated into the cells genome and expressed at the mRNA level. There was significant difference in cell proliferation between HL-7702/TGF-betai cells and HL-7702/T 0 RII -IgGi Fc cells, however, there was no significant difference between HL-7702/T 3 RII -IgGi Fc and HL-7702 cells. Both flow cytometry and TUNEL technology revealed that the apoptosis rates of HL-7702/T 0 RII -IgGi Fc cells and HL-7702 cells were much lower than those of HL-7702/TGF-betai cells. Furthermore, the apoptotic phenomena and apoptotic body were observed in HL-7702/TGF-betai cells under microscope, but not in HL-7702/T 0 RII -IgGi Fc and HL-7702 cells. The expression of TIMPs protein and mRNA in HL-7702/TGF-betal cells was higher than the HL-7702/T 0 RII -IgGi Fc and HL-7702 cells in vitro. The expression of TIMP-1 and TIMP-2 was not obviously changed in the HL-7702/T 0 RII -IgGi Fc and HL-7702 groups. Conclusion: Transforming growth factor-betai is a key mediator in establishing liver fibrosis. Therefore, TGF-betai as a causative agent may serve as a primary target for antifibrotic gene therapy approaches. We have shown that the retroviral delivery of pL(T 3 RII -IgGi

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