【作者】 吴乔；

【导师】 别平；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 目的:一、研究二氮嗪预处理对肝细胞缺氧-复氧后Bcl-2蛋白表达及细胞凋亡的影响,探讨二氮嗪对肝细胞缺氧-复氧损伤的保护作用及其可能机制。二、摸索小鼠肝移植动物模型中应用RNA干扰技术沉默Bcl-2基因表达的方法。三、研究二氮嗪预处理对小鼠肝移植缺血-再灌注损伤的保护作用及其与凋亡调控相关的可能机制。方法:1.建立小鼠肝细胞缺氧-复氧损伤模型,细胞随机分为A组(缺氧-复氧损伤组),B组(二氮嗪预处理组)和C组(正常对照组),分别于细胞缺氧-复氧损伤后6h、12h、24h、48h、72h 5个时相点采集标本,检测肝细胞缺氧-复氧后细胞培养液中谷丙转氨酶(ALT)、谷草转氨酶(AST)浓度,肝细胞Bcl-2 mRNA和蛋白表达及肝细胞凋亡率变化。2.建立小鼠同种异体原位肝移植模型,根据冷保存的时间随机分为A组(冷保存4h组),B组(冷保存8h组),C组(冷保存12h组),通过各组小鼠移植术后14d存活情况做生存曲线分析;建立小鼠肝移植RNA干扰模型,利用化学合成Bcl-2基因的SiRNA,经门静脉高压注射途径导入供体肝脏。动物分为D组(供肝导入Bcl-2SiRNA组),E组(供肝导入Bcl-2SiRNA 6h肝脏移植组),F组(供肝导入Bcl-2SiRNA 24h肝脏移植组)和G组(正常对照组)。D组、E组、F组内分别设导入non-sence siRNA组为阴性对照。D组于SiRNA导入后6h、24h、48h、72h4个时相点采集标本,E组、F组和G组于移植术后24h采集标本,采用实时荧光定量PCR的方法检测Bcl-2mRNA表达变化。3.供肝导入Bcl-2SiRNA 24h摘取供肝建立小鼠肝移植RNA干扰模型,动物分为A组(冷保存2h肝移植组)、B组(供肝导入Bcl-2 SiRNA 24h冷保存2h移植组)、C组(二氮嗪预处理冷保存2h肝移植组)、D组(供肝导入Bcl-2 SiRNA 24h二氮嗪预处理冷保存2h肝移植组)和E组(供肝导入non-sence SiRNA24h冷保存2h移植组)。每组小鼠6只,移植术后24h采集标本,检测肝移植术后肝细胞病理形态学改变、血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)浓度,肝组织Bcl-2 mRNA和蛋白表达及肝细胞凋亡率变化。结果:1.在小鼠肝细胞缺氧-复氧损伤模型中,A、B组细胞培养液中ALT、AST浓度与C组比较明显升高(P<0.01) ,A、B两组之间比较,细胞培养液中ALT、AST浓度在缺氧-复氧6h差异无统计学意义(P>0.05),12h、24h、48h和72h B组ALT、AST浓度明显低于A组(P<0.01)。A、B组肝细胞凋亡率均高于C组( P < 0.01),A、B两组之间比较,肝细胞凋亡率在缺氧-复氧6h差异无统计学意义(P>0.05),12h、24h、48h和72h B组肝细胞凋亡率明显低于A组(P<0.01)。肝细胞Bcl-2mRNA和蛋白表达,A组和C组中均较低,两组之间比较差异无统计学意义(P>0.05),B组中Bcl-2mRNA和蛋白表达明显高于A、C组,缺氧-复氧损伤6h、12h、24h、48h和72h B组与A组比较差异存在统计学意义( P <0.01)。2.在小鼠肝移植模型中,A组术后生存率明显高于B组和C组。在小鼠肝移植RNA干扰模型中,D组Bcl-2mRNA表达量在6h开始明显下降,24h达到高峰,持续到72h,6h、24h、48h和72h Bcl-2mRNA表达较正常对照组分别减少36.0%、66.2%、61.8%、58.1%。E组小鼠肝脏组织中Bcl-2mRNA表达较G组减少33.3%,F组较G组减少58.9%,F组与E组两组之间比较,F组肝脏组织中Bcl-2mRNA表达较E组减少38.1% ,两组之间差异有统计学意义(P<0.05)。3.在二氮嗪预处理小鼠肝移植实验中,C组Bcl-2 mRNA和蛋白表达水平较A组明显增高(P<0.01),Bcl-2mRNA和蛋白表达分别增加2.6倍和5.1倍;B组Bcl-2 mRNA和蛋白表达水平较A组明显降低(P<0.01),Bcl-2mRNA和蛋白表达分别被抑制57.1%和83.4%;D组Bcl-2 mRNA和蛋白表达水平较A组明显降低(P<0.01),Bcl-2mRNA和蛋白表达分别减少53.3%和67.6%;B组与D组两组之间比较Bcl-2 mRNA和蛋白表达水平差异无统计学意义(P>0.05)。C组中肝细胞病理损伤程度、凋亡指数、血清AST、ALT含量明显低于各组。B组和D组中肝细胞病理损伤程度、凋亡指数、血清AST、ALT含量明显高于各组。在各组中Bcl-2表达量与缺血再灌注损伤程度和肝细胞凋亡指数呈负相关。结论:1.二氮嗪预处理能通过抑制肝细胞凋亡,起到保护肝细胞缺氧-复氧损伤的作用,其保护机制可能与上调Bcl-2表达有关。2.采用门静脉高压注射途径向肝脏导入Bcl-2 siRNA 24h后摘取供肝的方法建立肝移植模型,能在小鼠肝移植体内实现Bcl-2基因的有效沉默。3.二氮嗪预处理可能通过诱导Bcl-2表达,抑制肝细胞凋亡,从而减轻小鼠肝移植缺血-再灌注损伤。更多还原

【Abstract】 Objectives:1. To investigate the impact of diazoxide preconditioning on the expression of Bcl-2 in liver cell and apoptosis during hypoxia-reoxygenation injury.2. To silence the expression of Bcl-2 gene by delivery of synthetic Bcl-2 siRNA in mice orthotopic liver transplantation model.3. To explore the effect of diazoxide preconditioning on attenuation ischemia- reperfusion injury in mice liver graft and to study the possible mechanism relating to apoptosis .Methods:1. The cultured hepatocytes were randomly divided into three groups with 6 cases each : groupA (anoxia -reoxygenation group) , groupB (diazoxide preconditioning group) and groupC (control group) . Samples were collected at 6h、12 h、24 h、48h and 72h after anoxia -reoxygenation . ALT、AST levels in nutritive medium were detected. Cell apoptosis were observed by TUNEL. The expressions of Bcl-2 gene and protein level were determined by RT-PCR and Western blot.2. Orthotopic liver transplantations in mice were performed , mice were randomly divided into three groups namely:group A (cold preservation of 4h), group B (cold preservation of 8h)and group C (cold preservation of 12h) ,the survival ratio of each group was analysised 14days after operation. The Bcl-2 gene was silenced in liver graft mice by RNAi.Synthetic Bcl-2 siRNA sequence was delivered in vivo by using a‘‘hydrodynamic’’transfection method.siRNA sequence was delivered into the portal vein with a 29-G needle. Mice were randomly divided into four groups namely: group D (donor group), group E (liver transplantation group after Bcl-2 siRNA sequence was delivered in donor 6h), group F (liver transplantation group after Bcl-2 siRNA sequence was delivered in donor 24h) and group G (control group). Samples in group D (donor group) were collected at 6h、24h、48h and 72h after Bcl-2 siRNA sequence was delivered. Samples in groupE、F、G were collected after liver transplantation 24h . The expression of Bcl-2 gene level was determined by RT-PCR.3. Orthotopic liver transplantations in mice were performed after Bcl-2 siRNA sequence was delivered in donor 24h, mice were randomly divided into five groups namely: groupA(the liver transplantation group),group B(Bcl-2 SiRNA sequence delivery liver transplantation group),group C(diazoxide preconditioning liver transplantation group), groupD(diazoxide preconditioning and Bcl-2 SiRNA sequence delivery liver transplantation group )and groupE(non-sense SiRNA sequence delivery liver transplantation group ).24h after liver transplantation, The pathological changes of liver tissue and the serum AST、ALT level were observed in each group .Cell apoptosis were observed by TUNEL.The expression of Bcl-2 gene and protein level were determined by RT-PCR and Western blot.Results:1.In the cultured hepatocytes ,the cell apoptosis index and the levels of AST、ALT in nutritive medium in group A、B were significantly higher than in group C (p<0.01), the cell apoptosis index and the levels of AST、ALT in nutritive medium in group B were significantly lower than in group A (p<0.01); The expression of Bcl-2 gene and protein level in group B were significantly higher than group A、C (p<0.01).2. In the Orthotopic liver transplantation groups,the survival ratio of group A was higher than in group B、C after liver transplantation 14d(p<0.01).In the RNAi groups , the expression of Bcl-2 mRNA in group D was decreased gradually at 6h after siRNA sequence delivery, arrived on the top at 24h and continued to decrease at 72h . The expression of Bcl-2 mRNA in group D was inhibited 36.0%、66.2%、61.8%、58.1%respectively ,at 6h、24 h、48h and 72h after Bcl-2 siRNA sequence delivery. The expression of Bcl-2 mRNA in group E and F was decreased 33.3% and 58.9%, respectively ,compared with group G. The expression of Bcl-2 mRNA existed significant difference between group E and group F, The expression of Bcl-2 mRNA in group F was decreased 38.1% compared with group E.3.In the diazoxide preconditioning liver graft groups, the levels of Bcl-2 mRNA and protein in group C were remarkably higher than group A (p<0.01). The expression of Bcl-2 in group C was improved by 2.6-fold and 5.1-fold, compared with group A, respectively at mRNA and protein levels. The levels of Bcl-2 mRNA and protein in group B were remarkably lower than group A (p<0.01),the expression of mRNA and protein were inhibited 65.7% and 83.4%,respectively. The levels of Bcl-2 mRNA and protein in group D were remarkably lower than group A (p<0.01),the expression of mRNA and protein were decreased 53.3% and 67.6%,respectively. There was no statistical difference of Bcl-2 mRNA and protein levels between group B and group D (p>0.05). The pathological injure degree of liver tissue、apoptosis index and the serum AST、ALT levels in group C were significantly lower than other groups; The pathological injure degree of liver tissue、apoptosis index and the serum AST、ALT levels in group B、D were significantly higher than other groups; There found negative correlation between expression of Bcl-2 and the degree of ischemia- reperfusion injury as well as hepatocyte apoptosis index in each group.Conclusions:1. Diazoxide preconditioning can attenuate the apoptosis of hepatocytes induced by anoxia -reoxygenation ,and the protective effect may be related to the expression of Bcl-2.2. The method of liver transplantation was operated 24h after siRNA sequence was delivered into the portal vein of donor can realize Bcl-2 gene silencing in vivo of liver graft mice.3. Diazoxide preconditioning can protect against ischemia-reperfusion injury in liver graft mice,with its possible mechanism of upregulating the expression of Bcl-2 and prohibiting hepatocyte apoptosis.更多还原