OptiPrep密度梯度分离液分离培养小鼠原代肝星状细胞的实验研究

Experimental study on isolation and culture of mouse primary hepatic stellate cells with OptiPrep density gradient separation solution

目的寻找分离培养小鼠原代肝星状细胞(hepatic stellate cells,HSC)的有效方法。方法采用OptiPrep密度梯度分离液对BALB/c雄性小鼠HSC进行原位手工灌注分离培养。倒置荧光显微镜观察HSC自发荧光、油红O染色,α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)及结蛋白(Desmin)免疫细胞化学染色鉴定原代细胞及传代培养的细胞。结果我们分离出高得率和高纯度的原代HSC,在倒置显微镜下328 nm波长的紫外光激发下自发蓝绿色荧光,油红O染色胞浆中脂滴呈红色,细胞核呈蓝色;在传2代培养的HSC中,α-SMA、Desmin染色阳性细胞胞浆为棕色颗粒,达100%阳性,全部活化。结论OptiPrep密度梯度分离液分离培养出高纯度、高得率的原代HSC。

Objective To find an effective method for isolating and culturing primary mouse hepatic stellate cells(HSC).Methods The HSC of BALB/c male mice were isolated and cultured by in situ manual perfusion with OptiPrep density gradient separation solution.HSC autofluorescence,oil red O staining,α-smooth muscle actin(α-SMA)and Desmin immunocytochemical staining were observed under inverted fluorescence microscope to identify primary cells and subcultured cells.Results Primary HSC with high yield and high purity were isolated and induced spontaneous blue-green fluorescence by ultraviolet light at 328 nm wavelength under inverted microscope.The lipid droplets in the cytoplasm of HSC were red and the nucleus was blue after oil red O staining.In the second generation of HSC,α-SMA and Desmin staining in the cytoplasm of HSC positive cells were brown granules,up to 100%positive and all activated.Conclusion Primary HSC with high purity and high yield can be isolated and cultured by OptiPrep density gradient separation solution.