利用NASH大鼠原代肝细胞与原代Kupffer细胞共培养建立NASH原代细胞模型

Isolation and co-culture of primary hepatocytes and primary Kupffer cells from rats with nonalcoholic steatohepatitis

目的:通过分离并提纯非酒精性脂肪性肝炎(NASH)大鼠原代肝细胞以及原代Kupffer细胞建立体外NASH原代细胞模型,为研究NASH提供可靠的细胞实验技术支持。方法:选择SD大鼠40只,随机分为2组(n=20):对照组和NASH组,对照组大鼠利用普通饲料喂养,NASH组大鼠利用高脂饲料(88%基础饲料+10%猪油+2%胆固醇)喂养,6~8周后,利用NASH评分表,病理观察下肝组织切片脂肪变+小叶内炎症+气球样变评分≥4分,表明大鼠NASH模型的成功建立,利用胶原酶原位灌注法分离并提纯NASH模型大鼠原代肝细胞以及原代Kupffer细胞,利用CK-18及CD68免疫荧光以及墨汁吞墨实验进行细胞鉴定,利用油红O染色、试剂盒测定谷丙转氨酶(ALT)、谷草转氨酶(AST)含量观察NASH大鼠原代肝细胞脂质累积和肝功情况,Western blot检测原代Kupffer细胞炎症因子表达情况,最后采用原代肝细胞:原代Kupffer细胞=6∶1比例共培养,显微镜下观察细胞状态。结果:实验成功分离并提纯NASH原代肝细胞以及原代Kupffer细胞,通过油红O染色,NASH组大鼠原代肝细胞存在明显的脂肪沉积,且NASH组大鼠原代肝细胞中AST、ALT明显高于对照组,存在明显肝损伤(P<0.05),Western blot测定原代Kupffer细胞TNF-α、IL-1β以及MCP-1,NASH组大鼠明显高于对照组(P<0.05)。结论:通过胶原酶原位灌注法可以成功分离NASH大鼠原代肝细胞以及原代Kupffer细胞,同时成功建立比例共培养大鼠体外原代细胞NASH模型。

Objective:By isolating and purifying primary hepatocytes and primary Kupffer cells from rats with nonalcoholic steatohepatitis(NASH),and establishing the primary cell model of NASH in vitro,to provide reliable technical support for cell experiment in the study of NASH.Methods:Forty SD rats were selected and randomly divided into the control group and the NASH group.The rats in the control group were fed with common feed,and the rats in the NASH group were fed with a high-fat diet(88%basal feed+10%lard+2%cholesterol).After 6-8 weeks,using the NASH score table,the liver tissue section steatosis+intralobular inflammation+ballooning degeneration score≥4 points under pathological observation,indicating that the rat NASH model was successfully established.And the primary hepatocytes of NASH rats were isolated and purified by collagenase in situ perfusion.Cells were identified by CK-18 and CD68 immunofluorescence and ink swallowing test.The lipid accumulation was tested by Oil red O staining,and the contents of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were determined to evaluate the liver function in primary hepatocytes of NASH rats.The expressions of inflammatory factors of primary Kupffer cells were detected by Western blot.Finally,primary hepatocytes and primary Kupffer cells were co cultured at the ratio of 6:1 and observed under microscope.Results:NASH primary hepatocytes and primary Kupffer cells were successfully isolated and purified.Compared with the control group,Oil red O staining showed that the primary hepatocytes of the NASH group had obvious fat deposition,and the AST and ALT levels in the primary hepatocytes of the NASH group were significantly higher than those of the control group,indicating obvious liver damage(P<0.05).The Western blot result showed that the levels of TNF-α,IL-1βand MCP-1 in primary Kupffer cells was significantly higher than the control group(P<0.05).Conclusion:The primary hepatocytes and primary Kupffer cells of NASH rats were isolated successfully by collagenase in situ perfusion.At the same time,a proportional co-culture rat in vitro primary cell NASH model was successfully established.