非酒精性脂肪性肝炎大鼠TLR4基因及其甲基化水平的相关性研究

目的 观察非酒精性脂肪性肝炎（NASH）大鼠肝脏中Toll样受体4（TLR4）基因的表达及其甲基化水平的 变化，并探讨两者在NASH发病中的相关性。方法 将20只大鼠采用随机数字表法分为正常组与模型组，每组10 只。正常组与模型组大鼠分别饲喂普通饲料和高脂高糖饲料，于24周后处死。测量大鼠体质量及肝质量，并取各组 大鼠血清及肝脏组织，于低温保存。通过病理染色观察肝细胞的形态变化、脂肪变性及炎性细胞浸润程度。采用酶 联免疫吸附测定（ELISA）法检测血清中丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）、三酰甘油（TG）及总胆固醇 （TC）水平；采用免疫组织化学染色法检测肝脏CD68蛋白的表达；采用蛋白免疫印迹法检测肝脏TLR4蛋白的表达； 实时荧光定量PCR检测肝脏TLR4基因mRNA的表达；亚硫酸氢测序PCR（BSP）检测肝脏TLR4基因甲基化水平。结 果 与正常组相比，模型组肝细胞体积增大，出现大量脂肪空泡，炎性细胞浸润明显；肝指数升高，血清中ALT、AST、 TC及TG明显升高（P＜0.01）；CD68蛋白的表达含量明显升高；TLR4蛋白、mRNA表达明显升高（P＜0.01）；甲基化水 平降低（P＜0.01）。TLR4的甲基化水平与其mRNA相对含量的表达呈负相关（P＜0.01）。结论 NASH模型大鼠肝 脏TLR4基因的表达与其甲基化水平呈负相关，TLR4基因甲基化水平的降低在NASH发病因素中发挥重要作用。

Correlation between TLR4 gene and its methylation level in non-alcoholic steatohepatitis rats

Objective To observe the expression of Toll-like receptor 4 (TLR4) gene and its methylation level in liver of non-alcoholic steatohepatitis (NASH) model rats, and explore the correlation between the two in the pathogenesis of NASH. Methods Twenty rats were randomly divided into normal group and model group,with 10 rats in each group. Rats in the normal group and the model group were fed with ordinary diet and high-fat and high-sugar diet. Rats were sacrificed after 24 weeks. The body weight and liver weight of the rats were measured. The serum samples and liver tissues were collected under low temperature conditions. The morphological staining was used to observe morphological changes,steatosis and inflammatory cell infiltration of hepatocytes. The serum levels of alanine aminotransferase (ALT), aspartic aminotransferase (AST),triglyceride (TG) and total cholesterol (TC) were detected by ELISA.The expression of CD68 protein in liver was detected by immunohistochemistry. The expression of TLR4 protein in liver was detected by Western blot assay. Real-time fluorescence quantitative PCR was used to detect the mRNA content of TLR4 in liver. Methylation of TLR4 gene in liver was detected by bisulfite sequencing PCR (BSP). Results Compared with the normal group，the volume of liver cells became larger, a large number of fat vacuoles appeared, and inflammatory cell infiltration was obvious; The liver index increased in the model group, the serum levels of AST,ALT,TG and TC increased significantly (P＜0.01). The expressions of TLR4 and CD68 protein increased significantly (P＜0.01). The mRNA content of TLR4 gene increased obviously (P＜0.01)， and its methylation level decreased (P＜0.01). Conclusion The expression of TLR4 gene is negatively correlated with its methylation level in the liver of NASH model rats. The reduction of TLR4 gene methylation level plays an important role in the pathogenesis of NASH.