兔脂肪干细胞与软骨细胞微球共培养成软骨分化最适诱导比例的实验研究  

An Experimental Study on Optimal Ratio of Chondrogenesis in Pellet Co-Culture of Adipose-Derived Stem Cells and Chondrocytes Differentiation in Rabbits

作　　者：赵明璨 杨午[1] 刘畅[1,2] 

机构地区：[1]吉林大学口腔医学院,长春130021 [2]广州医科大学附属口腔医院,广州口腔病研究所,口腔医学重点实验室,广州510140

出　　处：《中国生物医学工程学报》2016年第5期570-576,共7页Chinese Journal of Biomedical Engineering

基　　金：吉林省科技发展计划项目(20140204022SF);广东省教育厅省级高等教育“创新强校工程”专项资金(B16036087);长春市重大科技攻关项目(13KG45)

摘　　要：通过间接共培养ADSC与软骨细胞微球,得出使ADSC发生软骨向分化的最佳比例。体外分离培养新西兰大白兔ADSC和软骨细胞,制成微球分置于Tranwell上下室,实验分为阳性对照组,阴性对照组,生长因子对照组及不同比例(0.5∶1;1∶1;1∶2;1∶3;1∶5;1∶7)的ADSC和软骨细胞共培养组,培养28天后分别行组织形态学观察,GAG、COL-Ⅱ和COL-Ⅹ的定性定量检测。阿利新蓝-核固红染色及Ⅱ型胶原免疫组化染色显示比例为0.5∶1和1∶1的阳性较弱,而Ⅹ型胶原在生长因子对照组的表达最为强烈。DMMB法显示总GAG含量在共培养组较阴性对照组均有所增加(P<0.05),且增加量与软骨细胞的比例呈正相关,但是在ADSC:AC比例达到1∶5之后有所下降。OHP检测总胶原蛋白的变化同上,且1∶5组中沉积的胶原量比阴性对照组高出7.3倍。能够使ADSCs最大程度的表达软骨特异性细胞外基质,同时使COL-Ⅹ等软骨肥大标志无明显上调的ADSC与软骨细胞的比例为1∶5,共培养诱导较生长因子能够抑制ADSC软骨向分化后的肥大问题。The aim of this work was to figure out the optimal ratio of adipose-derived stem cells（ ADSCs） to articular chondrocyte（ AC）（ ADSCs / AC） in the chondrogenesis effect through indirectly co-culture pellets of ADSCs and AC. Isolate and cultivate ADSC and chondrocytes to form pellets that were placed into upper and lower layers of tranwell system. Experiments included positive control group,negative control group,growth factor control groupand ADSC and chondrocytes pellets in different ratios（ 0. 5∶ 1; 1∶ 1; 1∶ 2; 1∶ 3; 1∶ 5; 1∶ 7）groups. After 28 days of culture,histological morphology,protein level of GAG,COL-II and COL-X were examined. Results： Toluidine blue staining and collagen type II displayed 0. 5 ∶ 1 and 1 ∶ 1 groups showed weak positive staining. The collagen X in the growth factor group showed the most strong positive staining; DMMB showed that the total GAG content in co-culture groups increased when compared with that of the control group（ P 〈0. 05）,while decreased whenthe ratio of ADSC /AC reached 1 ∶ 5. Results obtained from OHP measurements howed the variation tendency of total collagen was similar to the change tendency of GAG.Conclusions： The optimal ratio that made adipose derived stem cells expressed the maximum cartilage specificextracellular matrix was 1 ∶ 5. The co-culture inhibited the hypertrophic problem when compared with growth factor induction.

关 键 词：脂肪干细胞 软骨细胞 共培养 生长因子 

分 类 号：R318[医药卫生—生物医学工程]