摘要
目的:探讨一种高效、稳定的小鼠肺微血管内皮细胞(PMVECs)原代培养方法。方法:选取45只1周龄Balb/c小鼠,随机分成3组,分别用酶消化法、组织贴块法、免疫磁珠分选法3种方法分离培养小鼠PMVECs,观察细胞形态学以及VIII因子相关抗原免疫荧光染色鉴定内皮细胞,计算各组原代培养成功率及从原代培养开始到首次传代所需时间,测定细胞纯度,MTT法绘制生长曲线。结果:3组原代培养的细胞在倒置显微镜下均能见到短梭形、多边形的微血管内皮细胞,血管内皮细胞特异性标志物VIII因子相关抗原表达阳性。免疫磁珠分选法组原代培养成功率及细胞活性显著高于其他2组,差异有统计学意义(P<0.01),从原代培养至首次传代所需的时间短于组织贴块法组,差异有统计学意义(P<0.05),细胞纯度显著高于酶消化法组,差异有统计学意义(P<0.01)。结论:相比于酶消化法与组织贴块法,免疫磁珠分选法原代培养小鼠PMVECs具有重复性好,分离速度快,细胞纯度高及活性好的优点。
Objective： To explore an efifcient and stable method for the mouse pulmonary microvascular endothelial cells （PMVECs） culture.Methods： Forty-ifve 1 week old Balb/c mice were randomly divided into three groups. Mouse PMVECs were isolated and cultured respectively by enzyme digestion method, explants method and immunimagnetic beads method. Cells were identiifed by morphological observation and VIII factor related antigen immunolfuorescence staining. The success rate of primary culture and the time required from pri-mary culture to the beginning of the ifrst passage was calculated, the cell purity was determinated, growth curves were drew by MTT method.Results： Short spindle and polygonal microvascular endothelial cells were observed in all of the three methods. Those cells were positive to the VIII factor related antigen. The success rate and cell activity in immunomagnetic beads method group increased signiifcantly compared with the other two groups （P〈0.01）. As for the time required from primary cultured to the beginning of the ifrst passage, it decreased in im-munomagnetic beads method group compared with the explant method group （P〈0.05）. And the purity increased significantly in immunomagnetic beads method group compared with the enzyme digestion method group （P〈0.01）.Conclusion： Immunomagnetic beads method is of higher repeatability, rapider separation, and obtains much higher purity and better activity mice PMVECs.
出处
《温州医科大学学报》
CAS
2016年第8期590-593,共4页
Journal of Wenzhou Medical University
基金
浙江省自然科学基金资助项目(LY12H01001)
关键词
细胞培养
原代培养
免疫磁珠
肺微血管内皮细胞
cell culture
primary culture
immunomagnetic beads
pulmonary microvascular endothelial cells
