从分化胚胎干细胞中分选血管构建种子细胞的实验研究

Purification of Vascular Cells with MACS and FACS for Constructing Vessel from Differentiated Mouse Embryonic Stem Cells

目的本研究主要比较了应用免疫磁珠(MACS)和流式细胞仪(FACS)两种分选手段,从ES分化细胞中获得血管平滑肌细胞和内皮细胞的效率。方法利用MACS和FACS从ES分化细胞中分选出FLK-1阳性细胞,分别在VEGF和PDGF诱导培养下获得血管平滑肌细胞和血管内皮细胞,运用MACS和FACS进行细胞性质和纯度鉴定,并对两种方法所获细胞的活力和细胞得率进行比较研究。结果利用MACS分选获得的FLK-1阳性细胞经过PDGF诱导培养后可以得到108～109量级和纯度超过90%的血管平滑肌细胞,可满足构建血管的需要,而利用FACS分选难以获得构建血管肌层所需的大量细胞。利用FACS分选的FLK-1阳性细胞经过VEGF诱导培养后的纯度约60%,表达vWF和吸收低密度脂蛋白(Ac-LDL)的血管内皮细胞;而利用MACS从ES细胞中分选获得的血管内皮细胞纯度较低,两种分选方法获得的细胞活力无明显差别。结论因组织工程血管构建所需要的平滑肌细胞数量巨大,宜选用MACS从ES分化细胞中分选获得;而血管内皮细胞宜选用FACS分选获得。

Objective To compare the efficiency between using FACS and MACS for purification of smooth muscle cells and endothelial cells induced from differentiated mouse embryonic stem cells.Methods FLK-1＋ cells were sorted from EBs by FACS and MACS,and were subsequently induced to vascular endothelial cells and smooth muscle cells with PDGF and VEGF respectively.Cells were then characterized by immunefluoresence staining and FACS.Results Large amount of vascular smooth muscle cells could be obtained from FLK-1 positive cells sorted by MACS.Over 90% of these cells were positive for smooth muscle markers,including SM α-actin,SM-SMC and Calponin.However,low output of smooth cells was observed by FACS sorting.Nearly 60% of cells expressed endothelial cell markers CD31 and vWF after VEGF induction of sorted cells by FACS,but low purity of endothelial cells was achieved from MACS sorting.Conclusion Large quantity of vascular smooth muscle cells with high purity can be obtained by MACS selection of FLK-1＋ cells from EBs while high quality of vascular endothelial cells could be achieved by FACS sorting.