基因沉默对肝衰竭内毒素血症小鼠肝细胞凋亡的实验研究

发布时间：2018-05-18 01:04

本文选题：SMAC + 肝衰竭　；参考：《华中科技大学》2014年博士论文

【摘要】：[背景与目的] 肝衰竭是各种原因导致的肝功能严重失代偿,出现以凝血功能障碍、腹水、肝性脑病、肝肾综合征和出血等为主要表现的一种临床症候群,死亡率高达80%,目前尚缺乏特异的治疗手段。研究发现肝细胞凋亡是肝衰竭发生发展的重要事件。肝衰竭时内毒素血症发生率高达80.0%,内毒素(lipopolysaccharide, LPS)可以介导肝细胞的凋亡。我们课题组前期试验发现第2个线粒体衍生的胱天蛋白酶激活剂(second mitochondria derived activator of caspase, SMAC)在体外可以单独介导LPS诱导的肝细胞凋亡,体内试验也发现肝衰竭内毒素血症大鼠肝细胞的凋亡与SMAC密切相关。研究发现SMAC是凋亡的发生发展所必须的。RNA干扰(RNA interference, RNAi)是一种高效、特异性阻断mRNA表达而导致转录后的基因沉默现象。应用RNAi抑制SMAC的表达,可能减轻肝衰竭内毒素血症小鼠肝细胞的凋亡,这将为肝衰竭内毒素血症的治疗提供一个新的途径,也有助于进一步明确SMAC在该凋亡中的作用。最后,研究HEV相关急性肝衰竭患者血清LPS水平,观察该血清对人肝细胞生存和凋亡的影响,并初步探讨内毒素核心多糖抗体的保护作用。 [实验方法] 一.细胞水平针对人SMAC基因设计、合成并筛选出特异小干扰RNA (small interferingRNA, siRNA),脂质体法将该特异干扰片段转染肝细胞,再用内毒素诱导细胞凋亡。转染后不同的时间点(24h、36h、48h、60h)收集肝细胞,Real time PCR法检测细胞SMAC mRNA。转染后48h, western检测肝细胞线粒体SMAC蛋白和胞浆caspase-9蛋白的表达,caspase-3活性检测试剂盒检测caspase-3活性,流式细胞仪检测肝细胞的凋亡。二.动物水平构建小鼠SMAC特异干扰慢病毒,测序鉴定构建的正确性并测定其滴度。通过尾静脉高压注射技术将慢病毒导入小鼠肝脏。应用D-半乳糖氨(D-galactosanine, D-GalN)和LPS腹腔注射制作急性肝衰竭小鼠模型,观察小鼠体征的变化并记录小鼠死亡情况,留取小鼠血液和肝脏组织。全自动生化分析仪检测小鼠血清谷丙转氨酶(alanine aminotransferase, ALT)、谷草转氨酶(aspartate aminotransferase,AST),鲎试剂检测试剂盒检测血清LPS水平,冰冻切片检测慢病毒的感染效率,HE染色评定肝组织病理学改变,real time PCR法检测肝组织SMAC mRNA, western检测肝组织线粒体和胞浆SMAC蛋白、caspase-9蛋白的表达,Tunel染色观察肝组织肝细胞的凋亡。三.人体水平收集13例HEV相关急性肝衰竭患者血清,采用鲎试剂基质显色法检测血清LPS水平,用该血清孵育肝细胞,流式细胞仪检测肝细胞凋亡率；再用内毒素核心多糖抗体和该急性肝衰竭患者血清与肝细胞共培养,流式细胞仪检测肝细胞凋亡率。四.统计学方法计量资料用均数±标准差(x±S)表示,应用统计学软件SPSS13.0进行多样本均数两两比较的q检验方差分析,比较各样本间的差异。P0.05为显著性差异界限。 [结果] 1.3条siRNA (siRNA1、siRNA2、siRNA3)对人肝细胞SMAC mRNA均有不同程度的抑制作用,其中siRNA2的抑制效应最强,抑制效应达68.0%。 2.在转染siRNA2的LPS诱导的肝细胞,线粒体SMAC蛋白、caspase-9蛋白、caspase-3酶活性均被抑制,肝细胞的凋亡率也被有效降低。 3.筛选并构建了特异的小鼠SMAC干扰慢病毒载体,测序表明其构建正确,慢病毒滴度为6E+8TU/ml。 4.荧光显微镜下,注射慢病毒的小鼠肝脏组织冰冻切片可见大量绿色荧光。 5.模型组小鼠血清ALT和AST水平较正常对照组明显上升,特异序列组小鼠ALT和AST水平较模型组显著降低。 6.模型组小鼠血清LPS水平较正常对照组明显升高,特异序列组小鼠血清LPS水平较模型组显著下降。 7.HE染色显示,正常对照组小鼠肝组织无炎性细胞浸润,无肝细胞坏死：模型组和无关序列组小鼠肝组织可见炎性细胞浸润和肝细胞坏死；而特异序列组小鼠肝组织炎性细胞浸润和肝细胞坏死较模型组明显减少。 8.在肝衰竭模型小鼠,肝组织SMAC mRNA的表达水平较正常对照组小鼠明显升高,线粒体SMAC蛋白表达降低,胞浆SMAC蛋白表达明显增多,肝组织胞浆caspase-9蛋白表达量较正常组明显增多,肝细胞凋亡增多,凋亡指数为33.0±±5.6%。而构建的特异慢病毒,可以显著降低肝组织SMAC mRNA的水平,下调线粒体和胞浆SMAC蛋白的表达,降低caspase-9的含量,减少肝组织肝细胞的凋亡。 9.HEV相关急性肝衰竭患者的血清LPS水平为0.26±0.02EU/ml,明显高于健康者。 10.HEV相关急性肝衰竭患者血清孵育肝细胞后,细胞的凋亡率为5.83±0.42%,较对照组明显增加,健康者的血清对细胞凋亡率没有明显影响。应用LPS抗体和急性肝衰竭患者血清共培养,细胞凋亡率下降,但没有统计学差异。 [结论] 1.通过抑制SMAC的表达,可以有效减少LPS诱导的肝细胞的凋亡及肝衰竭内毒素血症小鼠肝细胞的凋亡,为肝衰竭内毒素血症的治疗提供了新的手段和理论基础。同时也表明SMAC及其下游通路可能是LPS介导肝细胞凋亡的通路之一。 2.HEV相关急性肝衰竭患者血清含有较高浓度的LPS,该血清可以诱导肝细胞的凋亡。
[Abstract]:BACKGROUND & OBJECTIVE :

