裸鼠肝窦内皮细胞的分离、培养及鉴定

Isolation,culture and identification of liver sinusoidal endothelial cells in nude mice

背景:建立裸鼠肝窦内皮细胞的原代分离培养方法,进一步研究其生长特点及生物学特性。目的:建立裸鼠肝窦内皮细胞的原代分离、培养方法,并鉴定其纯度及观察其生物学特性。方法:无菌下摘除裸鼠肝脏,剪成1mm3大小,Ⅳ型胶原酶在37℃恒温下消化,应用CD146免疫磁珠分选肝窦内皮细胞,在倒置显微镜观察内皮细胞形态及生长特性,用von-Willebrand因子免疫细胞化学验证分选细胞的表面蛋白表达,鉴定肝窦内皮细胞的纯度,Hochest33342测定凋亡。结果与结论:分离的肝窦内皮细胞纯度达95%以上,活力好,光镜下可以看到典型的铺路石样形态,von-Willebrand因子免疫细胞化学染色阳性。肝窦内皮细胞在体外培养3周后凋亡。提示Ⅳ型胶原酶消化加CD146免疫磁珠分选肝窦内皮细胞纯度高,方法可靠。

BACKGROUND：It is necessary to establish an effective method of isolating and culturing primary liver sinusoidal endothelial cells（LSECs） from nude mice to intensively investigate the biological features and dynamic growth state of LSECs.OBJECTIVE：To establish a method to isolate and culture primary LSECs so as to indentify the purity and observe the biological characteristics of LSECs.METHODS：Liver tissues were removed from nude mice under sterile condition and cut into species of 1 mm3.Then,the species were digested with type Ⅳ collagenase to generate single cells suspension at 37 ℃.Single cells were subsequently captured by CD146 microbeads linked to LSECs with magnetic activated cells sorting.Under an inverted microscope,LSECs samples were examined to make sure the cells homogeneity and expression of von-Willebrand factor by immunocytochemistry according to LESCs＇ morphology and phenotype.In addition,apoptotic LESCs were labeled with Hochest33342 under a fluorescence microscope.RESULTS AND CONCLUSION：The captured cells from liver tissues with CD146 microbeads were positive for von-Willebrand factor and more than 95% cells were confirmed to be endothelial cells.All the cells were free from Trypan blue staining and were verified to be in good vitality.Under the inverted microscope,the LSECs showed a monophasic cobblestone feature as other endothelial patterns,and most of LSECs displayed apoptosis of Hochest33342 staining after 3 weeks.This isolation method is easy and reliable to separate LSECs from the liver of nude mice with a high purity.