| Liver fibrosis is the common pathologic basis and characteristics in the occurrence and development of chronic hepatic diseases. Hepatic stellate cells (HSC) play important role in hepatic fibrogenesis. Previous studies found that cyclic RGD could specifically combine with VI type collagen receptor in HSC surface. Small heterodimer partner (SHP), a nuclear receptor, serving as a key step in FXR-SHP regulatory enzyme chain, can transform the HSC to static phenotype when highly expressed in the cell, and promote apoptosis,exert the resolution of fibrosis. Polyenthylenimine (PEI), which is a recently developed positive ion polymer non-virus carrier, is modified by polyethylene glycol to increase its solubility. The cytotoxicity was decreased and circulation time in vivo could be prolonged.The main objective of this study is to construct the positive ion polymer carrier, RGD-PEG-PEI. which will be specifically targeting the HSC, and taking along the SHP gene. The anti-fibrosis effects of RGD-PEG-PEI/SHP will be evaluated both in vitro and in vivo experiment, and its mechanism of fibrisis remission will be explored and discussed.The main contents of this study include:To investigate RGD (Arg-Gly-Asp) modified PEG-PEI as gene cascade carrier; To evaluate the transfection effect of RGD-PEG-PEI/SHP complex in the activation of HSC; To research the therapeutic effect of RGD-PEG-PEI/SHP complex for rats liver fibrosis.Part one:To investigate RGD (Arg-Gly-Asp) polypeptide modified PEG-PEI as gene cascade carrierObjective:To address the synthetic procedure of RGD polypeptide modified PEG-PEI as gene therapeutic carrier, and its physical and chemical characteristics. To set up a gene therapeutic carrier system, specifically targeting HSC which contains plasmid DNA-SHP. Methods:The bifunctional gene product NHS-PEG-MAL was commercially produced by Nektar Co., USA. On the basis of different reaction activity of NHS and MAL RGD-PEG-PEI complex could be synthesized by simple chemical additive reaction. Mass spectral analysis was used to identify the product and calculate the reactive efficiency. To construct The SHP-pEGFP eukaryotic expression carrier was constructed, and RGD-PEG-PEI/SHP complex with different N/P ratio was synthesized. Entrapped-efficency of the polyplexes was determined by gel electrophoresis. Particle size and potential of the complex was analyzed with particle and potential measuring instrument. The cytotoxicity was evaluated with MTT method.Results:Two NHS-PEG-MAL active amine groups separately conjugated with RGD and PEC. Mass spectral analysis demonstrated the conjugation was successful and completed. The Entrapped efficiency of 1% RGD-PEG-PEI/SHP complex was over 99%. The particle size was 157nm to 160nm. The size decreased and the zeta potential increased with the increased N/P ratio of the polyplexes. The cytotoxicity of 1% RGD-PEG-PEI/SHP polyplexes were significantly lower than PEI/NDA (P<0.01) in different N/P ratio. Conclusion:The RGD polypeptide with targeting ability and PEG modified gene therapeutic carrier PEI was successfully conjugated. The evaluation of 1% RGD-PEG-PEI/SHP polyplexes characteristics showed high entrapped-efficiency, stable particle size and potential, with low cytotoxicity.Part Two:To evaluate the transfection efficiency of RGD-PEG-PEI/SHP polyplexes to activated HSCObjectives:To compare the targeting effect and anti-fibrotic efficiency with 1% RGD-PEG-PEI/SHP, PEG-PEI/SHP, PEI/SHP and SHP polyplexes in activated rats HSC. Methods:HSC were isolated from normal male SD rats (400-450g,14 weeks old) by continuous infusion with pronase E and type IV collagenase and purified with density gradient centrifugation. These HSC were cultured and passaged. The green fluorescent protein reporting gene was detected by flow cytometry to evaluate the in vitro transfection rate. The inhibition of?-SMA,?1 (?) procollagen. tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2, matrix metalloproteinase-2 (MMP-2) and transforming growth factor-?1 (TGF-?1) mRNA level in HSC was detected by fluorescent quantitative qRT-PCR. Western blot was used to compare the inhibition of?-SMA and type?collagen formation.Results:The flow cytometry demonstrated that the fluorescent intensity of 1% RGD-PEG-PEI/SHP trasfection activated HSC was significantly higher than other treated groups (P<0.01). SHP mRNA level in 1%RGD-PEG-PEI/SHP trasfection activated HSC was significantly increased comparing with other groups (P<0.01). While the intracellular mRNA levels of?-SMA, TIMP-1, TIMP-2, TGF-?1 and?1 (?) procollagen were decreased (P<0.01). There was no influence on the MMP-2 mRNA level (P>0.05). The SHP expression level of 1%RGD-PEG-PEI/SHP increased significantly (P<0.05). Moreover, the a-SMA and type I collagen expression decreased significantly comparing with other groups (P<0.05).Conclusion:In 1%RGD-PEG-PEI/SHP treated group, the transfection efficiency for activated HSC increased significantly. The SHP mRNA highly expressed, mRNA level of?-SMA, TIMP-1, TIMP-2, TGF-?1 and?1 (?) procollagen which inducing liver fibrosis, were down-regulated.?-SMA and type I collagen production was also decreased.Part Three:Therapeutic effect of RGD-PEG-PEI/SHP polyplexes on liver fibrosis in ratsObjectives:To evaluate the therapeutic effects of 1%RGD-PEG-PEI/SHP for liver fibrosis in rats and its mechanism.Methods:Liver fibrosis was induced in rats by injecting thioacetamide intraperitoneally for 10 weeks.60 rats were divided into 5 groups (n=12) according to injected agents: 1%RGD-PEG-PEI/SHP, PEG-PEI/SHP, PEI/SHP, SHP and normal saline as control. After injected these agents every 3 weeks and 6 weeks, the liver pathologic stage,?-SMA positive cell apoptotic rate and anti-fibrotic gene mRNA level were compared among each group.Results:Comparing with other treatment groups,1%RGD-PEG-PEI/SHP injection obviously relieved the liver fibrosis in rats. Fibrotic staging and inflammatory staging decreased significantly (P<0.01). Apoptotic rate of a-SMA positive cell induced by 1%RGD-PEG-PEI/SHP dramatically increased (P<0.01). After 1%RGD-PEG-PEI/SHP treatment, mRNA level of?-SMA, TIMP-1, TIMP-2, TGF-?1 and?1 (?) procollagen decreased significantly in liver tissue comparing with other treatment groups (P<0.01) There was no effect on MMP-2 mRNA level (P>0.05)Conclusion:1%RGD-PEG-PEI/SHP can induce the apoptosis of?-SMA positive cell, down-regulate the liver fibrosis gene expression, and reverse the liver fibrosis. It can promote targeting activity of anti-fibrosis in liver. |
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