骨质疏松大鼠骨髓基质细胞膜片的体外构建研究

Construction of osteoporotic rat bone marrow stromal cell sheet in vitro

目的探讨骨质疏松大鼠骨髓基质细胞膜片的构建及基本生物学特性。方法建立大鼠绝经后骨质疏松症(PMOP)模型,分离培养骨质疏松大鼠骨髓基质细胞(O-r BMSCs),并以O-rBMSCs为种子细胞构建细胞膜片。通过倒置显微镜、HE染色、扫描电镜及透射电镜对细胞膜片进行形态学检测;以免疫组织化学染色检测细胞外基质相关蛋白的表达情况。结果成功建立了大鼠PMOP模型,体外分离培养的O-rBMSCs具有多向分化潜能。显微镜下可见细胞复层生长,采用富含维生素C的培养基连续培养10 d左右即可获得白色膜样结构,该细胞膜片具有较好的弹性,HE染色及扫描电镜观察发现O-rBMSCs膜片由多层细胞及丰富的细胞外基质组成。透射电镜下可观察到细胞间连接。免疫组织化学染色示O-rBMSCs膜片高表达Ⅰ型胶原、纤维连接蛋白。结论 O-rBMSCs体外采用富含维生素C的培养基连续培养可构建细胞膜片,该细胞膜片富含细胞外基质,有望应用于骨质疏松机体的组织再生。

Objective To construct osteoporotic rat bone marrowstromal cell sheet and characterize its fundamental biological property. Methods We established postmenopausal osteoporotic（ PMOP） rat model,isolated and cultured osteoporotic rat bone marrowstromal cells（ O-rBMSCs） in vitro. Then we constructed O-rBMSCs sheet. Inverted microscope,HE staining,scanning electron microscopy（ SEM） and transmission electron microscopy（ TEM） were employed for morphological observation of the sheet,and the expression of extracellular matrix associated proteins were examined by immunohistochemical staining. Results PMOP rat model was successfully established. O-rBMSCs had multi-differentiation potential. O-rBMSCs were cultured in growth medium supplemented with vitamin C for 10 days,and a white membranaceous structure was formed,which possessed good flexibility. O-rBMSCs grewin multilayer under microscope. HE staining and SEM showed that O-rBMSCs sheet was formed by layers of cells and abundant extracellular matrix. Intercellular junctions were observed under TEM. Immunohistochemical staining showed that O-rBMSCs sheet highly expressed collagenⅠand fibronectin. Conclusion O-rBMSCs sheet can be constructed by in vitro culture of O-r BMSCs in vitamin C medium. The O-rBMSCs sheet was rich in extracellular matrix,and can be expected to be applied to tissue regeneration in osteoporosis.