摘要：
目的 优化制备合并缺氧的非酒精性脂肪性肝炎大鼠动物模型,探讨其纤维化形成与新生血管生成的关系. 方法 将清洁级SD大鼠32只随机均分为正常对照组(A组)、缺氧对照组(B组)、高脂组(C组)、高脂缺氧组(D组),分别给予正常饮食、腹腔内注射亚硝酸钠、高脂饮食、高脂饮食+腹腔内注射亚硝酸钠,每组共饲养16周,B、D两组在后8周每天腹腔内注射亚硝酸钠模拟缺氧.检测大鼠生物化学指标,进行肝组织非酒精性脂肪性肝炎活动度评分和纤维化评分、用Realtime PCR比较各组缺氧诱导因子1α、血管内皮生长因子1、血管内皮生长因子受体2、肿瘤坏死因子、白细胞介素1β、白细胞介素6.用免疫组织化学检测血管内皮生长因子受体2、CD34表达,以染色阳性细胞数评分和染色程度评分之积为总评分来定量分析表达强度.计量资料采用t检验,等级资料采用秩和检验.结果 C、D两组的非酒精性脂肪性肝炎活动度评分＞4,D组HE染色可见到非酒精性脂肪性肝炎的病理学特征,肝腺泡3区可见明显脂肪变性,小泡性为主混合部分为大泡性脂肪滴,合并肝细胞气球样变性,小叶内炎症明显.D组的Masson染色可见窦周纤维化合并门静脉周围纤维化,部分大鼠见到桥接纤维化.D组比C组的天冬氨酸氨基转移酶、缺氧诱导因子1α、血管内皮生长因子1、血管内皮生长因子受体2明显升高,差异均有统计学意义(P值均＜ 0.05),免疫组织化学检测结果显示血管内皮生长因子受体2、CD34表达总评分增加(P值均＜0.05). 结论 高脂饮食基础上给予腹腔内注射亚硝酸钠能够制备出合并缺氧的非酒精性脂肪性肝炎模型,模型的肝纤维化进展与缺氧诱导因子1α诱导新生血管的生成相关.%Objective To optimize the construction of combined hypoxia NASH rat model on the basis of preliminary work,and to explore the role of neovascularization in the process of hepatic fibrosis.Methods 32 rats were divided randomly to four groups that were null control group(A group),hypoxia group(B group),high fat diet group(C group) and high fat diet plus hypoxia group (D group),treated with null,Intraperitoneal injection of NaNO2,high fat diet and high fat diet plus Intraperitoneal injection of NaNO2 respectively.Every group was observed for 16 weeks,B and D group was treated with Intraperitoneal injection of NaNO2 20 mg/kg.d at the laster 8 weeks.Liver histology NASH activity score(NAS) and Fibro score(FibroS),biochemical index were detected in this combined hypoxia NASH rat model(D group),meanwhile the changes of HIF1α,inflammatory factor and neovascularization were measured by ELISA,realtime PCR and immunohistochemistry.Results Liver tissue NAS ＞ 4 was seen in C and D group.D group showed NASH characteristics,including significantly steatosis at liver acinar 3 area(mostly a microvesicular type fat droplets mixed with macrovesicular type),hepatocyte balloon degeneration,obvious lobular inflammation,while fibrosis score increased significantly,including visible hepatic sinusoid fibrosis,fibrosis around portal vein,and bridging fibrosis in a considerable portion of the rats.Compared with C group,biochemical indicators of aspartate aminotransferase (AST),HIF1α,neovascularization-related VEGFA,VEGFR2 mRNA level increased obviously and the expression of immunohistochemistry VEGFR2,CD34 enhanced markedly in D group(r ＜ 0.05).Conclusion A combined hypoxia NASH rat model can be established throught feeding 16 weeks' high-fat diet then intraperitoneal injection of NaNO2 20 mg / kg.d at the laster 8 weeks,meanwhile chronic hypoxia can accelerate this combined hypoxia NASH model liver fibrosis process.In this process neovascularization promoted the formation of hepatic fibrosis in this model.
