| Hepatic fibrosis is a common pathological process of chronic hepatic disease, which can lead to cirrhosis and increase risk for hepatocellular carcinoma. Advanced fibrosis and cirrhosis were generally considered to be irreversible conditions even after removal of the injurious agent. Over the past 15 years, substantial progress has been made in understanding the cellular and molecular regulation of hepatic fibrosis. It is now clear that the accumulation of extracellular matrix (ECM) in fibrotic diseases of the liver is not a static or unidirectional event but a dynamic and regulated process that is amenable to intervention. At present, the common sense is that cirrhosis could be prevented and hepatic fibrosis could be reversed effectively when the right therapeutic strategy is applied. With the development of the technology of gene therapy and deep study of the mechanism of hepatic fibrosis, the experimental gene therapy of hepatic fibrosis has been becoming the main strategy on treating hepatic fibrosis. Augmenter of liver regeneration (ALR) was originally cloned from liver tissue of neonatal rats by Hagiya in 1994. Many studies had revealed that ALR appeared to be an important regulator of liver regeneration and had trophic effects on regenerating liver and potent anti-hepatitis effects. The pcDNA3-ALR recombinant plasmid had been constructed by our laboratory. The present research was to observe the expression of ALR recombinant plasmid in live tissue of rats with immune hepatic fibrosis and the therapeutic effects of anti-fibrosis and to explore the molecular mechanism of anti-fibrosis from the expression of TGF-β1 and TIMP-1 on the level of molecule and protein. In addition, different carrier with different dose recombinant plasmid through different introducing routes can lead to different therapeutic effects. This research was also to screene more excellent carrier, fit dose and more appropriate introducing routes as well as their best combination through orthogonal test. Part 1 The preparation of rat ALR recombinant plasmid and expression in rat liver tissue Objective: To prepare pcDNA3-ALR recombinant plasmid and to observe the expression in rat liver tissue. Methods: Administering intraperitonealy with porcine serum developed the immune hepatic fibrosis model. The pcDNA3-ALR recombinant plasmid was extracted abundantly and purified from competent E.coli DH5 and then was administered the rats with immune hepatic fibrosis. The expression of ALR protein in liver and nephridium tissue was detected by immunohistochemistry staining and western bloting and the expression of ALR mRNA was detected by RT-PCR with two specific gene sequences T7 and SP6 consist in pcDNA3 plasmid as the primers. All results were analyzed statistically by SPSS 11.5 software. Results: ALR was expressed in all groups'rats liver tissue in relatively low abundance in normal group but relatively high abundance in ALR treat group. The immunohistochemistry results by image analysis for cell staining density were 0.109±0.01, 0.159±0.02 and 0.198±0.04 in normal group, model group and ALR group respectively. There were significant differences between model and normal group (P<0.01) as well as ALR group and model group (P<0.01). The results of western bloting were similar to immunohistochemistry. The expression of ALR was not found in nephridium tissue of all groups'rats. The specific 550bp gene fragment that was amplified by RT-PCR with a couple of especial primers was found in ALR group but not in normal and model group, which was also coincident with fragment amplified by PCR with recombinant plasmid extracted directly from competent E.coli DH5. NO amplified fragment was found in nephridium tissue of all groups'rats.Conclusion: The expression of ALR was relatively low abundance in normal rats liver tissue but increased markedly in immune injured liver tissue. The expression of exogenous ALR could be found in pcDNA3-ALR recombinant plasmid treat group rats liver tissue and could superpose on the expression of endogenous ALR. The expression of ALR was not found in nephridium tissue of all groups'rats. Part 2 Effects and molecular mechanisms of ALR recombinant plasmid on rats with experimental hepatic fibrosis Objective: To investigate the effects of ALR recombinant plasmid on rat hepatic fibrosis and to explore their molecular mechanisms. Methods: Administering intraperitonealy with porcine serum developed the immune hepatic fibrosis model. The all rats were divided randomly into 5 groups: normal group, model group, colchicine group, pcDNA3 (empty plasmid) group and pcDNA3-ALR (recombinant plasmid) group. Then saline, colchicine, pcDNA3 and pcDNA3-ALR recombinant plasmid were administered to them respectively. At the end of the 4th week, rats were euthanized. Serum markers of hepatic fibrosis and hydroxyproline (Hyp) of liver tissue were detected and histopathological changes were graded. Expression of type I, III collagen, TGF-β1 and TIMP-1 were detected by immunohistochemistry and expression of TGF-β1 mRNA and TIMP-1 mRNA was examined by RT-PCR. Results: The serum level of ALT, AST and ALB in model group, colchicine group and ALR group was 108.67±16.13, 201.22±22.84, 24.6±5.8; 73.20±12.55, 182.10±48.11, 34.2±4.1 and 78.40±14.60, 160.90±23.99, 37.3±4.6 respectively. Compared with model group, ALR and colchicine can significantly reduce the serum level of ALT, AST and increase evidently the serum content of ALB (P<0.05 or P<0.01). But there was not significantly difference between two treatment groups (colchicines group and ALRrecombinant plasmid group). The Hyp content of liver tissue was relatively high abundance in model group and was decreased markedly by treating with colchicine and ALR. As far as this effect, ALR was more excellent than colchicines. The level of four serum hepatic fibrosis markers in colchicine group HA(357.90±90.98),LN(41.00±9.74),PCⅢ(11.30±4.45),ⅣC(37.70±8.91) and in ALR group HA(319.30±74.11),LN(36.00±8.79),PCⅢ(5.72±2.14),ⅣC(38.35±16.34)was