摘要
目的改良人原代大隐静脉内皮细胞的提取方法,为冠状动脉旁路移植术后静脉移植物衰败的研究提供细胞模型。方法传统酶消法(酶消组)和刮取法(刮取组,刮取酶消法后的大隐静脉内皮并进一步酶消)从人大隐静脉中提取内皮细胞。获得的内皮细胞在含10%胎牛血清+1%内皮细胞生长补充剂+100 U/mL青霉素+100 mg/mL链霉素的内皮细胞培养基中传代培养。倒置显微镜观察内皮细胞形态和贴壁情况;血管性血友病因子(vWF)的免疫荧光实验鉴定内皮细胞特异性抗原;CD31流式细胞计数检测内皮细胞纯度;绘制不同观测日的内皮细胞生长曲线测定内皮细胞活性。结果显微镜下观察到两组大隐静脉内皮细胞贴壁汇合并且呈典型的鹅卵石样排列;免疫荧光实验证实培养的细胞vWF阳性表达;流式细胞计数结果显示,酶消组99.9%、刮取组89.2%的细胞阳性表达CD31;不同观测日的生长曲线结果显示,两组内皮细胞活性的主效应不显著(P=0.057)。两组内皮细胞均能够传至第7~8代。结论在传统酶消法的基础上增加刮取再消化的步骤,能够提取出数量更多且满足实验需要的原代人大隐静脉内皮细胞。
Objective To improve the extraction method of primary endothelial cells from human saphenous vein and to provide a cell model for the study of vein graft failure after coronary artery bypass grafting.Methods Endothelial cells were extracted from abandoned human saphenous vein by enzymatic digestion and scraping(scraping the vascular intima after enzymatic digestion for further enzymatic digestion).The extracted endothelial cells were cultured in the endothelial cell culture medium containing 10% fetal bovine serum+1% endothelial cell growth supplement+100 U/mL penicillin+100 mg/mL streptomycin.The morphology and adhesion of endothelial cells were observed under an inverted microscope.The specific antigen was identified with immune fluorescence assay of von Will brand Factor(vWF).The purity was detected with flow cytometry of CD31.The activity of endothelial cells was determined with the cell growth curve plotted on different observation days.Results Under the inverted microscope,endothelial cells converged on the wall and arrayed in a typical cobblestone shape.Immune fluorescence assay confirmed the positive expression of vWF.The results of flow cytometry showed that 99.9% and 89.2% of cultured cells in the enzymatic group and scraping group were positive for CD31.Data of the cell growth curve demonstrated that there was no significant difference in endothelial cell activity between the two groups(P=0.057).Endothelial cells from both groups were able to transmit to the 7th-8th generation.Conclusion Based on the traditional enzymatic digestion method,adding the scraping and re-digestion step can extract more primary human saphenous vein endothelial cells.
作者
于瑞明
邴卫东
刘春晓
孟祥斌
毕研文
YU Ruiming;BING Weidong;LIU Chunxiao;MENG Xiangbin;BI Yanwen(Department of Cardiovascular Surgery,Qilu Hospital of Shandong University,Jinan 250012,Shandong,China)
出处
《山东大学学报（医学版）》
CAS
北大核心
2023年第2期43-48,共6页
Journal of Shandong University:Health Sciences
基金
山东省自然科学基金(26010111670901)
山东大学科研发展基金(26010132007092)。
