血红素加氧酶-1基因修饰的骨髓间充质干细胞对大鼠减体积肝移植的影响及机制

The role of HO-1/BMMSCs in rat reduced-size liver transplantation

目的分析血红素加氧酶-1(HO-1)基因修饰的骨髓间充质干细胞(BMMSCs)对大鼠减体积肝移植的影响以及相关机制。方法选取雄性Brown Norway(BN)大鼠50只制备BMMSCs。雄性BN及Lewis大鼠各75只。BN大鼠随机分为模型组(n=25)、干细胞组(n=25)和联合组(n=25),"二袖套"法建立大鼠原位50%减体积肝移植急性排斥模型,术后即刻分别注射生理盐水1 ml、BMMSCs单细胞悬液1 ml、HO-1/BMMSCs单细胞悬液1 ml。术后即刻、第3天、第7天和第14天每组分批处死大鼠。鉴定BMMSCs及HO-1/腺病毒感染BMMSCs效率。HE染色观察肝组织病理改变,检测肝功能指标。检测术后第7天门静脉压力。酶联免疫吸附法(ELISA)检测血清中内皮素-1(ET-1)和一氧化氮(NO)。免疫组化染色、Western印迹检测移植肝ET-1的表达。结果获得高纯度BMMSCs且HO-1/BMMSCs感染成功。与模型组术后比较,干细胞组和联合组肝组织损伤、排斥反应减轻,肝功能改善,且联合组优于干细胞组。门静脉压力分别为模型组(21.3±0.2)mmHg、干细胞组(11.2 ±0.2)mmHg、联合组(10.1 ±0.1)mmHg(1 mmHg=0.133 kPa),三组呈降低趋势,差异有统计学意义(P<0.05)。术后第3天、第7天和第14天,与模型组比较,干细胞组和联合组ET-1表达水平降低,血清NO水平也降低,且联合组低于干细胞组,差异有统计学意义(P<0.05)。结论 HO-1/BMMSCs改善大鼠肝移植术后肝功能和门静脉压力,可能通过调节ET-1/NO平衡发挥保护作用。

Objective To explore the effect and mechanism of heme oxygenase-1 (HO-1) gene modified bone marrow mesenchymal stem cells (BMMSCs) on rat reduced-size liver transplantation. Methods 50 male Brown Norway (BN) rats were used to prepare BMMSCs. Male Lewis and BN rats were 75, respectively. BN rats were randomly divided into model group (n=25), stem cell group (n=25) and combined group (n=25). Acute rejection models following 50% reduced-size transplantaton were established in rats using two-cuff technique, 1 ml of normal saline, BMMSCs suspension, or HO-1/BMMSCs suspension were injected immediately after surgery. Rats were executed at an instant, 3rd, 7th and 14th day after surgery to identify BMMSCs and HO-1/adenovirus infection efficiency. Evaluated hepatic pathology by HE staining. Liver function indexes were detected. Portal vein pressure on 7th day after surgery was detected. The levels of endothelin-1 (ET-1) and nitric oxide (NO) in serum were detected using ELISA. The expressions of ET-1 in liver were detected by immunohistochemistry staining and Western blotting. Results High purity BMMSCs were obtained and HO-1/BMMSCs were successfully infected. Compared with model group, liver tissue injury and rejection were alleviated in stem cell group and combined group, liver function was improved, and the combined group was superior to stem cell group. The portal vein pressure in model group, stem cell group, and combined group were 21.3±0.2 mmHg, 11.2±0.2 mmHg, and 10.1±0.1 mmHg, respectively. The portal vein pressure in three groups showed a decreasing trend, difference was statistically significant (P<0.05). On the 3rd, 7th and 14th day after surgery, compared with model group, the expression levels of ET-1 and NO in the stem cell group and the combined group were decreased, and the combined group was significantly lower than stem cell group (P<0.05). Conclusion HO-1/BMMSCs improved liver function and portal vein pressure after reduced-size liver transplantation in rats, and may play a protective role by regulating ET-1/NO expression.