lower than in model group HA(436.00±40.25),LN(62.56±9.76),PCⅢ(19.56±6.84),ⅣC(125.11±45.47), but there was not significantly difference between the two treatment groups. The hepatic fibrosis histopathological grading was G0 in normal group, G3 and G4 in modal group and empty group, G2 and G3 in colchicines group, G1 and G2 in ALR group. The statistical analysis showed there was significantly difference in normal group compared with other every group (P<0.05). There was significantly difference between model group and two treatment groups (P<0.05), but no significantly difference between two treatment groups. Immunohistochemical staining showed that expression of type I, III collagen, TGF-β1 and TIMP-1 in two treatment groups was decreased obviously compared with model group (I collagen: 7.10±1.76, 5.80±1.66 and 10.83±3.58 in colchicines , ALR and model groups respectively; III collagen: 5.81±1.86, 4.50±1.67 and 10.25±2.61 respectively; TGF-β1: 1.21±0.22, 1.17±0.26 and 1.56±0.31 respectively; TIMP-1: 0.30±0.05, 0.20±0.06 and 0.53±0.12 respectively, P<0.05 or P<0.01). The expression level of TGF-β1 mRNA and TIMP-1 mRNA in the liver tissues was also markedly decreased in colchicines and ALR group compared with model group (TGF-β1 mRNA/β-actin: 1.13±0.12, 1.02±0.09 and 1.61±0.18 in colchicines, ALR and model group respectively; TIMP-1 mRNA/β-actin: 0.77±0.16, 0.65±0.11 and 1.36±0.11 respectively, P<0.05 or P<0.01). There was not significantly difference between colchicine group and ALR group and no effects were found in empty plasmid groups. No effects were found in empty plasmid groups. Conclusion: ALR recombinant plasmid can protect injured liver cells;lower the contents of four hepatic fibrosis serum markers and Hyp of liver tissue; reduce obviously content of type I,III collagen in immune hepatic fibrosis liver tissue and depress the expression of TGF-β1 and TIMP-1 in liver tissue, which play crucial role in rehabilitated process of hepatic fibrosis. ALR recombinant plasmid was similar as colchicines or a little better than colchicines in anti-fibrosis effects. It may be one of the molecular mechanism of anti-fibrosis to inhibit the expression of TGF-β1 and TIMP-1 in liver tissue. Part 3 Comparative studies of different dose, different carriers and different introducing routes of ALR recombinant plasmid on anti-fibrosis effects Objective: To select relatively excellent carrier, relatively appropriate introducing route and relatively adaptive dose as well as their optimal combination on ALR gene therapy. Methods: Administering intraperitonealy with porcine serum developed the immune hepatic fibrosis model, which were divided into 9 groups with orthogonal design L9(33) and each group represented one time of test. Three different dose (50mg/kg, 100mg/kg, 200mg/kg) pcDNA3-ALR recombinant plasmids carried by three different carriers (naked plasmid, liposomes and glyco-poly-L-lysine) were delivered on hepatic fibrosis rats with three different introducing routes (intramusclar, intravenous and intraperitoneal injection). The effects were assessed and compared with histopathological changes of liver tissue and four serum hepatic fibrosis markers. Results: The semiquantitative scoring system (SSS) of liver tissue were 7.99±6.7, 5.93±4.5 and 1.58±1.7 in 50mg/kg, 100mg/kg and 200mg/kg group respectively, which was significantly low in 200mg/kg group compared with 50mg/kg and 100mg/kg group, P<0.01 and P<0.05 respectively but no significantly difference between 50mg/kg and 100mg/kg group. The result of comparison to the general scoring of four serum fibrosis markers was similar to SSS. The expression of ALR mRNA in liver tissue was weaker in 100mg/kg group than 200mg/kg group but there were not significantly differencebetween 100mg/kg and 50mg/kg group and between 50mg/kg and 200mg/kg group. The SSS were 3.52±3.0, 5.44±6.0 and 6.52±6.4 in naked gene, liposomes and GPLL group respectively. The significantly difference wasn't found among three groups and between any two groups(P>0.05). The general scoring of four serum fibrosis markers were 14.42±4.27, 11.17±3.86 and 11.75±3.52 in naked gene, liposomes and GPLL group respectively, which were lower in liposomes and GPLL group compared with naked gene group but no significantly difference between liposomes and GPLL group. The values of expression of ALR mRNA in liver tissue were 0.88±0.07, 0.83±0.23 and 0.95±0.12 in naked gene, liposomes and GPLL group respectively, which was obviously higher in GPLL group than in liposomes and naked gene group but no significantly difference between liposomes and naked gene group. The SSS were 3.63±4.9, 5.43±5.3 and 6.41±5.9 in intramusclar, intravenous and intraperitoneal injection group repectively. There was not significantly difference among three groups and between any two groups(P>0.05). The general scoring of four serum fibrosis markers were 11.25±4.65, 11.75±3.05 and 14.33±3.89 in intramusclar, intravenous and intraperitoneal injection group repectively, which was higher in intraperitoneal injection group than in intramusclar and intravenous injection group but no significantly difference between intramusclar and intravenous injection group. The values of expression of ALR mRNA in liver tissue were 0.96±0.22, 0.87±0.14 and 0.83±0.17 in intramusclar, intravenous and intraperitoneal injection group respectively, which was obviously higher in intramusclar injection group compared with intravenous and intraperitoneal injection group but no significantly difference between intraperitoneal and intravenous injection group(P>0.05). Conclusion: The anti-fibrosis effects of 200mg/kg ALR recombinant plasmid were more available than 100mg/kg and 50mg/kg. The liposomes and GPLL was more excellent carrier than naked gene and the GPLL was more excellent than the liposomes on expression of ALR mRNA in liver tissue. The intramusclar and intravenous injection was more appropriate gene delivery...
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