【作者】 刘斌；

【导师】 郑勇；

【作者基本信息】 石河子大学 ， 内科学， 2008， 硕士

【摘要】 背景:在我国肝纤维化发生率高,是肝硬化发展的必经环节。目前研究认为肝纤维化属于可逆性病变,因此阻止和延缓肝纤维化的发生、发展则是治疗肝硬化的关键。研究发现在致肝纤维化的细胞因子中最重要是转化生长因子β(transforming growth factorβ,TGF-β),其致纤信号传导通路已阐明:首先活化了的TGF-β与II型TGF-β受体(TβRⅠI)结合并使之磷酸化而具有激酶活性;与TGF-β结合的TβRⅠI再结合I型TGF-β受体(TβRⅠ)形成I、II型受体复合物,并使TβRⅠ磷酸化具有激酶活性;最后,激活的TβRⅠ激活酶磷酸化其特殊的受体Smads (R-Smads) ,从而调节肝星形细胞(HSC)的转化和ECM的合成、降解。即TGF-β发挥作用必须借助其在细胞膜上的跨膜受体TβRⅠ和TβRⅠI。故从理论上,可推测选择性抑制TβRⅠ的表达,应可影响TGF-β信号传导,从而抑制TGF-β的促纤维化作用。在正常肝脏中,基质金属蛋白酶(matrixmetalloprotei-nases,MMPs)与基质金属蛋白酶组织抑制因子(tissue inhibitor of metallopropteinases,TIMPs)处于动态平衡的状态,维持了胶原的产生与降解之间的平衡。当有损伤因子作用于肝脏,使HSC活化,打破了MMPs与TIMPs的平衡,就会导致胶原纤维降解的减少,致使大量的胶原在肝组织中沉积。肝内主要存在的TIMPs有TIMP-1和TIMP-2,其中以TIMP-1表达更为显著;主要存在的MMPs在人类为MMP-1,在啮齿类动物为MMP-13;TIMP-1可与MMP-1/MMP-13特异性结合,抑制其活性。故推测,抑制TIMP-1的表达,可减轻TIMP-1对MMP-1/MMP-13的抑制作用,增加ECM的降解,减少ECM的沉积,延缓肝纤维化的发生、发展。本实验旨在前期研究的基础上,应用分子生物学检测技术RT-PCR和western–bloting(蛋白印迹)测定各实验模型组大鼠肝组织TβRⅠ,TβRⅡ和TIMP-1,MMP-13 mRNA含量及蛋白的表达,并对所得数据进行科学分析,从生物分子的角度阐述并验证本课题,反义转化生长因子受体与反义基质蛋白酶抑制因子联合作能够有效的干预肝纤维化的发生,发展。最终的目标是运用基因治疗技术寻找一种防止和延缓各种慢性肝病向肝纤硬化发展的有效方法,为今后人类肝纤维化基因治疗奠定基础。材料与方法:取-80℃低温保存的实验大鼠肝脏:正常对照组12例,模型对照组12例、空质粒组13例、反义TIMP-1真核表达质粒组13例,反义TβRⅠ治疗组13例,反义TβRⅡ治疗组13例,反义TβRⅠ+反义TIMP-1治疗组13例:反义TβRⅡ治疗组+反义TIMP-1治疗组13例。1.运用RT-PCR半定量检测肝组织内TβRⅠmRNA、TβRⅡmRNA、TIMP-1mRNA、MMP-13 mRNA测定。2.western blot蛋白印记技术检测大鼠肝组织中TβRⅠ、TβRⅡ、TIMP-1、MMP-13蛋白表达。3.进行系统科学数据分析,论证。结果:与模型对照组和空质粒组相比,反义TβRⅠ治疗组,反义TβRⅠ+反义TIMP-1治疗组、反义TIMP-1治疗组、反义TβRⅡ治疗组、反义TβRⅡ+反义TIMP-1各治疗组中的TβRⅠ、TβRⅡ及TIMP-1 mRNA含量及蛋白表达均有下降,各组比对均有统计学意义(P<0.05)。在正常对照组与各实验治疗组之间TβRⅠ、TβRⅡ及TIMP-1 mRNA含量及蛋白表达均有显著性差异(P<0.05);反义TβRⅠ+反义TIMP-1治疗组较反义TβRⅠ治疗组、反义TIMP-1治疗组比对有统计学意义(P<0.05);反义TβRⅡ+反义TIMP-1各治疗组较反义TβRⅡ治疗组,反义TIMP-1治疗组比对有统计学意义(P<0.05)模型对照组和空质粒对照组之间TβRⅠ、TβRⅡ及TIMP-1 mRNA含量及蛋白表达则无显著差异(P>0.05)。结论:1、反义TβRⅠ、TβRⅡ真核细胞表达质粒通过阻断TGF-β1的作用通道,减少细胞外间质(ECM)的合成,减轻肝纤维化的发展;通过实验也验证了TGF-β信号通路的存在。2、反义TβRⅠ、TβRⅡ真核细胞通过阻断TGF-β1的作用通道,对MMP-13产生抑制作用,对TIMP-1的促进作用,间接使细胞外间质(ECM)降解增加,抑制肝纤维化的进程。3、反义TIMP-1真核细胞通过拮抗TIMP-1对MMP-13的抑制作用,使细胞外间质(ECM)降解增加。4、两种重组质粒协同作用,可产生叠加效应,达到有效抑制延缓肝纤维化发生的目的。最终的目的是寻求一种防止和延缓各种慢性肝病向肝硬化发展的有效方法。更多还原

