靶向SMAD3基因的shRNA重组慢病毒对大鼠肝再生的作用

Lentiviral-mediated delivery of shRNA targeting the SMAD3 gene promotes liver regeneration in rats

目的:应用大鼠肝大部切除肝再生模型,将前期构建好的靶向SMAD3基因的shRNA重组慢病毒注射入大鼠体内,观察SMAD3 shRNA对大鼠肝再生的影响.方法:将60只♂Wistar大鼠随机分为3组:SMAD3 shRNA组(20只)、shRNA对照组(20只)、生理盐水对照组(20只),通过脾脏注射方法给药,慢病毒给药剂量为1.0×108TU/只,生理盐水对照组给予等体积500L生理盐水.给药96h后行2/3肝大部切除,构建大鼠肝再生模型;肝大部切除术后96h各组分别除死大鼠7只,剩余大鼠于144h处死,收集肝脏标本,Real-Time PCR、免疫组织化学检测肝组织SMAD3表达,免疫组织化学检测肝组织Ki67表达,测定大鼠肝质量/体质量,观察SMAD3 shRNA对肝再生的影响.结果:Real-Time PCR检测显示,通过脾脏注射慢病毒,在96、144h处死时间点,SMAD3 shRNA组SMAD3 mRNA分别较shRNA对照组平均下降73%、63%.免疫组织化学检测显示SMAD3蛋白表达明显下降.Ki67免疫组织化学结果显示,肝大部切除术后96、144h,SMAD3 shRNA组Ki67表达阳性细胞数均明显多于生理盐水对照组及shRNA对照组,表明抑制SMAD3表达后肝细胞增殖活跃.大鼠肝质量与体质量比值显示,SMAD3 shRNA组分别较生理盐水对照组及shRNA对照组有增加趋势(96h:4.50±0.43vs3.97±0.55vs3.98±0.40,144h:4.66±0.54vs4.15±0.51vs4.20±0.34),但没有统计学意义(P>0.05).结论:SMAD3 shRNA在大鼠体内可一定程度上促进肝细胞增殖,但对肝再生的促进作用尚弱.

AIM：To investigate the effect of lentiviral-mediated delivery of shRNA targeting the SMAD3 gene on liver regeneration in rats.METHODS：Sixty male rats were randomly divided into three groups：SMAD3 shRNA group （n = 20）, shRNA control group （n = 20）, and normal saline group （n = 20）. Intrasplenic injection of lentivirus carrying the SMAD3 shRNA or control shRNA at a dose of 1.0 × 108 TU per rat was performed in rats of the SMAD3 shRNA group and shRNA control group, respectively, while the normal saline group was given equal volume of normal saline. After 96 h, 2/3 partial hepatectomy was performed to develop a rat model of liver regeneration. Seven rats of each group were sacrificed at 96 h, and the rest were sacrificed at 144 h after hepatectomy. Liver tissue specimens were collected. SMAD3 expression was detected using real-time PCR and immunohistochemistry. Proliferation of hepatocytes was evaluated by detecting Ki67 by immunohistochemistry. The ratio of liver weight to body weight was determined to observe its role in liver regeneration in vivo.RESULTS：At 96 h and 144 h after hepatectomy, SMAD3 mRNA expression decreased by 73% and 63% in the SMAD3 shRNA group compared to the shRNA control group. The expression of SMAD3 protein also decreased obviously. More active proliferation of hepatocytes was observed after SMAD3 down-regulation. The liver weight/body weight in the SMAD3 shRNA group was higher than those in the normal saline control group and shRNA control group （96 h： 4.50 ± 0.43 vs 3.97 ± 0.55 vs 3.98 ± 0.40, 144 h： 4.66 ± 0.54 vs 4.15 ± 0.51 vs 4.20 ± 0.34）, but there was no statistically significance between them （all P 0.05）. CONCLUSION：Down-regulation of SMAD3 expression could moderately promote liver regeneration in rats.