猪骨髓基质细胞的分离培养及生物学特性研究  

Isolation,cultivation and biological characteristics of bone marrow stromal cells of swine in vitro

作　　者：熊奎州[1] 张彦明[1] 王文秀[1] 温元鹏[1] 解林红[1] 张浩[1] 邓文[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2008年第10期19-23,共5页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金资助项目(30471290)

摘　　要：【目的】探讨猪骨髓基质细胞（bone marrow stromal cells,BMSCs）的体外分离培养方法,并对猪BM-SCs的生物学特性进行研究,旨在建立一种稳定的体外培养猪BMSCs的方法体系。【方法】分离刚出生猪的BMSCs进行体外培养,传代,分别观察原代及传代细胞的形态和分化特征,绘制生长曲线,测定细胞分裂指数和贴壁率。采用细胞免疫化学染色对碱性磷酸酶（ALP）进行检测,以确定成骨分化能力,并通过免疫组化法对表面分子CD44、CD34进行染色鉴定。【结果】猪BMSCs体外生长良好,细胞接种后1-2 d生长缓慢,2-5 d进入对数生长期,6 d后细胞数开始下降;猪BMSCs在第4天分裂指数最高,约为10%;猪BMSCs传代后10 h贴壁率最高,达90%以上。猪BMSCs ALP染色阳性率达到75%;CD44阳性率可达98.83%,而CD34在第2代后消失;猪BMSCs在第5代以前生长形态稳定,培养至7代后,衰老现象严重。【结论】该方法分离的猪BMSCs早期生长性状稳定,增殖速度快,并获得了高纯度的猪BMSCs,据此建立了一种稳定的体外培养猪BMSCs的方法体系。[Objective] The study was to explore and establish a stable culturing system of swine bone marrow stromal cells （BMSCs） in vitro. [Method] BMSCs were isolated from the newborn pigs,primarily cultured and passaged. Growth of cells was observed and curves of cells were drawn to determine the divisional index and cells adhesive rate. Alkaline phosphatase （ALP） of cells was detected by using cytochemistry staining and CD44 and CD34 cells were detected by using immunohistochemistry staining. [Result] The swine BMSCs grew well in vitro. On the 1st and 2nd day,cell grew slowly,while between the 2nd and 5th day,they reached logarithmic growth phase. And the amount of cells declined after the 6th day. The division index reached the highest of 10% on the 4th day of culturing. 10 hours after passaged,the adhesive rate of swine BMSCs climbed to 90% or above. Positive rate of ALP staining was 75%. And the positive rate of CD44 staining reached the highest of 98.83%,while CD34 could not be detected after passage 2. Within the previous five generations, cells stayed stable in shape, whereas, they aged dramatically after the 7th generation. [Conclusion] All the results above indicated that pig BMSCs isolated from newborn pigs grew well at the early culturing,and cells proliferated quickly. High purification of pig SBMSCs were obtained. Therefore an in vitro culture system of swine BMSCs was established.

关 键 词：骨髓基质细胞 分离培养 猪 生物学特性 

分 类 号：Q813.11[生物学—生物工程]