犬骨髓基质干细胞体外定向分化为软骨细胞

背景:骨髓基质干细胞的多向分化性不利于向软骨细胞单一方向分化,植入体内后成骨系细胞分泌的骨形态发生蛋白作用于非定向分化的前体细胞,使其向成骨细胞分化,而已定向分化的细胞则不受骨形态发生蛋白的影响,形成相应的组织.目的:体外诱导犬骨髓基质干细胞定向分化为软骨细胞,探讨体外诱导成软骨的方法和条件.设计、时间及地点:单一样本观察,于2005-03/2006-01在中山大学组织工程实验室完成.材料:选取4个月龄雄性家犬1只,骨髓基质干细胞取自犬肋骨.方法:自犬肋骨取骨髓2.0～3.0mL行体外原代骨髓基质干细胞分离培养.8～11d细胞接近融合时,加入胰酶消化,用含体积分数10%胎牛血清的L-DMEM合成培养液终止消化,收集细胞悬液,离心后,用培养液悬浮细胞,按1:3接种传代.取第3代细胞培养扩增,换液时加入10μg/L碱性成纤维细胞生长因子2mL,换液2次后,再加入1mg/L转化生长因β1 2mL诱导骨髓基质干细胞向软骨细胞分化.主要观察指标:行甲苯胺蓝、阿新蓝染色检测软骨基质的分泌,免疫组织化学染色检测软骨特异性Ⅱ型胶原表达.结果:骨髓基质干细胞体外行原代和传代培养至P4代生长良好,经碱性成纤维细胞生长因子和转化生长因β1扩增诱导的细胞甲苯氨蓝异染性、阿新蓝染色阳性;Ⅱ型胶原免疫组织化学检测阳性,显示被诱导的骨髓基质干细胞具备软骨细胞特性.结论:应用碱性成纤维细胞生长因子和转化生长因β1体外可以诱导犬骨髓基质干细胞分化为软骨细胞,诱导的软骨细胞可作为软骨组织工程较理想的种子细胞.

In vitro chondrocyte differentiation of canine bone marrow stromal stem cells

BACKGROUND:Bone marrow stromal stem cells(BMSCs)do not allow for single differentiation of chondrocytes due to their multi-directional differentiation,bone morphogenetic protein secreted from osteoblasts affect the non-differentiated precursor cells and promote their osteoblast differentiations,while those differentiated cells are bound to form tissues.OBJECTIVE:To in vitro induce canine BMSCs differentiate into chondrocytes,and to investigate the method and conditions of chondrocyte differentiation in vitro.DESIGN,TIME AND SETTING:Single sample observation was performed in the Laboratory of Tissue Engineering,Sun Yat-sen University between March 2005 and January 2006.MATERIALS:One male dog,aged 4 months,was involved to harvest BMSCs from the rib.METHODS:Rib BMSCs extracted from bone marrow of 2.0-3.0 mL were cultured in vitro. When cells reached a confluence at 8-11 days,trypsinization was conducted and then halted with L-DMEM synthesis culture solution containing 10% fetal bovine serum. Cellular suspension was collected and centrifuged,cells were rssuspended and incubated at a ratio of 1:3. The third generation of cells were cultured and amplified,10 μg/L basic fibroblast growth factor 2 mL was added to replenish culture medium twice,then 1 mg/L transforming growth factor β1 of 2 mL was applied to induce BMSCs differentiation into chondrocytes.MAIN OUTCOME MEASURES:Toluidine blue and alcian blue stains were applied to determine cartilage matdx secretion,immunohistochemistTy was used for the detection of cartilage specific Ⅱ collagen expression.RESULTS:After BMSCs were primarily cultured and subcultured in vitro,they were shown to grow well at the fourth generation,those induced by basic fibroblast growth factor and transforming growth factor β1 were positive for toluidine blue and aician blue staining;immunohistochemistry showed a positive outcome for type Ⅱ collagen,indicating the induced BMSCs exhibited chondrocyte's characteristics.CONCLUSION:Utilizing basic fibroblast growth factor and transforming growth factor β1,the induced canine BMSCs could differentiate into chondrocytes,which is considered as an ideal seed cells for cartilage tissue engineering.