细胞间直接接触诱导BMSC软骨分化的初步研究  

The Study on the Role of the Cell-cell Contact in Inducing Bone Marrow Stromal Cells Chondrogenesis

作　　者：刘霞 周广东[2] 刘伟[2] 曹谊林[1,2] 

机构地区：[1]中国医学科学院整形外科医院,北京市100144 [2]上海交通大学医学院附属第九人民医院整复外科,上海市200011

出　　处：《组织工程与重建外科杂志》2010年第2期70-74,共5页Journal of Tissue Engineering and Reconstructive Surgery

基　　金：国家重点基础研究发展规划项目(2005CB522702);国家高技术发展计划项目(2006AA02A126);国家自然科学基金项目(30801192;30973131;30772264)

摘　　要：目的探讨细胞间直接接触是否能够单独诱导骨髓基质干细胞(Bone Marrow Stromal Cells,BMSC)软骨分化。方法体外培养扩增猪BMSC与关节软骨细胞,将1.0×106的细胞总量以2000r/min离心8min,制成细胞团块,即Pellet培养。实验分为5组,实验组:经0.5%多聚甲醛固定的软骨细胞与BMSC以1∶1混合;对照组1:同等数量的未经多聚甲醛固定的软骨细胞与BMSC1:1共培养制成Pellet;对照组2:同等数量的单纯软骨细胞制成Pellet;对照组3:同等数量的单纯BMSC制成Pellet;对照组4:同等数量的单纯经多聚甲醛固定的软骨细胞制成Pellet。每3天换液一次,每组3例。培养4周后,以大体观察、组织学、免疫组织化学、RT-PCR等方法对Pellet进行全面评价。结果培养4周后,实验组形成的Pellet,明显缩小,形状略不规则,颜色灰暗,柔软且没有弹性,组织学检测提示主要为纤维性成分及死细胞样结构,Safranin O染色及Ⅱ型胶原免疫组化均为阴性,RT-PCR检测未表达Ⅱ型胶原。对照组1和对照组2均形成圆盘状组织块,表面光滑,触之有一定弹性,组织学检测软骨陷窝形态规则,Safranin O染色及Ⅱ型胶原免疫组化均有阳性表达,RT-PCR检测Ⅱ型胶原高表达。对照组3的组织块明显收缩,呈褐色,无弹性。对照组4,细胞松散,不能形成Pellet。对照组3和4的各种软骨特异性相关检测均为阴性。结论细胞间直接接触不能够单独诱导BMSC向软骨分化,不是软骨细胞与BMSC混合共培养中发挥诱导作用的主要因素。Objective To explore the possibility of cell-cell contact in initiating chondrogenic differentiation of BMSCs. Methods Porcine BMSCs and chondrocytes were respectively in vitro expanded. And then chondrocytes were inactivated by 0.5% paraformaldehyde and co-cultured with BMSCs at a ratio of 1：1. 1.0×10^6 mixed cells were centrifuged at 2 000 r/min for 8 rain to form a cell pellet. The mixture of normal chondrocytes and BMSCs and the pure normal chondrocytes were respectively centrifuged likewise as positive controls （Ctrl 1 and Ctrl 2）. The BMSCs and inactivated chondrocytes were respectively centrifuged likewise as negative controls （Ctrl 3 and Ctrl 4）. All the pellets were cultured in regular media for 4 weeks and then evaluated for chondrogenic phenotype by staining with HE and Safranin O, and immunohistochemically with type II collagen. Expression of cartilage specific genes was detected by reverse transcription polymerase chain reaction （RT-PCR）. There were 3 specimens in each group. Results the specimens of BMSCs plus fixed chondrocytes failed to form cartilage- like tissue with negative expression of cartilage specific matrices at both gene and protein levels. The pellets in positive controls formed the mature lacuna structures and metachromatic matrices. Collagen II expression could be observed by immunohistochemistry and RT-PCR examination. The specimens in negative controls formed fibrous tissues. Conclusion The cell-cell contact alone between chondrocytes and BMSCs was not enough to initiate chondrogenic differentiation of BMSCs.

关 键 词：骨髓基质细胞 软骨细胞 细胞间直接接触 多聚甲醛 软骨形成 

分 类 号：Q813.12[生物学—生物工程]