体外分离培养人脐静脉内皮及内皮下间充质干细胞

Isolation and culture of mesenchymal stem cells from human umbilical vein endothelium and subendothelium in vitro

目的:建立人脐静脉内皮及内皮下间充质干细胞体外培养和扩增的方法,探讨其生物学特性,建立间充质干细胞体外培养扩增体系。方法:实验于2006-04/2006-09在辽宁医学院解剖学实验室完成。解剖细胞培养室为无菌百级培养间。正常健康产妇顺产或剖宫产的新生儿脐带由辽宁医学院附属第一医院提供,产妇及其家属均知情同意,并经医院伦理委员会批准。实验方法:①体外分离和培养贴壁细胞:无菌条件下取正常健康产妇分娩或剖宫产脐带,将其用预热PBS充分洗涤去血渍后,从脐静脉一端插入留置针,用预热PBS冲净静脉腔血后,用止血钳夹闭另一端,注入经预热至37℃的Ⅰ型胶原酶,置于37℃水浴箱中消化,30min后放出胶原酶,并用PBS冲洗血管腔,收集消化液和冲洗液,400r/min离心10min,吸弃上清液,重悬于M199培养基(含体积分数为0.15的胎牛血清,2mmol/L谷氨z酰胺,2μg/L碱性成纤维细胞生长因子,100U/mL青霉素,100U/mL链霉素)。以5×108L-1密度接种于6孔培养板中,置于37℃、体积分数为0.05的CO2饱和湿度培养箱中培养,48h后全量换液,以后每3d全量换液。待细胞80%融合时,0.25%胰酶消化,按1×108L-1传代培养。②间充质干细胞生长曲线的测定:取传代培养细胞,按2×107L-1密度接种于24孔培养板内,每天取3孔,将细胞消化计数,连测8d,绘制间充质干细胞生长曲线。③间充质干细胞表面抗原检测:在24孔塑料培养板内放置无菌的盖玻片,每孔中种植108L-1第2代细胞悬液1mL。采用免疫细胞化学方法进行细胞表面抗原检测。结果:①间充质干细胞的形态学观察:接种的细胞48h后细胞完全贴壁生长,其镜下形态有呈椭圆形、多角形的内皮细胞以及呈梭形的成纤维样细胞,有的形成漩涡状生长的集落。②间充质干细胞生长曲线的分析:传代培养的潜伏期约为24～36h,细胞倍增时间约为30～36h,对数增殖期约为二三天,对数增殖期后第5天进入平台期。③间充质干细胞表面抗原特性:免疫细胞化学分析结果显示,间充质干细胞表面抗原cd166、cd44阳性,而vWF阴性,说明分离获得的细胞具有间充质干细胞的特点。结论:所建立的分离和培养方法可获取人脐静脉黏附细胞中一组独特的细胞群,具有间充质干细胞的生物学特性。

AIM： To establish a method of in vitro culture and amplification of mesenchymal stem cells （MSCs） from endothelial and subendothelial cells of human umbilical vein and to explore their biological properties and establish a system of MSCs culture and amplification in vitro. METHODS： The experiment was conducted in the Laboratory of Anatomy, Liaoning Medical University between April and September 2006. Cell culture room was a sterile culture room. The infant umbilical cords were obtained from healthy lying-in women with natural deliveries or caesarean birth from First Affiliated Hospital, Liaoning Medical College after each mother and their family numbers signed informed consent approved by Hospital Ethics Committee. ①in vitro isolation and culture of adherent cells： The umbilical cords were obtained from healthy lying-in women with natural deliveries or caesarean birth. The umbilical vein was catheterized and washed internally with warm PBS and its distal end was clamped with hemostatic forceps. By coUagenase Ⅰ digestion at 37℃, collagenase was released 30 minutes later, and vessel lumen was washed with PBS. Digestive juice and rinse solution were collected, centrifuged for 10 minutes at 400 r/min. Supematant was removed. Re-suspension was done in M199 medium （containing fetal bovine serum of 0.15 volume fraction, 2 mmol/L glutamine, 2 p,g/L basic fibroblast growth factor, 100 U/mL penicillin, 100 U/mL streptomycin）. Cultures were maintained at 37 ℃ in 6-well plate at the density of 5×10^8 L^-1 and a humidified atmosphere containing CO2 of 0.05 volume fraction. After 48 hours of culture, the medium was changed and with a change of culture medium every 3 days. When the cells reached 80% confluence and with 0.25% trypsin, the cells were passaged in a new well for further expansion at 1×10^8 L^-1.②Growth curve determination of MSCs： The passaged cells were harvested and inoculated onto 24-well plate at the density of 2×10^7 L^-1, and then analyzed growth curve before the cells were counted 3 wells every day for successively 8 days. ③Determination of the MSCs antigen：The sterile cover glass was placed in 24-well plate. The 1 mL cell suspension of the second generation was plated at 10^8 L^-1. Immunohistochemical method was applied to analyze the antigen of the cells. RESULTS： ①Morphology observation of the MSCs： As soon as 48 hours after culture, the cells almost adhered to the wall and appeared to be ellipse, polygonal endothelial cell and spindle-shape fibroblast-like, and some cells formed whirlpool-like colony.②Analysis of the MSCs growth curve： The cells in delitescence were 24-36 hours, and the double expansion time were 30-36 hours, and the logarithm expansion time were 2-3 days. After 5 days, the cells were in platform phase. ③Charactenstics of the MSCs antigen： Immunohistochemistry staining showed that the cells were negative for vWF and were positive for CD44 and CD166. It indicated that the isolated cells were with the characteristics of MSCs. CONCLUSION： A specific cell mass with the biological properties of MSCs can be obtained from adhered cells of human umbilical cord vein by established and cultural method.