中文摘要：

背景：研究骨髓间充质干细胞的培养方法，以获取大量高纯度的骨髓间充质干细胞，对应用组织工程技术重建眼部组织治疗眼部疾病具有重要意义。目的：应用密度梯度离心结合贴壁法进行成年大鼠骨髓间充质干细胞体外分离培养，观察其生长特性及大量繁殖的可能性。设计：完全随机分组设计，重复测量实验。单位：中山大学中山眼科中心病理科实验室。材料：选用6周龄SD大鼠4只，雌雄不限，清洁级2级，体质量约250g，只，由中山大学动物中心提供，许可证号码SCSK（粤2004／0011）。DMEMIF12培养基（美国GIBCo公司）．胎牛血清（杭州四季青生物工程公司）、胰蛋白酶、依地酸二钠、淋巴细胞分离液。纤维连接蛋白、CD44、CD34、CD31单克隆抗体，免疫组化二步法试剂盒（北京中杉生物技术公司）。方法：实验于2005—09／2006—01在中山大学中山眼科中心病理科实验室完成。①取SD大鼠，断颈处死后于750g／L酒精浸泡10min。无菌条件下，分离并暴露骨髓腔，用注射器吸人应用液直接插入股骨腔，用含肝素的培养液将骨髓腔里的细胞冲洗出来作为细胞混悬液，密度梯度离心结合贴壁培养法分离纯化大鼠骨髓问充质干细胞，观察活体细胞生长情况。②将第2代细胞接种于预置在培养板内的无菌盖玻片上，当细胞基本融合时，取出作原位甲醇固定10min，苏木精一伊红染色。原位丙酮固定10min，按二步法作免疫组织化学纤维连接蛋白、CD44、CD34、CD31反应，3，3’-二氨基联苯胺显色，最后将盖玻片反扣在载玻片上，封固、观察。③将细胞按4．25×10^7L^-1的浓度接种于96孔板中，每孔200μL，置培养箱中，分别于接种后1，2，3，4，5，6d各于两排孔中加入20μL/孔四甲基偶氮唑盐，继续培养4h后吸去上清液，每孔加200μL二甲基亚砜，振荡5min后，于570nm波长测吸?

英文摘要：

BACKGROUND： It is important to study the methods of culturing bone marrow-derived mesenchymal stem cells （MSCs） to obtain a great amount of high purity MSCs for applying ocular tissues constructed by tissue engineering technique to treat eye diseases. OBJECTIVE： To separate and culture in vitro MSCs from bone marrow of the adult rats by density gradient centrifugation combined with adherence culture, and observe the growing characteristics and the possibility of mass multiplication. DESIGN ： A completely randomized grouping design/repetitive measuring experiment SEI-FING： Pathological laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University MATERIALS： Four six-week-old SD rats about 250 g, grade Ⅱ of cleaning, were provided by the Animal Cen- ter of Sun Yat-sen University [certificate number： SCSK（Yue）2004/0011], about 250 g each rat and there was no limit to the sex. The main reagents and instruments included low sugar Dulbecco modified Eagle culture medium （DMEM/F12, American Gibco Corporation）, trypsin （fetal bovine serum （FBS, Hangzhou Sijiqing Bfo-En- gineering Material Research Institute）, American Gibco Corporation）, disodium edetate, lymphocyte separating medium, fibronectin, CD44, CD34, CD31 monoclonal antibodies, two-step-method kit for immunohistochemistry （Beijing Zhongshan Biotechnology Corporation）. METHODS ： This experiment was conducted at the Key laboratory of Ophthalmology （Sun Yat-sen University）, Ministry of Education, Zhongshan Ophthalmic Center. ~） The SD rats were put to death by breaking their necks, and then put into ethanol （750 g/L） for 10 minutes, under aseptfc condition, the meduUaris cavitas was exposed, the syringe containing ap- plication medium was directly punctured into the femoral cavity, the ceils in the medullaris cavitas were washed out with the culture medium containing heparin and taken as the cell suspension. The bone marrow-derived MSCs were separated and purified by density gradient centrifugation combined with ad