小鼠胎盘间充质干细胞的生物学特性

Biological characteristics of mesenchymal stem cells derived from mouse placenta

目的:胎盘中包含大量能够自我更新、具有多向分化潜能的细胞,其低免疫原性几乎不引起移植物抗宿主反应疾病。观察体外培养的小鼠胎盘来源间充质干细胞的生物学特性。方法:实验于2006-01/11在首都医科大学组织胚胎学教研室实验室(国家中医药管理局三级实验室)完成。①实验方法:孕10dICR小鼠100只,取出子宫收集胎盘实体组织,经0.1%IV型胶原酶消化获得细胞,以3×106接种于25cm2培养瓶中,加入含体积分数0.1胎牛血清、200U/mL青链霉素的高糖DMEM培养基进行体外培养。②实验评估:每天在倒置显微镜下观察细胞形态;MTT检测细胞生长状况,记录生长曲线;流式细胞术分析细胞周期;免疫细胞化学方法分析胎盘间充质干细胞的免疫表型表达。结果:①小鼠胎盘间充质干细胞的形态学特征:原代培养初期仅有少量细胞贴壁,3d后贴壁细胞逐渐增多,培养过程中细胞呈梭形,散在、集落式分布,细胞集落随着增殖而逐渐融合。②小鼠胎盘间充质干细胞的生长特性:接种3d后细胞增殖速度加快,随后保持稳定增殖速度,第9天速度大幅增长达峰值,10d后进入平台期。G0/G1期、S期、G2期的比例分别为64.4%,25.2%,10.4%。③小鼠胎盘间充质干细胞免疫表型检测:原代培养的小鼠胎盘间充质干细胞CD105、CD44、波形蛋白均呈阳性表达。结论:小鼠胎盘来源的间充质干细胞可成功在体外培养并大量扩增,生物学性状稳定,是组织工程优良的种子细胞来源之一。

AIM： A mass of cells with self-renewal and multi-directional differentiation capacity are seen in placenta, and its low immunogenicity hardly cannot induce graft versus host reaction disease. This article is designed to observe the biological characteristics of mouse placenta-derived mesenchymal stem cells （mPLMSCs） cultured in vitro. METHODS： The experiment was conducted at the Laboratory of Histology and Embryology （Three-level Laboratory of State Administration of Traditional Chinese Medicine of the People＇s Republic of China）, Capital Medical University from January to November 2006. ①100 ICR mice those were pregnant for 10 days were selected. The placentas were extracted from the mice and digested by 0.1% Ⅳ collagenase to separate placenta cells. Then the cells were cultured in 25 cm^2 culture flask at the density of 3×10^6 in high glucose DMEM medium containing fetal calf serum of 0.1 volume fraction and 200 U/mL mycillin. ②Morphology of mPLMSCs was observed with inverted microscope every day. The growth kinetics of mPLMSCs was detected by MTT and the growth curve was drawn. Cell cycle was measured by flow cytometry. Immunocytochemical method was used to detect the immunophenotype of mPLMSCs. RESULTS：①Morphological characteristics of mPLMSCs： In the initial stage, there was small quantity primary culture cells adhered, which was increasing gradually 3 days later. Cells were fusiform shape, scattered and colony distribution. Cell colonies were fusing gradually followed by its proliferation. ②Growth characteristics： The proliferation of mPLMSCs speeded up 3 days after inoculation, maintaining its speed in the following days, reached the peak at the 9^th day and went into platform stage 10 days later It was 64.4%,25.2% and 10.4% in the G0/G1 phase, S phase and G2 phase, respectively. ③lmmunocytochemistry： Primary culture mPLMSCs expressed CD105, CD44 and Vimentin. CONCLUSION： Because of its biological characteristics stabilization, mPLMSCs can be cultured and amplified generously in vitro, and regarded as another superordinary seed cell resource of tissue-engineering.