角膜基质细胞体外长期培养过程中生物学特性的改变  

Changes in biological characterization of corneal stromal fibroblasts during long term in vitro cultivation

作　　者：王爱丽[1] 胡晓洁[1] 李卿[1] 崔磊[1] 刘伟[1] 曹谊林[1] 

机构地区：[1]上海第二医科大学第九人民医院整复外科上海市组织工程研究重点实验室,上海200011

出　　处：《上海第二医科大学学报》2004年第4期307-310,318,共5页Acta Universitatis Medicinalis Secondae Shanghai

基　　金：国家重点基础研究发展规划资助项目("973"项目)(1999054309).

摘　　要：目的 研究兔角膜基质细胞在体外长期培养传代过程中,细胞增殖能力和胞外基质表达水平的改变。方法 取体外培养兔角膜基质细胞,由光镜对各代细胞的形态学变化进行观测。通过对各代细胞生长曲线的绘制,得出其各自的群体倍增时间;MTT法比较各代细胞增殖能力的改变。RT-PCR和Western杂交检测各代细胞中Ⅰ、Ⅲ型胶原表达水平的改变;爱茜蓝法检测糖胺聚糖(GAG)的含量。结果 体外培养兔角膜基质细胞随着传代的进程,逐渐显现衰老的形态特征。细胞群体倍增时间由第7代起显著延长(P<0.01);MTT比色法的结果亦显示,第7代细胞的增殖能力开始大幅下降(P<0.01)。第9-11代时,Ⅰ、Ⅲ型胶原在mRNA和蛋白水平的表达都有明显减少(P<0.01),降至第1代细胞的30％左右;GAG的含量也降至40％左右。结论 兔角膜基质细胞经历长期体外培养后,细胞增殖能力和胞外基质表达水平显著下降,逐渐丧失原有的功能。但作为构建组织工程化角膜的种子细胞,在它的生物学特性未发生改变之前,经3次传代培养,获取的细胞量达到组织块消化所得细胞的240倍左右,已足以用于构建之用。Objective To determine the changes in proliferation potential and extracellular matrix production during long term in vitro culture of rabbit corneal stromal cells. Methods Morphological changes of corneal stromal cells were observed by microscopy while proliferation potential was measured by growth curve and MTT assay. Expression of collagen Ⅰ and collagen Ⅲ was evaluated with RT-PCR and Western blotting and production of GAG was analyzed with Alcian Blue precipitation. Results Population doubling time of corneal stromal cells prolonged significantly since passage 7. MTT assay also showed cell viability of passage 7 decreased to 60% of passgage 1 (P < 0. 01 ). Expression of GAG, collagen Ⅰ and collagen Ⅲ declined dramatically from passage 9 to passage 11 (P < 0. 01) . Conclusion Corneal stromal cells lost proliferation potential and normal function when cultured in vitro for a long time. Howerver, enough stromal cells can be obtained via in vitro culture for stromal engineering before they become senescent.

关 键 词：角膜基质细胞 细胞培养 生物学特性 细胞外基质 细胞增殖 MTT比色法 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]