人肋软骨细胞体外培养中生长代谢及功能的变化  

Age-related changes in growth and metabolism function of human costal chondrocytes cultured in vitro

作　　者：张艳[1] 柴岗[1] 刘伟[1] 崔磊[1] 曹谊林[1] 

机构地区：[1]上海市第九人民医院整复外科,上海市组织工程重点实验室,上海市组织工程研究与开发中心,200011

出　　处：《中华整形外科杂志》2004年第5期372-376,共5页Chinese Journal of Plastic Surgery

基　　金：国家重点基础研究发展规划资助项目("973"项目)(G19990 5 43 0 4);上海市科委:软骨组织工程的临床应用 ( 0 0DJ14 0 0 1 1)

摘　　要：目的　通过检测体外培养的人肋软骨细胞老化过程中Ⅰ、Ⅱ型胶原和蛋白聚糖(aggrecan)的表达变化 ,为组织工程软骨构建的种子细胞选择适宜的回植时机。方法　取体外培养的P1 ～P5人肋软骨细胞 ,通过观察细胞形态 ,细胞增殖率 ,Alcianblue法定量检测每代Aggrecan中GAG含量 ,免疫组化蛋白水平观察Ⅰ、Ⅱ型胶原表达及RT PCR从mRNA水平分析Ⅰ、Ⅱ型胶原及Aggrecan基因表达量。结果　人肋软骨细胞从第 3代起逐渐向成纤维样细胞形态转换 ;每代软骨细胞Aggrecan中GAG含量随传代次数逐渐降低 ,第 3代后处于较低水平 ;Ⅰ、Ⅱ型胶原的表达在蛋白水平及mRNA水平基本一致 ,第 2代以前Ⅱ型胶原表达较强 ,Ⅰ型胶原表达较弱 ,之后随传代Ⅱ型胶原表达减弱 ,Ⅰ型胶原表达逐渐增强 ;而Aggrecan在第 3代以前表达较高 ,从第 4代后明显下降。结论　人软骨细胞体外培养老化过程中综合细胞扩增及细胞功能因素 ,第 2代的软骨细胞 (体外扩增约 18 32 6倍 )可作为构建人组织工程软骨的种子细胞。Objective To investigate the expressive changes of collagen Ⅰ,Ⅱ and aggrecan during the aging of the human costal chondrocytes cultured in vitro,in order to select the best chondrocytes for cartilage engineering. Methods The human costal chondrocytes from the different passages (P_1～P_5) were harvested. The morphological changes and cell proliferation rate were observed,and the quantity of glycosaminoglycans(GAGs) was examined in each passage with alcian blue precipitation. The expression of collagen Ⅰ,Ⅱ and aggrecan was measured with immunohistochemistry and RT-PCR method. Results From the third passage,the human costal chondrocytes were transformed into fibroblast-like cell morphology;and the GAGs content in each passage was decreased and became significantly low in the third passage. The expression of the collagen Ⅰ,Ⅱ was consistent in the protein level with that in the mRNA level. Before the second passage,the expression of collagen Ⅱ was high while the collagen Ⅰ was low. After the second passage,the expression of collagen Ⅱ decreased while the collagen Ⅰ increased. The expression of aggrecan was high before the third passage,and then became low in the fourth passage. Conclusion With the examinations of the cell proliferating rate and cell functions in human chondrocytes cultured in vitro,the chondrocytes of the second passage seem suitable for human cartilage engineering. ;

关 键 词：Ⅱ型胶原 表达量 肋软骨 软骨细胞 体外培养 组织工程软骨 Ⅰ型胶原 MRNA水平 传代 生长代谢 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]