【作者】 朱传龙；

【导师】 宁琴；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 博士

【摘要】 【背景及目的】HBV感染在发展中国家和亚太地区的流行非常严重,我国是HBV感染高发地区,人群携带率约9~10%,由HBV引起的重型肝炎是常见的传染病。重型肝炎是急骤发生广泛性肝细胞坏死而引起重度肝功能障碍,出现以肝性脑病为主的肝功能衰竭症状之高危疾病。该病病情严重,发展迅猛,发病机制尚不明了,临床缺乏上特异、有效的治疗靶点和干预手段,除非实施紧急肝移植,绝大部分患者预后不良。因此探索重型肝炎肝细胞死亡机制,并开发针对其发病机制、阻断病理衍变过程的基因治疗手段正在成为该领域的研究热点和发展方向。新近发现的纤维介素(fibroleukin)或fgl2凝血酶原酶(简称fgl2)蛋白属于纤维蛋白家族中的一员,由活化的巨噬细胞、血管内皮细胞表达,具有凝血酶原酶的活性,能催化凝血酶原转化为凝血酶,启动凝血过程。近来在重型肝炎患者和小鼠模型的实验研究中,发现纤维介素(fgl2)与重型肝炎的发生发展密切相关。基因水平干预基因“有害”高表达可以更好地阐明该基因在疾病的发生发展中重要作用,也为探索人类重型肝炎临床治疗方法提供了新的思路。使外源基因如何高效快捷导入肝脏一直是该领域研究的瓶颈之一。研究发现尾静脉高压注射可以使质粒DNA在小鼠肝脏获得高效表达。尾静脉高压注射,就是将大体积的裸质粒DNA溶液经小鼠尾静脉快速注入,大量的质粒溶液导致循环血量的急剧增加,超过心脏负荷,血液积聚在肝窦中不能回流,延长了质粒DNA在肝窦中的停留时间,从而被肝组织细胞摄取。该方法可以广泛应用于肝脏疾病基因干预的研究。肝细胞凋亡在肝病的发生过程中起重要作用,各种原因导致的肝衰竭都存在肝细胞的凋亡。在LPS + D-GalN的FHF模型中,LPS的毒性实际上是由于严重的凋亡性肝损伤和肝细胞完全破坏所引起的。在重型肝炎的发病过程中,Fas与TNF系统的激活,Bcl-2家族调控蛋白比例的失衡等因素都会影响肝细胞凋亡的发生。另外,mfgl2在肝细胞凋亡的发生过程中有重要作用,IFN-γ和TNF-α在小鼠体内诱导的肝细胞凋亡依赖于mfgl2的表达。然而,有关肝细胞凋亡在MHV-3诱导的小鼠重型肝炎肝衰竭中的重要作用还未见相关文献报道,因此研究肝细胞凋亡及其调控机制对重型肝炎肝损伤发生机理的理解具有重要意义。具体研究目的如下:1.建立一简单可靠的重型肝炎小鼠模型,用于重型肝炎发病机制和干预治疗的研究。2.针对重型肝炎发病关键基因mfgl2构建一反义质粒,抑制小鼠巨噬细胞系Raw264.7细胞mfgl2的表达。3.研究mfgl2反义质粒对重型肝炎小鼠体内mfgl2表达的改变,以及对小鼠重型肝炎病情发展的影响。4.研究凋亡相关蛋白在重型肝炎小鼠模型肝组织中的表达及其促肝细胞凋亡的作用。【方法】1.采用MHV-3感染Balb/cJ小鼠制造重型肝炎动物模型;重型肝炎小鼠肝组织HE染色进行HAI评分,并分析血清学指标;免疫组化检测mfgl2的肝组织表达和纤维蛋白的沉积;构建mfgl2反义质粒和对照正义质粒,细胞水平转染mfgl2反义质粒,并通过Real-time PCR、Western-blot技术检测mfgl2反义质粒的体外干预效应,通过检测细胞PCA活性证实mfgl2反义质粒的干预后效应;通过尾静脉高压注射将目的基因导入小鼠肝脏,并检测目的基因在肝脏的表达效率;mfgl2反义质粒高压注射后,检测小鼠肝组织病理、血清生化学变化,并观察重型肝炎小鼠生存率的改变;通过Real-time PCR、免疫组化、原位杂交技术检测mfgl2反义质粒干预后的小鼠肝脏mfgl2的表达情况;2. TUNEL法检测MHV-3诱导的重型肝炎小鼠模型肝细胞凋亡情况,并计算凋亡指数;3.免疫组化技术检测重型肝炎小鼠肝细胞Fas和TNFR1的表达情况;Western-blot检测重型肝炎小鼠肝细胞Bax和Bcl-2的表达水平,并计算Bax/Bcl-2比值。【结果】1. MHV-3感染后Balb/cJ小鼠肝组织出现大片肝细胞坏死和炎性细胞浸润,HAI评分逐步升高,72h达到最高水平(16.0±1.4);MHV-3感染后Balb/cJ小鼠血清酶ALT、AST,以及血清TBil逐步升高,72h达到最高水平(8192.3±224.4 IU/L, 9410.3±954.4 IU/L,11.6±0.75 umol/L),血清白蛋白逐步降低,72h达到最低水平(19.6±1.25 g/L)。2.成功构建mfgl2反义质粒和对照正义质粒,并鉴定无误;mfgl2反义质粒细胞水平显著抑制mfgl2的表达,并抑制Raw264.7细胞在IFN-γ刺激后的PCA活性;尾静脉高压注射可以将目的基因导入小鼠肝脏,24h重复注射可以使表达效率提高到40%。mfgl2反义质粒高压注射后,重型肝炎小鼠生存率从0提高到33.3%,并显著改善肝组织病理学变化和血清学指标;mfgl2反义质粒高压注射后,显著抑制mfgl2在重型肝炎小鼠模型体内的表达,并显著减少纤维蛋白在肝脏的沉积。3. MHV-3诱导的重型肝炎小鼠模型与对照组(MHV-3感染A/J鼠)相比,肝细胞出现大片凋亡,凋亡指数逐步升高,72h达到最高水平(79.8±7.4%);MHV-3诱导的重型肝炎小鼠模型与对照组相比,肝细胞表面Fas和TNFR1表达强阳性,同时Bax蛋白表达升高,而Bcl-2蛋白表达降低,Bax/Bcl-2比值升高。【结论】1.本研究成功地建立了MHV-3诱导的重型肝炎小鼠模型,并通过尾静脉高压注射技术将外源基因高效导入小鼠肝脏,为今后实验研究提供了一个重要技术平台。2.本研究构建的mfgl2反义质粒在体内外实验中可以有效且特异地下调目的基因的表达,为今后重型肝炎、器官移植急性排斥反应、SLE等fgl2参与的疾病治疗提供了一条新途径。3.我们首次发现MHV-3诱导的重型肝炎小鼠模型存在大量肝细胞凋亡现象,因此重型肝炎基因治疗的靶点需包含与肝细胞凋亡相关的多个关键基因。更多还原

【Abstract】 【BACKGROUND&OBJECTIVE】In developing countries and the Asia-Pacific region, HBV infection is seriously epidemic,the incidence rate of HBV infection is high as 9~10%。In china severe hepatitis caused by HBV infection become one of the common infectious diseases. The hallmark of this severe disease is the extreme rapidity of the necromicroinflammatory process, resulting in widespread or total hepatocellular necrosis in weeks or even days, and occurrence of hepatic encephalopathy. The mechanisms for virus-induced severe hepatitis are still not fully understood. Due to the lack of specific and effective clinical treatment, unless an emergency liver transplant, the majority of patients are with poor prognosis. Therefore investigating the mechanism of hepatocytes death in severe hepatitis, and developing gene therapeutic means with which we can block the pathological process of this servere disease has become a serious issue in this