转化生长因子-β诱导骨髓基质细胞团体外形成软骨的观察

Experimental Study on the Chondrogenesis Potentiality of Marrow Stromal Cell under the Induction of Transforming Growth Factor-β

目的 观察骨髓基质细胞 (MSCs)在TGF_β诱导下形成软骨的能力 ,以探讨其作为软骨组织工程种子细胞的可能性。方法 体外分离、培养、扩增兔MSCs,消化后移入离心管中 ,离心使之成为细胞团 ,每个细胞团含 1× 10 6 个MSCs,将细胞团暴露于含 2 0ng mlTGF_β的培养液中 7d ,然后进行连续培养 ,观察细胞团的变化 ,于培养的 14d、2 8d取出细胞团 ,通过体视显微镜、光镜、透射电镜观察软骨的形成情况。结果 TGF_β作用后约 14d ,细胞团出现收缩 ,在离心管底部呈圆球状 ,以后逐渐增大 ,2 8d后直径最大可达 1 8mm ,表面光滑 ,为亮白色 ;HE染色显示 14d可见大量活细胞 ,并有细胞外基质形成 ,某些局部可见细胞位于陷窝内 ,2 8d可见较多细胞位于陷窝中 ;透射电镜观察显示细胞周围有大量胶原基质形成。结论 MSCs在TGF_β作用下可以形成软骨团块 ,表明其可以向软骨细胞定向分化 。

Objective\ Seed cell study is an essential area in the research of tissue engineering To evaluate the potentiality of marrow stromal cell(MSCs) as seed cell in the regeneration of tissue engineered cartilage, formation of cartilage nodules by culture expanded MSCs pellets under the induction of TGF_β was investigated.Methods\ MSCs were cultured and expanded in vitro. Cell pellets containing 1×10\+6 MSCs were obtained by centrifuging MSCs solution at 1 000 r/min in 5 ml centrifugation tube Pellets were exposed to cell culture media containing 20 ng/ml TGF_β for 7 days and then cultured for another 7 and 21 days The nodules were moved out of the tube and cartilage formation was observed by stereomicroscope, light microscope and electronic microscope Results\ 10 days after exposure to TGF_β, pellets contracted and formed small and round nodules on the bottom of the tubes The nodules grew bigger slowly and reached maximal diameter of 1 8mm in 28 days The surface of the nodules was smooth and bright white Histological examination showed that extra cellular matrix formed in 14 days and in some areas cells situated in lacuna In 28 days′ specimens, a lot of cells situated in lacuna could be observed and the histological appearance looked much similar to cartilage Electronic microscope observation demonstrated that in 28 days′ specimens a large amount of collagen fiber existed Conclusion\ Under the induction of TGF_β, MSCs could differentiate into chondrogenesis cell and form cartilaginous nodules in vitro. This indicated that MSCs could be the potential seed cells in the regeneration of cartilage employing method of tissue engineering.\;