论文部分内容阅读
目的探讨肝脏靶向表达rIL-10基因对猪血清诱导的大鼠肝纤维化的抑制作用。方法 rIL-10基因通过尾静脉快速大容量注射法转移至大鼠体内,RT-PCR法检测大鼠肝、肾、脾和肺组织rIL-10 mRNA的表达及S-P免疫组化法检测rIL-10蛋白在肝脏中表达。30只体质量100～120 g清洁级雄性SD大鼠按随机数字表法分为正常对照组(n=6)、纤维化模型对照组(n=8)、rIL-10基因干预组(n=8)和空质粒对照组(n=8)。正常对照组与其余3组大鼠腹腔分别注射生理盐水和猪血清,0.5 ml/只,每周2次。第5周开始造模同时,rIL-10基因干预组和空质粒对照组大鼠分别从尾静脉注射pcDNA3-rIL-10和pcDNA3空质粒,每周1次。第8周末处死全部大鼠收集组织和血清备用。HE、天狼星红染色分别检测各组肝组织病理形态学和Ⅰ、Ⅲ胶原的改变,生化检测血清ALT和AST含量。结果 RT-PCR和免疫组化证实rIL-10基因经尾静脉快速大容量注射转染到大鼠体内后主要在肝组织中转录及表达;纤维化模型和空质粒对照组:HE染色显示大鼠肝细胞脂肪变性,炎性细胞浸润,大量胶原沉积形成粗细不等的纤维隔,部分假小叶形成;天狼星红染色显示肝组织中大量Ⅰ、Ⅲ型胶原纤维沉积;血清中ALT和AST表达水平较正常对照组明显升高(P<0.01)。与对照组比较,rIL-10基因干预组大鼠HE染色显示肝细胞变性坏死程度减轻、炎细胞的浸润减少,胶原沉积明显减少,天狼星红染色显示Ⅰ、Ⅲ型胶沉积面积显著下降(P<0.01),血清中ALT和AST表达水平显著降低(P<0.01)。结论 尾静脉快速大容量注射法可使rIL-10基因在肝组织靶向表达,靶向表达的rIL-10有效抑制猪血清诱导的大鼠肝纤维化形成。
Objective To investigate the inhibitory effect of liver targeting rIL-10 gene on pig serum induced liver fibrosis in rats. Methods The rIL-10 gene was transferred to the rat by rapid mass injection via the tail vein. The expression of rIL-10 mRNA in liver, kidney, spleen and lung was detected by RT-PCR and the expression of rIL-10 The protein is expressed in the liver. 30 male SD rats of 100-120 g body weight were divided into normal control group (n = 6), fibrosis model control group (n = 8) and rIL-10 gene intervention group (n = 8) and empty plasmid control group (n = 8). Normal control group and the other three groups were injected with normal saline and porcine serum, 0.5 ml per mouse respectively, twice a week. At the same time, the rats in rIL-10 gene group and empty plasmid control group were injected with pcDNA3-rIL-10 and pcDNA3 empty plasmid respectively from the tail vein once a week. At the end of the 8th week, all the rats were sacrificed to collect the tissue and serum. Hematoxylin and eosin (HE) and Sirius red staining were used to detect the histopathological changes of liver tissues and the changes of collagen Ⅰ and collagen Ⅲ. The biochemical changes of serum ALT and AST were detected. Results RT-PCR and immunohistochemistry confirmed that the rIL-10 gene was transcribed and expressed mainly in the liver tissue after transfection into the rat by rapid tail vein injection. The fibrosis model and empty plasmid control group: HE staining showed that the rat Hepatic steatosis, inflammatory cell infiltration, a large number of collagen deposition thickness of the fibrous septa formation, the formation of false lobules; Sirius red staining showed a large number of type Ⅰ, Ⅲ collagen deposition in the liver; serum ALT and AST expression levels The normal control group was significantly higher (P <0.01). Compared with the control group, hematoxylin and eosin staining showed that the degree of degeneration and necrosis of hepatocytes, infiltration of inflammatory cells and the deposition of collagen in the rIL-10 gene-treated group were significantly decreased, and the deposition area of type I and type III collagen was significantly decreased by Sirius red staining (P < 0.01). The levels of ALT and AST in serum were significantly decreased (P <0.01). Conclusion The tail vein injection of rIL-10 gene can be rapidly expressed in the liver tissue by rapid high-volume injection. Targeted expression of rIL-10 effectively inhibits porcine serum-induced liver fibrosis in rats.