field.Fibroleukin/fibringogen-like protein 2(fgl2) or fgl2 prothrombinase which was found recently belongs to profibrinogen superfamily. fgl2 expressed in activated macrophage and vascular endothelial cell induces prothrobin to thrombin, and activates the coagulation process. Recently we and our colleges demonstrated that fgl2 expression was closely related to the development of severe hepatitis from studies with both severe hepatitis patients and animal models.Target gene silence can be applied to evaluate the role and importance of the target gene(s) in the pathogenesis of interested disease. This technology also provides us a novel means for clinical intervention of disease development. However efficient transformation of foreign gene(s) into liver remains a“bottleneck”and limits the application of this modern technique. Currently high levels of gene transfer to mouse liver could be achieved by tail vein hydrodynamic injection of plasmid DNA solution in a large volume. It has been proposed that the injected DNA solution accumulates mainly in the liver because of its flexible structure and blood flow cease induced by transient heart failure, which can accommodate large volume of solution, and the hydrostatic pressure forces plasimid DNA into the liver cells before it is mixed with blood. This technology has been extensively used in gene therapy for mouse studies.Hepatocytes apoptosis occurred in the liver plays an important role in the process of many liver diseases, especially hepatic failure caused by various reasons. Actually LPS toxicity is due to serious apoptosis that further induce liver injury and destruction in the FHF model induced by LPS/D-GalN. In the process of severe hepatitis, Fas and TNF system activated, Bcl-2 protein family imbalances and other factors will affect the severity of hepatocytes apoptosis. Previous studies have demonstrated that mfgl2 plays important role in the development of hepatocytes apoptosis. IFN-gamma and TNF-alpha induced hepatocytes apoptosis in the mice liver are dependent on the expression of mfgl2. However the contribution of hepatocytes apoptosis to the development of murine hepatitis virus strain 3 (MHV-3) induced hepatic failure in Balb/cJ mice has never been explored, which in turn might be one of the important aspects for understanding the pathogenesis of hepatocytes injury.Therefore the purposes of this study are as the follows:1. To establish a simple and reliable model of fulminant viral hepatitis in mice, for studying the pathogenesis and treatment of fulminant viral hepatitis.2. To construct a mfgl2 antisense plasmid which can inhibit the expression of mfgl2 in Raw264.7 cells, a mouse macrophage derived cell line.3. To investigate the effect of mfgl2 antisense plasmid on mfgl2 expression in vivo and the disease progress in MHV-3 induced fulminant hepatitis mice model.4. To investigate the expression of apoptosis-related proteins and their correlation with hepatocytes apoptosis in MHV-3 induced fulminant hepatitis mice model. 