骨髓间质干细胞的分离培养鉴定与生物学特性

Isolation, cultivation, identification and basic biological properties of Bone Marrow - derived Mesenchymal Stem Cells in Vitro

目的观察贴壁法分离提取大鼠乳鼠骨髓间充质干细胞（mesenehymal stem cells，MSCs）并进行体外扩增培养的可行性并探讨其生物学特性，为进一步实验研究作基础。方法实验2008—01／06于南昌大学第一附属医院泌尿外科研究所完成。贴壁法分离培养SD大鼠乳鼠MSCs，经CD29、CD34、CD44、CD45鉴定MSCs，并绘制不同代数细胞生长曲线和贴壁率曲线分析其生物学特性。结果①原代细胞生长潜伏期略长，孵育24h后小部分圆形单核细胞开始贴壁，48h可见巢状克隆集落形成，贴壁细胞呈多形性，贴壁3d后增殖加快，9d后基本融合。②生长曲线及贴壁率曲线提示第2至第6代细胞接种存活率基本相同，生长曲线基本一致，而第7代以后细胞的生长速度渐缓，贴壁时间延长，细胞接种存活率也明显降低，并出现衰化现象。③免疫组化鉴定结果显示CD29、CD44表达基本阳性，而CD34、CD45表达阴性。结论贴壁法比较容易分离培养MSCs，为组织工程的应用提供了足够的种子来源，MSCs有一定的增殖能力但并不是无限的，而是随传代扩增逐渐衰化，因此了解和掌握MSCs的生物学特性为进一步研究应用打下基础。

Objectives To investigate the feasibility of isolating and culturing bone marrow - derived mesenchymal stem cells （MSCs） in vitro by adherence method. Methods The experiment was conducted at institute of Urology , First Affiliated Hospital of Nanchang University from January to June 2008. Neonate SD rats were selected. MSCs were isolated and cultured by the plastic adherence method in vitro and identified by CD29, CD34, CD44, CD5. Growth curve and the adhesive rate curve were drawn and biological properties of MSCs of different passages were analysed. Results ①The incubation period of primary generation was a bit longer. MSCs were round at the beginning and adhered to the wall in 24 hours. Most adherent cells were mainly monocytes and gradually changed into multiple cellular shapes 48 hours after primary culture. MSCs began to proliferate at day4 and coalesced 9 days later.②The growth curve and the adhesive rate curve demonstrated that the growth velocity and adhesive rate were similar before passage 7. After passage 7, the growth velocity of MSCs was significantly dropped, the adherence rate also decreased, and apolexis emerged. ③In vitro expanded MSCs are positive for CD29, CD44 and negative for CD34, CD45. Conclusions The adherence method can be manipulated conveniently. It is an ideal method to culture MSCs that provide sufficient source for tissue engineering. MSCs have limited proliferative capacity and showed apolexis with the increase of the passage. The study provides us with further understanding and mastering of biological characteristics and allows us to study the application of mesenchymal stem cells.