线粒体转录因子A在重组人肝再生增强因子对梗阻性黄疸大鼠肝功能保护中的作用机制研究
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摘要
一、目的
首先构建好4组针对目的基因序列的RNA干扰慢病毒载体;在体外目的细胞上筛选出有明显干扰效果的靶点并进行慢病毒大量包装。然后建立大鼠梗阻性黄疸动物模型,并进行活体RNA干扰实验,检测目的基因的干扰效果及大鼠线粒体功能和肝功能的变化情况;探讨目的基因在rhALR对大鼠梗阻性黄疸肝功能保护中的作用,为rhALR的临床应用提供理论基础。
二、方法
1、设计4个针对目的基因TFAM的RNA干扰序列,制备双链DNAoligo,然后通过双酶切慢病毒载体,连接及转化反应制备克隆;最后挑取克隆进行PCR鉴定并挑选阳性克隆测序以确认慢病毒载体构建成功。
2、进行RNA干扰慢病毒包装,对其进行浓缩、纯化并测定病毒滴度。
3、利用包装好的RNA干扰慢病毒,采用Real time PCR的方法在体外目的细胞中筛选出有明显干扰效果的慢病毒载体,并利用Western blot的方法在蛋白水平上验证干扰靶点的有效性;最后将有效的干扰靶点进行病毒大量包装以用于下一步的动物实验。
4、建立梗阻性黄疸大鼠动物模型。将健康Wistar大鼠随机分为5组。第1组为假手术组,第2组为梗阻性黄疸、胆道再通及rhALR治疗组,第3组为梗阻性黄疸、胆道再通、rhALR治疗,同时进行活体RNA干扰组,第4组为梗阻性黄疸、胆道再通、rhALR治疗及阴性对照病毒组,第5组为梗阻性黄疸、胆道再通、rhALR治疗及生理盐水对照组。分别于第一次术后4、7、14、18、21、28天采集标本,保证每时相点存活大鼠6只。
5、利用Real time PCR和Western blot的方法,检测大鼠活体RNA干扰的效果。
6、观察各组大鼠肝脏大体和病理改变,检测其肝功能和线粒体功能的变化情况。
三、结果
1、将制备好的RNA干扰慢病毒载体进行测序,结果证实4个靶点均成功连接入载体中,可以用于后续的筛选实验。
2、进行RNA干扰慢病毒包装后,测定4组靶点的病毒滴度依次为8E+7、7E+7、9E+7和1E+8 TU/ml
3、体外筛选结果显示3号靶点在目的细胞中对目的基因TFAM的干扰效率达到了55%左右,在所有的干扰靶点中效率最高;Western blot实验也证实了其干扰的有效性。
4、胆道梗阻后,除第1组外的其余各组大鼠肝脏均出现了不同程度的病理改变,其中第3组大鼠病理改变最明显;胆道再通后,上述各组大鼠肝脏病理改变逐步减轻,组间比较发现第3组大鼠恢复速度最慢。
5、胆道梗阻后第2～5组大鼠肝功能和线粒体功能均出现了不同程度的损害(与第1组比较,P<0.05),其中第3组大鼠的损害程度明显重于第2、4、5组大鼠(P<0.05);胆道再通后,上述各组大鼠肝功能和线粒体功能逐步恢复,其中第3组大鼠的恢复速度明显慢于第2、4、5组大鼠(P<0.05)。
6、活体RNA干扰实验结果显示,第3组大鼠肝脏中目的基因TFAM mRNA的含量出现了明显下降,而阴性病毒对照组(第4组)、生理盐水对照组(第5组)及未进行RNA干扰的第2组中目的基因TFAM mRNA的含量相比无明显差异。Western blot实验结果显示第3组大鼠目的基因TFAM的蛋白表达也明显降低。
四、结论
1、本研究成功构建了针对目的基因TFAM的RNA干扰慢病毒载体,在体外实验中具有良好的干扰效率,能有效降低目的基因TFAM mRNA的表达;在体内实验中同样具有良好的干扰效果,且无明显的“脱靶效应”和毒性反应。
2、在rhALR保护及改善梗阻性黄疸大鼠的肝功能,促进胆道再通后肝功能的恢复过程中,TFAM可能发挥了重要的作用;其机制可能通过诱导肝细胞TFAM的表达,提高线粒体DNA(mitochondrial DNA,mtDNA)的拷贝数,修复损伤的mtDNA,最终保护和改善梗阻性黄疸肝细胞线粒体功能和肝功能,促进胆道再通后肝功能的恢复。
Objectives: To study the change of mitochondrial and hepatic function in rats with obstructive jaundice after RNA interference of target gene and to explore the role of gene TFAM in the process of protecting mitochondrial and hepatic function by rhALR,and finally,to provide assistance for the clinic application of rhALR.
