【摘要】 目的 改进大鼠肝星状细胞的分离方法,提高肝星状细胞分离的纯度和活力。方法 SD大鼠在静脉注射1 ml含二氯亚甲基二磷酸盐的脂质体3 d后,用含100 U/ml肝素的D-Hanks液灌注肝脏10～15 min,改用含0.05％胶原酶溶液的D-Hanks液灌注25～30 min,将肝脏细胞悬液置于0.025％胶原酶、0.005％DNase Ⅰ的溶液中振荡消化30 min,细胞悬液过200目筛网,50×g离心2 min去除肝细胞,300×g离心10 min得到肝脏非实质细胞沉淀,将细胞悬浮于Nycodenz使终浓度为11.5％,1400×g离心17 min,吸取Nycodenz上层的细胞即为肝星状细胞、台盼蓝拒染实验鉴定细胞活力,自发荧光、Desmin免疫细胞化学染色鉴定细胞纯度,内源性过氧化物酶染色检测库普弗细胞。结果 肝星状细胞得率每只大鼠约3×10~7个,细胞活力在95％以上,初分离的肝星状细胞在328 nm激发光下自发蓝绿色荧光,Desmin免疫细胞化学染色鉴定纯度达到90％,未检测到内源性过氧化物酶阳性细胞。结论 建立了一种无库普弗细胞混杂的大鼠肝星状细胞分离方法,可以用于原代肝易状细胞的生物学行为的研究。更多还原

【Abstract】 Objective To present an improved method to obtain pure, viable, freshly isolated hepatic stellatecells. Methods Adult male SD rats were used. All procedures were performed with the animals under sodium pentobarbitalanesthesia. Three days after the single intravenous administration of 1 ml liposome-encapsulated CL2MDP, which hasselective cytotoxicity of Kupffer cells, livers were perfused with D-Hank’s solution containing 100 U/ml heparin for 10to 15 minutes, and then with 0.05% collagenase dissolved in D-Hank’s solution for 25 to 30 minutes. The liver was thengently homogenized and further incubated in 0.025% collagenase, and 0.005% DNAase I for 30 minutes at 37℃ underconstant stirring. This suspension was filtered through stainless steel gauze and centrifuged for 2 minutes at 50×g toremove parenchymal cells. Sinusoidal cells in the supernatant were recovered by centrifugation for 10 minutes at 300×g. The cells were resuspended in the presence of 28.7% Nycodenz stock solution. The final concentration of Nycodenzat this stage was 11.5%. Following centrifugation for 17 minutes at 1400×g, The cells at the top of this Nycodenzsolution were collected. Cells were resuspended in Dulbecco’s modified Eagle medium supplemented with 10% fetal calfserum, The cells were seeded in 50 ml culture flask at a density of 500 000 cells/ml, The cell viability was determined bytrypan blue exclusion staining, the purity of hepatic stellate cells was identified by the expression of Desmin usingimmunocytochemistry method. Endogenous peroxidase staining was used to detect Kupffer cells. Results The yield rateof hepatic stellate cells was 3×10~7 per rat, the cell viability was more than 95%, the desmin positive cell rate was 90%,no endogenous peroxidase positive cells were detected. Conclusion The method for the isolation of hepatic stellatecells was developed without kupffer cells confusion. The availability of highly purified stellate cells will facilitate theinvestigation of their functions in primary culture..更多还原