一种改进的肝纤维化大鼠肝星状细胞分离方法  

An improved Method for the Isolation of Hepatic Stellate Cells in Rats with Hepatic Fibrosis

作　　者：朱永建 舒建昌 Zhu Yongjian(Zhaoqing medical college,Zhaoqing Guangdong 526020)

机构地区：[1]肇庆医学院,广东肇庆526020 [2]暨南大学医学院附属广州市红十字会医院,广东广州510220

出　　处：《黑龙江医药》2024年第6期1263-1267,共5页Heilongjiang Medicine journal

基　　金：广州市科技计划项目(编号:201707010356)。

摘　　要：目的:建立一种简便、经济、高效的肝纤维化大鼠肝星状细胞的分离方法,为肝纤维化的相关研究奠定基础。方法:利用腹腔注射四氯化碳(CCL4)诱导SD大鼠肝纤维化模型,将肝纤维化SD大鼠经门静脉肝素化后,剪破下腔静脉,D-Hanks液灌注肝脏至土黄色,封闭上下腔静脉,缓慢注入5mL 37℃含0.100%链霉蛋白酶、0.050%Ⅳ型胶原酶的D-Hanks液,静置10min后,继续缓慢注入5mL该液后摘除肝脏,在37℃培养箱静置30min,将肝脏匀浆化后加入0.050%Ⅳ型胶原酶、0.020%链霉蛋白酶和0.010%DNA酶I溶液中振荡消化30min,18%Nycodenz密度梯度离心获得肝星状细胞。采用台盼蓝拒染实验鉴定细胞活力,自发荧光、Desmin及α-SMA免疫细胞化学染色鉴定细胞纯度。结果:肝星状细胞得率每只大鼠约5×10^(7)个/鼠,细胞活力在90%以上,肝星状细胞在328nm荧光下自发蓝绿色荧光,Desmin、α-SMA免疫细胞化学染色鉴定纯度达到90%以上。结论:建立了一种简便、经济、高效的肝纤维化大鼠肝星状细胞分离方法。Objective:To establish a simple,economical and efficient method of isolating hepatic stellate cells from rat with liver fibrosis for lay a foundation in the study of hepatic fibrosis.Methods:The liver fibrosis model of SD rats was induced by intraperitoneal injection of carbon tetrachloride(CCL 4).After hepatic fibrosis of SD rats through portal vein heparinization,the inferior vena cava was cut out,and D-hanks fluid was infused into the liver to the earthy yellow color.Then closed the inferior vena cava,slowly injected 5mL 37℃containing 0.100%pronase,0.050%Ⅳcollagenase D-Hanks fluid,let stand for 10 min.And continue to slowly remove liver after injected 5mL of the fluid.After in 37℃incubator standing for another 30 min,adding 0.050%Ⅳcollagenase,0.020%pronase and 0.010%DNases I solution digest liver homogenate 30 min.HSCs were obtained by 18%Nycodenz density gradient centrifugation.Cell viability was determined by trypan blue rejection assay,and cell purity was determined by spontaneous fluorescence,Desmin andα-SMA immunocytochemistry stain.Results:the rate of HSCs was about 5×10^(7)/rat,and the cell activity was more than 90%.Hepatic stellate cells spontaneously fluoresce blue-green at 328 nm.The purity was up to 90%by Desmin andα-SMA immunocytochemistry staining.Conclusion:a simple,economical and efficient method for isolating HSCs in rats with hepatic fibrosis was established.

关 键 词：肝纤维化 原代肝星状细胞 细胞分离 

分 类 号：R575[医药卫生—消化系统]