Methods:
1、We designed 4 siRNAs targeting gene TFAM and constructed the lentivirus-siRNA vector system.
2、Lentiviral vector was generated by co-transfecting 293T cell with pGC-LV recombinant plasmid, pHelper1.0 and pHelper2.0 plasmids,and then the titer of LV was determined on 293T cell.
3、To test the efficacy of designed siRNAs in knocking down TFAM expression,we transfected HSC-T6 cell(a rat liver stellate cell line)with lentivirus carrying siRNA or control vectors.According to the results of RT-PCR and Western blot analysis, we picked out the most effective siRNA and it was going to be used to downregulate the expression of target gene in vivo.
4、One hundred and eighty Wistar rats were randomized into five groups,namely groupⅠ(sham operation),groupⅡ(OJ+RBF+rhALR),groupⅢ(OJ+RBF+rhALR+LV),groupⅣ(OJ+RBF+rhALR+control vector) and groupⅤ(OJ+RBF+rhALR+NaCl).Samples were collected on the 4th,7th,14th,18th,21st and 28th day after the first operation.
5、The efficacy of RNAi in vivo was determined by Real time PCR and Western blot analysis.
6、We observed the pathological change of liver from each group and detected the changes of hepatic function and mitochondrial function.
Results:
1、The results of sequencing showed that designed siRNAs were successfully integrated into the lentivirus-siRNA vector system.
2、Titers of lenti-target1#, lenti-target2#, lenti-target3# and lenti-target4# were 8E+7 TU/ml, 7E+7 TU/ml, 9E+7 TU/ml, and 1E+8 TU/ml respectively.
3、The result of RNAi of target gene in vitro showed that lenti-target3# was the most effective one,and the result of Western blot analysis also proved its potency.
4、After bile duct ligation,the livers in all groups except groupⅠexhibited various degrees of pathological changes,and groupⅢshowed the most obvious pathological change among all groups.With the restoration of bile flow,the pathological changes abated gradually,and the restoration in groupⅢwas slowest among all groups.
5、After bile duct ligation,there were various degrees of damage to hepatic function and mitochondrial function in groupⅡ, groupⅢ, groupⅣand groupⅤ(P<0.05,compared with groupⅠ),and the damage to groupⅢwas more severe than those to other groups(P<0.05,compared with groupⅡ, groupⅣand groupⅤ). After the restoration of bile flow,mitochondrial function and hepatic function of all groups recovered gradually,but the recovery speed in groupⅢwas slower than those of other groups(P<0.05,compared with groupⅡ, groupⅣand groupⅤ).
6、The result of RNAi in vivo showed that target gene expression in gourpⅢdecreased obviously in mRNA and protein levels and the difference among groupⅡ, groupⅣand groupⅤwas not statistically significant.
Conclusion:
1、In this study,we successfully construced a lentiviral-siRNA vector system. The results of RNAi in vitro and in vivo proved its efficacy in knocking down target gene expression without obvious“off-target”effect and toxic reaction.
2、Target gene TFAM may played a crucial role in the process of protecting mitochondrial and hepatic function by rhALR. We hypothesized that rhALR could induce the expression of TFAM, protect and repair damaged mtDNA, increase its replication number in hepatocyte, and as a result, improve mitochondrial and hepatic function,and finally promote the recovery of hepatic function after restoration of bile flow in rats with obstructive jaundice.
