CAJ | 학술논문

组织工程%血管内皮细胞%人胎盘微血管内皮细胞%早期绒毛组织%原代培养%免疫细胞荧光化学%细胞鉴定%胰酶消化%纯化%FⅧ因子相关抗原%CD31%MTT法%国家自然科学基金組織工程%血管內皮細胞%人胎盤微血管內皮細胞%早期絨毛組織%原代培養%免疫細胞熒光化學%細胞鑒定%胰酶消化%純化%FⅧ因子相關抗原%CD31%MTT法%國傢自然科學基金
조직공정%혈관내피세포%인태반미혈관내피세포%조기융모조직%원대배양%면역세포형광화학%세포감정%이매소화%순화%FⅧ인자상관항원%CD31%MTT법%국가자연과학기금
placenta%endothelial cel s%blood vessels%cel s%fluorescent antibody technique

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 Percol 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 Percol 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。
배경：건립고순도적인태반미혈관내피세포체외배양체계대연구태반공능시십분필요적。목전，국내외연구다채용삼보매소화법결합면역자주법분리태반미혈관내피세포，연이보취과우번쇄차대세포손상교대。인이，여하간화인태반미혈관내피세포분리보취，동시우능제고목표세포순도적체외배양방법성위연구열점。 　　목적：탐색일충간편고효적종조기융모조직중분리인태반미혈관내피세포적체외배양방법，관찰세포생장상태병진행감정。 　　방법：이용건강잉6-8주잉부행인공유산후적융모조직，경량보매소화법화비련속 Percol 밀도제도리심득도인태반미혈관내피세포，전대시이용이매소화법화반복첩벽법대세포진행순화。 　　결과여결론：실험성공획취인태반미혈관내피세포，원대배양적인태반미혈관내피세포재배양24 h후기본완전첩벽，제10천진입대수생장기，제12지13천세포농도체80%，전대세포교원대세포생장활약，배양5-7 d가장만배양병저，정“포로석양”배포。면역형광화학결과현시，배양적세포중 FⅧ인자여 CD31상관항원쌍형광염색정양성，세포양성솔체100%。MTT법측득배양제5대인태반미혈관내피세포적생장곡선정도“S”형。결과증실，응용량보매소화법、비련속 Percol 밀도제도리심법분리세포，병이용이매소화법화반복첩벽법순화세포，가획득대량고순도적인태반미혈관내피세포。
BACKGROUND:Establishment of in vitro culture system of human placental microvascular endothelial cel s with high purity is very important. In recent studies, some scholars have successful y obtained a large number of placental microvascular endothelial cel s by three-stepenzyme digestion and magnetic separation method, but the procedures were extremely complex and it had great damage to the cel s. Therefore, how to separate human placental microvascular endothelial cel s easily and obtain high-purified cel s has become a research hotspot. OBJECTIVE:To investigate an efficient method to isolate and purify human placental microvascular endothelial cel s from early vil us microvessels, observe the cel growth and identify the cel s. METHODS:The vil i from normal early pregnancies (6-8 weeks) after artificial abortion were col ected aseptical y. Using two-step digestion procedure and discontinuous Percol density gradient centrifugation method, human placental microvascular endothelial cel s were obtained. Then the cel s were identified by trypsin digestion method and repeated adherence method. RESULTS AND CONCLUSION:Human placental microvascular endothelial cel s were isolated successful y from early vil i. The primary cel s adhered to the wal s after inoculated for 24 hours and entered logarithmic phase at 10 days. 80%of the cel s achieved a confluence at 12-13 days after inoculating. The subculture cel s grew swiftly with the typical cobblestone appearance. Immunofluorescence staining showed that, cultured human placental microvascular endothelial cel s demonstrated a strong positive reaction to von Wil ebrand factor antigen and CD31, accounting for 100%. MTT assay results showed that, human placental microvascular endothelial cel s at passage 5 exhibited an S-shaped growth curve. High-purity human placental microvascular endothelial cel s can be obtained by proteolytic enzymes digestion and discontinuous Percol density gradient centrifugation method, and the purity is detected by trypsin digestion method and repeated adherence method.