重组质粒pcDNA3.1/C-sis对大鼠FHF的影响

目的 探讨重组质粒peDNA3.1/C-sis导入大鼠对暴发性肝功能衰竭(FHF)的影响.方法 利用流体力学的方法将重组质粒pcDNA3.1/C-sis导入大鼠肝脏,48 h后用内毒素(LPS)+D-半乳糖胺(D-GalN)诱导大鼠FHF;利用荧光定量PCR及蛋白质印迹法(Western blotting)检测C-sis表达;利用苏木精-伊红(HE)染色及测定半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)活性检测凋亡;利用Western Blotting检测Bcl-2和Bax的表达变化;计算大鼠24 h观察期内的死亡率.结果 FHF+C-sis质粒组的C-sis mRNA和蛋白表达水平较正常对照组和FHF+空载质粒组明显增加,差异均有统计学意义(P＜0.01).与正常对照组相比,FHF+林格液注射组和FHF+空载质粒组肝脏细胞凋亡增多;与FHF+空载质粒组相比,FHF+C-sis质粒组肝脏细胞凋亡减少.与正常对照组相比,FHF+林格液注射组大鼠肝脏组织中Caspase-3的表达增多(P＜0.01);与FHF+空载质粒组相比,FHF+C-sis质粒组大鼠肝脏组织中Caspase-3的表达减少(P＜0.05).与正常对照组相比,FHF+林格液注射组大鼠肝脏组织中Bcl-2表达明显减少(P＜0.01),Bax表达明显增多(P＜0.01);而与FHF+空载质粒组相比,FHF+C-sis质粒组大鼠肝脏组织中Bcl-2表达增多(P＜0.05),Bax表达减少(P＜0.05).在24 h观察期内,正常对照组大鼠均存活,FHF+林格液注射组及FHF+空载质粒组大鼠死亡率分别为70.0％和80.0％;而FHF+C-sis质粒组大鼠仅2o.0％死亡.结论 C-sis基因可减弱LPS+ D-GalN诱导的大鼠FHF.

The effect of the recombinant plasmid pcDNA3.1/C-sis on FHF of rats

Objective To explore the effect of the recombinant plasmid pcDNA3.1/C-sis on fulminant hepatic failure (FHF) in rats.Methods 48 h after recombinant plasmid pcDNA3.1/C-sis being imported into rat liver by using the method of fluid mechanics,FHF in rats was induced by endotoxin (LPS)+D-galactosamine (D-GaIN).With fluorescence quantitative PCR and Western blotting,C-sis expression was tested.The apoptosis of rat liver was detected by using HE staining and measuring Caspase-3 activity.The expression changes of Bcl-2 and Bax were examined through using Western blotting.The mortality rate of rats was calculated during 24 h observation period.Resnlts Compared with the normal control group and FHF+ empty plasmid group,C-sis mRNA and protein expression levels were increased significantly in the FHF+C-sis plasmid group,there were statistically significant differences (P＜0.01).Compared with the normal control group,the apoptotic hepatocytes were increased in the FHF + Ringer's solution injection group and FHF+ empty plasmid group;compared with FHF+ empty plasmid group,the apoptotic hepatocytes in the FHF+C-sis plasmid group were decreased.Compared with the normal control group,Caspase-3 expression level was increased in the FHF+ Ringer's solution injection group (P＜0.01);compared with the FHF+ empty plasmid group,Caspase-3 expression level in the FHF+C-sis plasmid group was decreased (P＜0.05).Compared with the normal control group,Bcl-2 expression level was decreased significantly (P＜0.01),and Bax expression level was increased significantly (P＜0.01) in the FHF+Ringer's solution injection group;compared with the FHF+ empty plasmid group,the Bcl-2 expression level was increased (P＜0.05),and Bax expression was decreased (P＜0.05)in the FHF+C-sis plasmid group.During the 24 h observation period,all rats in the normal control group were alive;the mortality rates of the FHF+ Ringer's solution injection group and FHF+ empty plasmid group were 70.0％ and 80.0％ respectively,while that of the FHF+C-sis plasmid group was only 20.0％.Conclusion C-sis gene could inhibit FHF in rats induced by LPS+D-GalN.