首先构建好4组针对目的基因序列的RNA干扰慢病毒载体;在体外目的细胞上筛选出有明显干扰效果的靶点并进行慢病毒大量包装。然后建立大鼠梗阻性黄疸动物模型,并进行活体RNA干扰实验,检测目的基因的干扰效果及大鼠线粒体功能和肝功能的变化情况;探讨目的基因在rhALR对大鼠梗阻性黄疸肝功能保护中的作用,为rhALR的临床应用提供理论基础。
二、方法
1、设计4个针对目的基因TFAM的RNA干扰序列,制备双链DNAoligo,然后通过双酶切慢病毒载体,连接及转化反应制备克隆;最后挑取克隆进行PCR鉴定并挑选阳性克隆测序以确认慢病毒载体构建成功。
2、进行RNA干扰慢病毒包装,对其进行浓缩、纯化并测定病毒滴度。
3、利用包装好的RNA干扰慢病毒,采用Real time PCR的方法在体外目的细胞中筛选出有明显干扰效果的慢病毒载体,并利用Western blot的方法在蛋白水平上验证干扰靶点的有效性;最后将有效的干扰靶点进行病毒大量包装以用于下一步的动物实验。
4、建立梗阻性黄疸大鼠动物模型。将健康Wistar大鼠随机分为5组。第1组为假手术组,第2组为梗阻性黄疸、胆道再通及rhALR治疗组,第3组为梗阻性黄疸、胆道再通、rhALR治疗,同时进行活体RNA干扰组,第4组为梗阻性黄疸、胆道再通、rhALR治疗及阴性对照病毒组,第5组为梗阻性黄疸、胆道再通、rhALR治疗及生理盐水对照组。分别于第一次术后4、7、14、18、21、28天采集标本,保证每时相点存活大鼠6只。
5、利用Real time PCR和Western blot的方法,检测大鼠活体RNA干扰的效果。
6、观察各组大鼠肝脏大体和病理改变,检测其肝功能和线粒体功能的变化情况。
三、结果
1、将制备好的RNA干扰慢病毒载体进行测序,结果证实4个靶点均成功连接入载体中,可以用于后续的筛选实验。
2、进行RNA干扰慢病毒包装后,测定4组靶点的病毒滴度依次为8E+7、7E+7、9E+7和1E+8 TU/ml
3、体外筛选结果显示3号靶点在目的细胞中对目的基因TFAM的干扰效率达到了55%左右,在所有的干扰靶点中效率最高;Western blot实验也证实了其干扰的有效性。
4、胆道梗阻后,除第1组外的其余各组大鼠肝脏均出现了不同程度的病理改变,其中第3组大鼠病理改变最明显;胆道再通后,上述各组大鼠肝脏病理改变逐步减轻,组间比较发现第3组大鼠恢复速度最慢。
5、胆道梗阻后第2～5组大鼠肝功能和线粒体功能均出现了不同程度的损害(与第1组比较,P<0.05),其中第3组大鼠的损害程度明显重于第2、4、5组大鼠(P<0.05);胆道再通后,上述各组大鼠肝功能和线粒体功能逐步恢复,其中第3组大鼠的恢复速度明显慢于第2、4、5组大鼠(P<0.05)。
6、活体RNA干扰实验结果显示,第3组大鼠肝脏中目的基因TFAM mRNA的含量出现了明显下降,而阴性病毒对照组(第4组)、生理盐水对照组(第5组)及未进行RNA干扰的第2组中目的基因TFAM mRNA的含量相比无明显差异。Western blot实验结果显示第3组大鼠目的基因TFAM的蛋白表达也明显降低。
四、结论
1、本研究成功构建了针对目的基因TFAM的RNA干扰慢病毒载体,在体外实验中具有良好的干扰效率,能有效降低目的基因TFAM mRNA的表达;在体内实验中同样具有良好的干扰效果,且无明显的“脱靶效应”和毒性反应。
2、在rhALR保护及改善梗阻性黄疸大鼠的肝功能,促进胆道再通后肝功能的恢复过程中,TFAM可能发挥了重要的作用;其机制可能通过诱导肝细胞TFAM的表达,提高线粒体DNA(mitochondrial DNA,mtDNA)的拷贝数,修复损伤的mtDNA,最终保护和改善梗阻性黄疸肝细胞线粒体功能和肝功能,促进胆道再通后肝功能的恢复。
Objectives: To study the change of mitochondrial and hepatic function in rats with obstructive jaundice after RNA interference of target gene and to explore the role of gene TFAM in the process of protecting mitochondrial and hepatic function by rhALR,and finally,to provide assistance for the clinic application of rhALR.
