Akt基因治疗大鼠肝硬化门静脉高压症

目的:探讨肝硬化时肝组织内Akt和eNOS的活化是否受到抑制以及腺病毒介导的Akt基因治疗门静脉高压症的可行性.方法:以细胞内同源重组法构建复制缺陷型重组腺病毒Ad-myr-HA-Akt和Ad-EGFP.采用四氯化碳复合法制备肝硬化门静脉高压症大鼠模型.取10只正常大鼠作为对照,另取40只肝硬化大鼠随机均分为4组:未处理组、Akt治疗组、EGFP组和生理盐水组.后3组分别经尾静脉分别注射Akt重组腺病毒、Ad-EGFP和生理盐水后,3 d后,分别测定各组的门静脉压力、平均动脉压和心率.用免疫印迹法检测各组大鼠肝组织内Akt,p-Akt,eNOS,p-eNOS蛋白的表达;硝酸还原酶法检测各组肝内NO含量.EGFP组于转染后3 d处死大鼠,取肝、心、肺、肾、脑、脾和睾丸,快速冰冻切片,观察绿色荧光蛋白的表达情况.结果:Ad-myr-HA-Akt和Ad-EGFP经纯化后滴度分剐为5.5×1011vp/mL和6.0×1011 vp/mL.肝硬化大鼠肝内Akt和eNOS的活化均受到抑制,肝内NO生成减少,门静脉压力升高.Akt重组腺病毒治疗能使受到抑制的Akt和eNOS磷酸化得到恢复,同时增加肝内NO含量,降低门静脉压力.而经Ad-EGFP和生理盐水处理组,Akt和eNOS磷酸化不能恢复,肝内NO含量和门静脉压力与未经治疗大鼠相似.EGFP组在Ad-EGFP转染后3 d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:肝硬化时,大鼠肝内Akt和eNOS的活化均受到抑制,导致NO生成减少而肝内血管阻力增加,表现为门静脉压力升高.以腺病毒介导的Akt基因治疗门静脉高压症可行.

Akt gene therapy for cirrhotic rats with portal hypertension

OBJECTIVE: To determine whether there is an impaired Akt and eNOS activation in cirrhotic livers, and to investigate the feasibility of transferring adenovirus-mediated Akt gene to the liver for portal hypertension. METHODS: Recombinant adenovirus Ad-myr-HA-Akt and Ad-EGFP were produced by homologoas recombination in 293 cells . The Methods of compound factor, carbon tetrachloride (CCl4), corn flour, and cholesterol plus alcohol were used to construct the hepatic cirrhosis rat models. Ten normal rats were served as a normal control group, and 40 cirrhotic rats were divided into 4 groups randomly: an untreated group, an Ad-myr-HA-Akt treated group, an Ad-EGFP group, and a saline group. Ad-myr-HA-Akt, Ad-EGFP, and saline were transduced into the Ad-myr-HA-Akt treated group, Ad-EGFP group, and saline group via the tail vein respectively. Portal vein pressure, mean arterial pressure, and heart rate were measured in all rats. Protein abundance and phosphorylation status of Akt and eNOS were examined by Western blot. Spectrophotometry was used to measure the NO level. Frozen sections of the liver, heart, lung, kidney, brain, spleen, and testis were made to examine the expression of enhanced green fluorescent protein (EGFP) by fluorescence microscopy on Day 3 in the Ad-EGFP group. RESULTS: The concentration of recombinant adenovirus Ad-myr-HA-Akt after the purification was 5.5 x 10(11)vp/mL and that of Ad-EGFP was 6.0 x 10(11)vp/mL. Akt and eNOS phosphorylations in the liver of cirrhotic rats were obviously impaired. Adenoviral delivery of myr-Akt restored eNOS phosphorylation, increased the NO level and decreased the portal pressure after 3 days of adenoviral infection. In contrast, the livers infected with Ad-EGFP and saline were not changed. The EGFP expression was mainly found under the fluorescence microscopy on the frozen section of liver. Very little fluorescence was detected in the lung and kidney; and there was no detectable EGFP in other organs. CONCLUSION: There is an impaired Akt and eNOS activation in the cirrhotic livers; myr-Akt gene therapy can restore the Akt activation and NO production in the cirrhotic liver, suggesting that this therapy may be helpful in treating portal hypertension.