应用琼脂糖微球抗体筛选原代乳鼠肺微血管内皮细胞新方法的建立  

A new method for screening pulmonary microvascular endothelial cells of primary neonatal mouse by agarose microsphere antibody

作　　者：刘姗 刘艳霞[2] 闫承慧[2] 田孝祥[2] 刘丹[2] 刘美丽[2] 张坡[2] 韩雅玲[2] LIU Shan;LIU Yan-xia;YAN Cheng-hui;TIAN Xiao-xiang;LIU Dan;LIU Mei-li;ZHANG Po;HAN Ya-ling(Graduate School, Jinzhou Medical University, Jinzhou, Liaoning 121000, China;Department of Cardiology, General Hospital of Shenyang Command/Laboratory of Cardiovascular Disease Research Institute of the Shenyang Command General Hospital of Chinese PLA/Key Laboratory of Cardiovascular Emergency Treatment of Chinese PLA/Key Laboratory for the Research of Cardiovascular Disease Transformation Medicine in Liaoning, Shenyang 10016. China)

机构地区：[1]锦州医科大学研究生学院,辽宁锦州121000 [2]沈阳军区总医院心内科/全军心血管病研究所实验室/全军心血管急重症救治重点实验室/辽宁省心血管病转化医学研究重点文验室,沈阳110016

出　　处：《解放军医学杂志》2018年第5期424-428,共5页Medical Journal of Chinese People's Liberation Army

基　　金：辽宁省自然科学基金计划重点项目(20170540965);辽宁省自然科学基金指导计划项目(201602804,20170540929)

摘　　要：目的建立一种简便、高效的原代乳鼠肺微血管内皮细胞(PMVECs)培养新方法。方法选取小于1周龄的C57BL/6乳鼠,采用琼脂糖微球抗体筛选法分离、培养原代PMVECs。倒置显微镜观察细胞形态,采用CD31、血管性血友病因子(vWF)相关抗原免疫荧光染色以及基质胶体外成血管实验鉴定内皮细胞,采用CCK-8法绘制细胞生长曲线。结果倒置显微镜下观察培养的原代PMVECs呈短梭形、类圆形,单层融合呈铺路石样排列。CD31相关抗原、vWF免疫荧光染色阳性,阳性率分别为91.35%±3.06%、92.99%±2.67%,证实培养的细胞为PMVECs。PMVECs接种在基质胶上8h后,有血管腔结构形成。CCK-8法绘制的生长曲线显示,琼脂糖微球抗体筛选法提取PMVECs培养2~5d后细胞处于对数生长期。结论琼脂糖微球抗体筛选法分离、培养乳鼠原代PMVECs简便、高效,是一种值得推广的分离培养乳鼠PMVECs的新方法。Objective To establish a new, simple and efficient method for culturing pulmonary microvascular endothelial cells（PMVECs） of primary neonatal mouse. Methods The C57BL/6 mice aged less than one week were sacrificed. The primary PMVECs were isolated and cultured with agarose microsphere antibody screening method. Inverted microscope was used to observe the cell morphology, immunofluorescence staining and matrigel angiogenesis test with CD31 related antigen and vascular Willebrand factor（vWF） related antigen were performed to identify the cells, and CCK-8 method was employed to draw cell growth curve. Results Under the inverted microscope, the primary PMVECs showed short spindle and round in shape, and integrated in monolayer as paved stone like. CD31 related antigen and v WF immunofluorescence staining were observed in endothelial cells, the positive rates were 91.35%±3.06% and 92.99% ± 2.67%, respectively. The vascular lumen was formed 8 h after inoculation of PMVECs on the matrigel. The growth curve drawn by CCK-8 method showed that PMVECs extracted with agarose microsphere antibody screening method were in the logarithmic growth period 2-5 days after cultivation. Conclusion Agarose microsphere antibody screening method is a simple and efficient method to isolate and cultivate the primary PMVECs of neonatal mouse, and is a worthwhile approach.

关 键 词：乳鼠 肺微血管内皮细胞 原代培养 

分 类 号：R331[医药卫生—人体生理学]