TGF-β3诱导骨髓间充质干细胞膜片成软骨分化

TGF-β3 induces chondrogenic differentiation of bone marrow mesenchy⁃mal stem cell sheet

目的:探索转化生长因子β3(TGF-β3)诱导骨髓间充质干细胞(BMSCs)细胞膜片成软骨分化的潜能。方法:体外分离培养兔骨髓间充质BMSCs细胞,用含抗坏血酸培养基构建干细胞膜片,添加TGF-β3对干细胞膜片成软骨诱导分化。光镜、HE染色和Masson染色观察细胞形态、大体结构及组织学改变,阿利新蓝染色、Ⅱ型胶原(ColⅡ)免疫组化染色和RT-qPCR检测成软骨标志物ColⅡ和蛋白聚糖(PG)的mRNA表达及蛋白合成情况。结果:TGF-β3诱导成软骨分化14 d,镜下间充质干细胞生长良好,可用镊子钳夹起膜片状结构,可卷缩成团再展开成膜。HE染色显示细胞质及细胞外基质成分均被染成粉红色,其胞核为蓝色,细胞间呈分层状排列结构。Masson染色可见胞核染成蓝黑色,其细胞质染成粉红色,周围包绕着大量染成蓝色的胶原成分。阿利新蓝染色与ColⅡ免疫组化染色均为阳性,对照组阴性。实验组高表达软骨细胞特异性ColⅡ和PG的mRNA(P<0.05)。结论:抗坏血酸诱导构建的干细胞膜片经TGF-β3诱导,能成功向软骨分化及构建软骨细胞膜片。

AIM:To explore the feasibility of chondrogenic differentiation of bone marrow mesenchymal stem scBMMSCs cell sheet induced by transforming growth factor-β3(TGF-β3).METHODS:Rabbit bone marrow mesenchymal stem cells were isolated by differential adherence method,and stem cell sheets were constructed with ascorbic acid medium.The stem cell sheets were induced for chondrocyte differentiation by TGF-β3.The cell morphology,gross structure and histological changes were observed by light microscopy,HE staining and Masson staining.Alcian blue staining,collagen type Ⅱ(Col Ⅱ)immunohistochemical staining and RT-qPCR were used to detect chondrocyte-specific markers Col Ⅱ and proteoglycan(PG)mRNA expression and protein synthesis.RESULTS:TGF-β3 induced cartilage differentiation for 14 d,microscopic observation indicated that mesenchymal stem cells grew well.The sheet structure was clamped with forceps,which was rolled into clumps and then expanded into a film.With HE staining,the cytoplasm and extracellular matrix components were stained pink,the nuclei were blue,and the cells were arranged in a layered arrangement.Masson staining showed that the nucleus was stained blue-black,while the cytoplasm was stained pink,surrounded by a large amount of collagen stained in blue.The Alcian blue staining and Col Ⅱ immunohistochemical staining of the chondrogenic differentiation sheet were positive,and those of the uninduced stem cell sheet were negative.Compared with uninduced control group,chondrocyte-specific Col Ⅱ and PG mRNA levels were highly expressed in experimental group after inducedby TGF-β3.CONCLUSION:Ascorbic acid-induced stem cell sheets stimulated by TGF-β3 successfully differentiated into cartilage and constructed chondrocyte sheets.