Liver failure is a clinical symptom caused by various causes , such as coagulation dysfunction , ascites , hepatic encephalopathy , hepatorenal syndrome , hemorrhage , etc .

EXPERIMENTAL METHOD

I . Cell level

Specific small interfering RNA ( siRNA ) was synthesized and screened for human SMAC gene . The specific interference fragment was transfected into hepatocytes by liposome method . After transfection , the cells were transfected with LPS . After transfection , the expression of SMAC protein and caspase - 9 protein were detected by real time PCR . The caspase - 3 activity was detected by caspase - 3 activity detection kit , and the apoptosis of hepatocytes was detected by flow cytometry .

II . Animal level

Mouse liver was induced by intraperitoneal injection of D - galactosanine ( D - GalN ) and lipopolysaccharide ( LPS ) by intraperitoneal injection of D - galactosanine ( D - GalN ) and LPS .

III . Human body level

The serum levels of LPS were measured by the method of chromogenic method in 13 patients with HEV - related acute liver failure , and the hepatocyte apoptosis rate was detected by incubating the hepatocytes with the serum .
then the serum of the endotoxin core polysaccharide antibody and the patient of the acute liver failure are co - cultured with the liver cells , and the apoptosis rate of the hepatocytes is detected by flow cytometry .

IV . Statistical method

The mean 卤 standard deviation ( x 卤 S ) of the measurement data showed that the statistical software SPSS 13.0 was used to carry out the q - test variance analysis of the two comparisons of the number of samples , and the difference between the samples was compared .

The result is not valid .

1.3 siRNA ( siRNA1 , siRNA2 , siRNA3 ) inhibited the expression of SMAC mRNA in human hepatocytes , in which siRNA2 had the strongest inhibitory effect and the inhibitory effect was 68.0 % .

2 . After transfection of siRNA2 - induced LPS - induced hepatocytes , mitochondrial SMAC protein , caspase - 9 protein and caspase - 3 activity were inhibited , and the apoptosis rate of hepatocytes was also effectively reduced .

3 . The specific mouse SMAC was screened and constructed to interfere with the slow virus vector . The sequencing showed that it was constructed correctly and the titer of slow virus was 6E + 8TU / ml .

4 . Under fluorescence microscope , a lot of green fluorescence was observed in frozen section of mouse liver tissue injected with slow virus .

5 . The serum ALT and AST levels in the model group were significantly higher than those in the normal control group , and the ALT and AST levels in the specific sequence group were significantly lower than those in the model group .

6 . The level of serum LPS in the model group was higher than that of the normal control group , and the level of LPS in the specific sequence group was significantly lower than that in the model group .

7 . HE staining showed that the liver tissues of normal control group had no inflammatory cell infiltration and no hepatocellular necrosis : inflammatory cell infiltration and hepatocellular necrosis were seen in liver tissues of model group and unrelated sequence group .
The inflammatory cell infiltration and necrosis of hepatocytes in the liver tissues of the specific sequence group were significantly reduced compared with the model group .

8 . In the model of liver failure , the expression level of SMAC mRNA in liver tissue was higher than that of normal control group , the expression of SMAC protein decreased , the expression of caspase - 9 protein in liver tissue was significantly increased , the apoptosis index was 33.0 卤 5.6 % .

9 . The level of serum LPS in patients with HEV related acute liver failure was 0.26 卤 0.02 EU / ml , which was significantly higher than that of healthy persons .

10 . After incubation of hepatocytes , the apoptosis rate of cells was 5.83 卤 0.42 % , which was significantly higher than that in the control group . There was no significant difference in the apoptosis rate of the healthy subjects .

Conclusion

1 . By inhibiting the expression of SMAC , the apoptosis of liver cells induced by LPS can be effectively reduced and the apoptosis of liver cells in mice with hepatic failure can be effectively reduced . It provides a new means and theoretical basis for the treatment of hepatic failure endotoxaemia . It also shows that SMAC and its downstream pathway may be one of the pathways of LPS - mediated apoptosis .

2 . Patients with HEV - related acute hepatic failure had higher concentrations of LPS , which could induce apoptosis of hepatocytes .
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R575.3

【参考文献】