【Abstract】 Background: In China the high incidence of liver fibrosis is an essential link of cirrhosis. The current studies show Liver fibrosis is a kind of reversible disease, so the key to treat cirrhosis is to prevent and delay the occurrence of liver fibrosis.Studies find that the cells of liver fibrosis are the most important factor in transforming growth factorβ(transforming growth factorβ, TGF-β), the signal transduction pathway to the MFA has been stated: First, the combination of the activation TGF-βand type II TGF -βreceptor (TβRⅠI) has a kinase activation due to phosphorylation. Type I, II receptor complex will be formed by recombining TβRⅠI with the combination of TGF-βwith type I TGF-βreceptor (TβRⅠ), which phosphorylates TβRⅠto make it have kinase activation. Finally, the activated TβRⅠactivates the special receptor Smads (R-Smads) 0f phosphorylation. Thereby the transformation of hepatic stellate cells (HSC) and the synthetic and degradation of ECM are regulated. That is , the role of TGF-βmust be based on the transmembrane receptor on the cell membrane TβRⅠand TβRⅠI. Therefore, inhibiting the expression of TβRⅠcan be inferred to affect TGF-βsignal transduction in theory, and thus inhibit TGF-βto promote fibrosis.In normal liver, the MMP (matrixmetalloprotei-nases, MMPs) and MMP inhibitor Organization (tissue inhibitor of metallopropteinases, TIMPs) are in a state of dynamic equilibrium to maintain the balance between the formation of collagen and its degradation. When damaged factors act on the liver, activate HSC, break the balance beween MMPs and TIMPs, degradation of collagen fibers will be reduced, then a lot of collagen in the liver tissue will be deposited. the main TIMPs in the liver is TIMP-1 and TIMP-2, TIMP-1 expresses more significantly. MMPs in the human is mainly MMP-1, in the rodents is MMP-13. The binding of TIMP-1 and the specifics of MMP-1/MMP-13 can inhibit its activity. It can be inferred that inhibiting the expression o TIMP-1 will reduce the effect of TIMP-1 to MMP-1/MMP-13, increase the degradation of ECM, reduce ECM deposition, delay the occurrence and development of liver fibrosisThis study aims to mensurate the rat’s tissue, TβRⅡand TIMP-1, the con tent of MMP-13 mRNA content and expression of its Protein in the experimental models on the basis of application of molecular biology detection technology RT-PCR and western-bloting (Western blot), and to analyze the collected data scientifically and verify this issue from the perspective of biological molecules. The cooperation role of anti-transforming growth factor receptor and anti-protease inhibitor-matrix can intervene the occurrence of liver fibrosis and the development of Liver fibrosis effectively. The ultimate goal is to find effective ways to prevent and delay the chronic liver to hardening of the liver defibrillators disease with gene therapy and to lay the foundation of clinical application for future human gene therapy of liver fibrosis.Material and methods:Take experimental rat’s liver preserved below the low-temperature of -80℃: 12 cases of the normal control group, 12 cases of the model control group, 13 cases of P group, 13 cases of TIMP-1 antisense expression vector eukaryotic group, 13 cases of anti-sense therapy group TβRⅠ, 13 cases of anti-sense TβRⅡgroup, 13 cases of anti-sense TβRⅠand anti-sense TIMP-1 treatment group, 13 cases of antisense TβRⅡtreatment group and anti-TIMP-1-treated group.1. Use semi-quantitative RT-PCR detection to test TβRⅠmRNA, TβRⅡmRNA, TIMP-1mRNA, MMP-13 mRNA in liver tissue.2. Detect the expression of TβRⅠ, TβRⅡ, TIMP-1, MMP-13 protein in rat liver with the technology of western blot.3. Analyze the statistics and verify the proof systematically and scientifically.Results:Compared with the control group and air plasmid group, TβRⅠ, TβRⅡand the content and protein expression of TIMP-1 mRNA are dropped in the treatment group of anti-sense TβRⅠ, treatment group of anti-sense TβRⅠand anti-sense TIMP-1, the anti-TIMP-1-treated group, treatment group of anti-sense TβRⅡ, treatment group of anti-sense TβRⅡand anti-sense TIMP-1. The comparison of every group has statistical significance (P <0.05).TβRⅠ, TβRⅡand the content and protein expression of TIMP-1 mRNA have significant differences (P <0.05) between the control group and the experimental treatment group, anti-sense TβRⅠ+ anti-TIMP-1-treated group has more statistical significance than the anti-sense therapy group of TβRⅠ, Anti-TIMP-1-treated group (P <0.05); anti-sense TβRⅡ+ anti-TIMP-1-treated group has more statistical Significance than the anti-sense TβRⅡtreatment group and the anti-TIMP-1-treated group (P <0.05).TβRⅠ, TβRⅡand content and protein expression of TIMP-1 mRNA haven’t significant difference between model control group and the air plasmid control group (P> 0.05).Conclusion:1. Extracell matrix (ECM) is reduced, the development of liver fibrosis is lowered by anti-sense TβRⅠ, TβRⅡeukaryotic cells blocking the acting access of TGF-β1. Experiments also show the existence of TGF-βsignaling pathway.2.The MMP-13 inhibits the TIMP-1, outside of cells (ECM) degradation increases, inhibit the process of liver fiber by anti-sense TβRⅠ, TβRⅡeukaryotic cells blocking TGF-β1 acting channel,3.Extracell matrix (ECM) increases by the inhibition of TIMP-1 to MMP-13 through antagonism of anti-sense TIMP-1 eukaryotic cells.4. Two recombinant synergies can generate stack effect to achieve the goal to delay the effective suppression of liver fibrosis. The ultimate goal is to seek a way to delay and prevent all kinds of chronic liver disease to become cirrhosis.更多还原