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背景:建立高纯度的人胎盘微血管内皮细胞体外培养体系对研究胎盘功能是十分必要的。目前,国内外研究多采用三步酶消化法结合免疫磁珠法分离胎盘微血管内皮细胞,然而步骤过于繁琐且对细胞损伤较大。因而,如何简化人胎盘微血管内皮细胞分离步骤,同时又能提高目标细胞纯度的体外培养方法成为研究热点。目的:探索一种简便高效的从早期绒毛组织中分离人胎盘微血管内皮细胞的体外培养方法,观察细胞生长状态并进行鉴定。方法:利用健康孕6-8周孕妇行人工流产后的绒毛组织,经两步酶消化法和非连续Percoll密度梯度离心得到人胎盘微血管内皮细胞,传代时利用胰酶消化法和反复贴壁法对细胞进行纯化。结果与结论:实验成功获取人胎盘微血管内皮细胞,原代培养的人胎盘微血管内皮细胞在培养24 h后基本完全贴壁,第10天进入对数生长期,第12至13天细胞浓度达80%,传代细胞较原代细胞生长活跃,培养5-7 d可长满培养瓶底,呈“铺路石样”排布。免疫荧光化学结果显示,培养的细胞中FⅧ因子与CD31相关抗原双荧光染色呈阳性,细胞阳性率达100%。MTT法测得培养第5代人胎盘微血管内皮细胞的生长曲线呈倒“S”形。结果证实,应用两步酶消化法、非连续Percoll密度梯度离心法分离细胞,并利用胰酶消化法和反复贴壁法纯化细胞,可获得大量高纯度的人胎盘微血管内皮细胞。
Abstract BACKGROUND: To establish an in vitro culture system of high purity human placental microvascular endothelial cells is very necessary for the study of placental function. At present, many studies at home and abroad use three-step enzymatic digestion combined with immunomagnetic beads method to separate placental microvascular endothelial cells, however, the steps are too complicated and have a greater damage to the cells. Therefore, how to simplify the step of human placental microvascular endothelial cell separation, while improving the purity of the target cells in vitro culture methods become a hot topic. OBJECTIVE: To explore a simple and efficient method for isolating human placental microvascular endothelial cells from early villi in vitro and to observe the cell growth status and identify. Methods: The human placental microvascular endothelial cells were obtained by two-step enzymatic digestion and discontinuous Percoll density gradient centrifugation using healthy pregnant women aged 6-8 weeks. After passage, trypsin digestion and repeated adherent method The cells are purified. RESULTS AND CONCLUSION: Human placental microvascular endothelial cells were successfully obtained in this experiment. Primary cultured human placental microvascular endothelial cells almost completely adhered 24 h after culture, and entered the logarithmic growth phase on the 10th day. The cell density reached 80 on the 12th to 13th day %, Subculture cells more active than the primary cell culture, cultured 5-7 d can be filled with bottle bottom, was “paving stone-like ” arrangement. The results of immunofluorescence showed that the FⅧ and CD31-related antigens in the cultured cells were double-stained, and the positive rate was 100%. The MTT assay showed that the growth curve of the 5th generation human placental microvascular endothelial cells was inverted “S” shape. The results confirmed that a large number of high purity human placental microvascular endothelial cells could be obtained by two-step enzymatic digestion and discontinuous Percoll density gradient centrifugation, and purified by trypsin digestion and repeated adherent method.