Methods:
1、We designed 4 siRNAs targeting gene TFAM and constructed the lentivirus-siRNA vector system.
2、Lentiviral vector was generated by co-transfecting 293T cell with pGC-LV recombinant plasmid, pHelper1.0 and pHelper2.0 plasmids,and then the titer of LV was determined on 293T cell.
3、To test the efficacy of designed siRNAs in knocking down TFAM expression,we transfected HSC-T6 cell(a rat liver stellate cell line)with lentivirus carrying siRNA or control vectors.According to the results of RT-PCR and Western blot analysis, we picked out the most effective siRNA and it was going to be used to downregulate the expression of target gene in vivo.
4、One hundred and eighty Wistar rats were randomized into five groups,namely groupⅠ(sham operation),groupⅡ(OJ+RBF+rhALR),groupⅢ(OJ+RBF+rhALR+LV),groupⅣ(OJ+RBF+rhALR+control vector) and groupⅤ(OJ+RBF+rhALR+NaCl).Samples were collected on the 4th,7th,14th,18th,21st and 28th day after the first operation.
5、The efficacy of RNAi in vivo was determined by Real time PCR and Western blot analysis.
6、We observed the pathological change of liver from each group and detected the changes of hepatic function and mitochondrial function.
Results:
1、The results of sequencing showed that designed siRNAs were successfully integrated into the lentivirus-siRNA vector system.
2、Titers of lenti-target1#, lenti-target2#, lenti-target3# and lenti-target4# were 8E+7 TU/ml, 7E+7 TU/ml, 9E+7 TU/ml, and 1E+8 TU/ml respectively.
3、The result of RNAi of target gene in vitro showed that lenti-target3# was the most effective one,and the result of Western blot analysis also proved its potency.
4、After bile duct ligation,the livers in all groups except groupⅠexhibited various degrees of pathological changes,and groupⅢshowed the most obvious pathological change among all groups.With the restoration of bile flow,the pathological changes abated gradually,and the restoration in groupⅢwas slowest among all groups.
5、After bile duct ligation,there were various degrees of damage to hepatic function and mitochondrial function in groupⅡ, groupⅢ, groupⅣand groupⅤ(P<0.05,compared with groupⅠ),and the damage to groupⅢwas more severe than those to other groups(P<0.05,compared with groupⅡ, groupⅣand groupⅤ). After the restoration of bile flow,mitochondrial function and hepatic function of all groups recovered gradually,but the recovery speed in groupⅢwas slower than those of other groups(P<0.05,compared with groupⅡ, groupⅣand groupⅤ).
6、The result of RNAi in vivo showed that target gene expression in gourpⅢdecreased obviously in mRNA and protein levels and the difference among groupⅡ, groupⅣand groupⅤwas not statistically significant.
Conclusion:
1、In this study,we successfully construced a lentiviral-siRNA vector system. The results of RNAi in vitro and in vivo proved its efficacy in knocking down target gene expression without obvious“off-target”effect and toxic reaction.
2、Target gene TFAM may played a crucial role in the process of protecting mitochondrial and hepatic function by rhALR. We hypothesized that rhALR could induce the expression of TFAM, protect and repair damaged mtDNA, increase its replication number in hepatocyte, and as a result, improve mitochondrial and hepatic function,and finally promote the recovery of hepatic function after restoration of bile flow in rats with obstructive jaundice.
引文
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