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重组Akt腺病毒对CCl4诱导的肝硬化门静脉高压症大鼠模型的影响

Effects of Ad-myr-HA-Akt on cirrhotic portal hypertension in rats

摘要:

目的 研究重组Akt腺病毒对CCl4诱导的大鼠肝硬化门静脉高压症的影响及其作用机制.方法 以细胞内同源重组法构建重组腺病毒Ad-myr-HA-Akt;采用四氯化碳复合法制备肝硬化门静脉高压症大鼠模型.将大鼠模型分为二部分:用于组织学检测的大鼠,在制备大鼠模型的第2周和第6周自尾静脉导入Ad-myr-HA-Akt.第8周时应用Western blot法检测各组大鼠肝组织内Akt,p-Akt,Fas,DR5蛋白的表达.用于检测肝硬化门静脉高压症大鼠,在肝硬化模型的第9周自尾静脉导人生理盐水和Ad-myr-HA-Akt,3 d后测定各组的门静脉压力、平均动脉压和心率.结果 Ad-myr-HA-Akt治疗后Akt组组织学病变减轻,Fas蛋白的表达明显低于肝硬化组、生理盐水组和增强型绿色荧光蛋白(enhance green fluorescent protein,EGFP)组,血清ALT和AST及羟脯氨酸(hydroxyproline,Hyp)均显著低于其他各组,p-Akt蛋白的表达明显高于其他各组,Fas和HSC活化的指标DR5蛋白含量降低.EGFP组在Ad-EGFP转染后,肝组织中可见大量绿色荧光,肺和肾组织中仅见少量荧光,而其他实质器官未见EGFP表达.结论 Akt重组腺病毒能够阻断大鼠肝硬化的发展,可能是防治肝硬化的一条有效途径.

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abstracts:

Objective To probe the effects of recombinant adenovirus containing Akt on carbon tetrachioride-induced rat liver cirrhosis and portal hypertension. Methods Cirrhosis was induced in rats by a complex method of carbon tetrachloride. Recombinant adenovirus Ad-myr-HA-Akt was produced by homologous recombination in 293 cells. Rats received Ad-myr-HA-Akt via the tail vein at the second and the sixth week respectively. The pathological changes in liver tissues were observed after Van Gieson (VG) staining. Fas antigen in rat livers were determined by immunohistochemical method. The levels of alanine minotransferase( ALT), aspartate aminotransferase ( AST), albumin( ALB ) and hydroxyproline (Hyp) were measured. Fas antigen in rat livers were determined with immunohistochemical method. Expression of Akt, p-Akt, Fas and DR5 were evaluated by Western blotting. Frozen sections of the liver, heart,lung,kidney, brain,spleen and testis were made to examine the expression of enhance green flourescent protein (EGFP) by fluorescence microscopy in EGFP group. After 8-week CCl4 treatment, portal hypertensive rats in the saline group and Ad-Akt group received saline and Ad-myr-HA-Akt via the tail vein respectively. Portal vein pressure, mean arterial pressure and heart rate were measured in all rats on Day 3. Results In comparison with other cirrhosis rats, the pathological changes in the Akt group was markedly attenuated, and the levels of ALT, AST and Hyp were significantly lowered. Western blotting showed that the protein expression of p-Akt in the Akt group was higher significantly as compared with those in the negtive control group, saline group and EGFP group. Western blot also showed that the protein expression of Fas and DR5 in the Akt group was lower significantly. EGFP expression was mainly demonstrated by fluorescence microscopy on the frozen section of liver, very little fluorescene were detected in lung and kidney and there was no detectable EGFP in the other organs. Conclusions Ad-myr-HA-Ak inhibits CCl4-induced liver cirrhosis and is a potential pharmacological target for gene therapy in liver cirrhosis.

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