【METHODS】1. Fulminant viral hepatitis animal model was established by MHV-3 infection of Balb/cJ. HE staining was performed to observe the pathological change of live tissue for evaluation of HAI scores. The expression of mfgl2 and fibrin deposition was observed by immunohistochemistry staining. mfgl2 antisense plasmid and mfgl2 sense plasmid as a control were constructed. Raw 264.7 cells were transfected with mfgl2 antisense, intervention effect of mfgl2 antisense in vitro was detected by Real-time PCR, Western-blot. And functional PCA assay. Target gene was introduced into mice liver by hydrodynamic injections, and expression efficiency of target gene was detected; After hydrodynamic injection of mfgl2 antisense plasmid, the survival rate of mice, hepatic pathological change and serum biochemical disorder were examined and compared between mice with/without mfgl2 antisense plasmid intervention. The expression of mfgl2 was detected by Real-time PCR, immunohistochemistry staining, and in-situ hybridization.2. The TUNEL method was used to detect hepatocytes apoptosis in MHV-3 induced fulminant hepatitis and then AI (apoptotic index) was evaluated3. The expression of Fas and TNFR1 in hepatocytes was observed by immunohistochemistry staining. Western-blot assay was performed for measurement of Bax and Bcl-2 expression, and ratio of Bax/Bcl-2 was calculated.【RESULTS】1. Massive inflammatory cells infiltration and hepatocytes death were evident in the hepatic lobulbe in MHV-3 induced fulminant hepatitis in Balb/cJ mice. HAI scores gradually increased, with the highest score of 16.0±1.4 at 72 hours post MHV-3 infection. The level of ALT, AST, and TBil gradually increased, with the highest level of 8192.3±224.4 IU/L, 9410.3±954.4 IU/L and 11.6±0.75 umol/L at 72 hours post MHV-3 infection,while serum ALB gradually decreased, with the lowest level of 19.6±1.25 g/L at 72 hours post MHV-3 infection.2. The mfgl2 antisense plasmid and mfgl2 sense plasmid was successfully constructed as evidenced by the restriction enzyme mapping as shown and further confirmed by sequence analysis. Target gene was introduced into mice liver by hydrodynamic injections, and repeated injection can improve the efficiency of expression up to 40% 24 hours later. A dose dependent inhibitory effect of mfgl2 expression by mfgl2 antisense plasmid was observed in IFN-gamma treated Raw246.7 cells. By hydrodynamic delivery, mfgl2 anti-sense plasmid significantly reduced mfgl2 expression in vivo, markedly ameliorates inflammatory infiltration, fibrin deposition and hepatocytes death, prolonged the survival time period and elevated the survival rate from 0 up to 33.3% in Balb/cJ mice with MHV-3 induced fulminant hepatitis.3. Compared with the control group (A/J mice with MHV-3 infection), a large number of hepatocytes apoptosis were emerged, and AI increased gradually, with the highest level of 79.8±7.4% at 72 hours post MHV-3 infection of Balb/cJ mice. The upregulated expression of TNFR1, Fas and Bax and down regulated expression of Bcl-2 (an increasing ratio of Bax/Bcl-2) was observed in mice with MHV-3 infection.【CONCLUSION】1. In this study we have successfully re-established the fulminant viral hepatitis model induced by MHV-3 and demonstrated that hydrodynamic tail vein injections efficiently introduced target gene into mice liver, which provide fundamental techniques for further studies.2. Efficient and specific mfgl2 gene silence targeted by the constructed mfgl2 antisense plasmid sheds light on the future investigation of gene therapeutic strategies for patients with fulminant viral hepatitis and disease such as acute rejection of xeno- or allograft transplantation and SLE, which fgl2 gene has been shown to play a key role in the disease development.3. Hepatocytes apoptosis was also first evidenced in MHV-3 induced fulminant viral hepatitis model, thus the therapeutic targets should also cover the key genes involved in the process of hepatocytes apoptosis.更多还原