兔骨髓间充质干细胞体外诱导复合藻酸钙成软骨实验研究

发布时间：2018-12-15 01:00

【摘要】：目的：观察兔骨髓间充质干细胞(BMSCs),经转化生长因子-β1(TGF-β1)等向软骨细胞诱导8d、11d、14d、17d、20d后,与藻酸钙凝胶复合体外培养,细胞在三维条件下增殖情况及共同构建组织工程化软骨情况。 方法：穿刺新西兰大耳白兔股骨抽取骨髓,密度梯度离心法分离、培养BMSCs。将传至第3代的BMSCs予以含TGF-β1、地塞米松等成份的无血清诱导液定向向软骨细胞诱导。根据不同的诱导时间,实验组分成为A、B、C、D、E5组：A组：诱导至第8d;B组：诱导至11d；C组：诱导至14d；D组：诱导至17d：E组：诱导至20d。通过细胞形态、TB染色、Ⅱ型胶原免疫组化、培养液内多聚蛋白聚糖(AG)含量,比较各实验组定向诱导成软骨细胞情况。分别将以上各组成软骨细胞复合藻酸钙凝胶,以1×107/ml的密度制作为细胞-藻酸钙凝胶珠,继续体外培养14d。培养过程中观察细胞在藻酸钙凝胶中生长、增殖情况,并通过Ⅱ型胶原免疫组化、阿利新蓝比色法测定各组Ⅱ型胶原表达和多聚蛋白聚糖(AG)的含量,综合比较各实验组构建组织工程化软骨的情况。 结果：密度梯度离心法分离纯化的BMSCs定向成软骨细胞诱导8d时形态有较明显改变,14d时具有明显的软骨细胞形态。诱导培养液在诱导4d时即可检出少量AG,8d时AG浓度出现明显升高,并保持缓慢升高至20d。在14d时,诱导的细胞爬片TB染色可见异染颗粒；Ⅱ型胶原免疫组化明显阳性表现。与藻酸钙复合后,各组成软骨细胞在凝胶3D支架中均保持分裂增殖,AO染色见细胞能增殖成“葡萄串”样。各实验组材料切片Ⅱ型胶原免疫组化均可见阳性表达,以C组、D组为明显；AG含量检测显示各实验组、阳性对照组间AG含量具有差异(p0.05),C组、D组均高于阳性对照组,以C组AG含量为最高。 结论：1.采用密度梯度离心法获取的兔BMSCs,在体外经过14d的定向诱导后基本向软骨细胞分化;2.藻酸钙凝胶与种子细胞生物相容性良好,可以采用注射方式植入诱导14d后的成软骨细胞在藻酸钙凝胶内增殖良好,分泌软骨细胞外基质功能较强。以BMSCs为种子细胞、藻酸钙为支架的可注射式软骨组织工程方法具有可行性。
[Abstract]:Aim: to observe the effect of transforming growth factor 尾 1 (TGF- 尾 1) on rabbit bone marrow mesenchymal stem cells (BMSCs),) induced by transforming growth factor 尾 1 (TGF- 尾 1) into chondrocytes for 8 days, 11 days, 14 days and 17 days, and then cultured in vitro with calcium alginate gel. The proliferation of cells in three-dimensional condition and the construction of tissue engineered cartilage. Methods: bone marrow was extracted from the femur of New Zealand big ear white rabbit, and BMSCs. was isolated by density gradient centrifugation. The third passage of BMSCs was induced to chondrocytes by serum-free medium containing TGF- 尾 1 and dexamethasone. According to the different induction time, the experimental group was divided into two groups: group A: induced to the 8th day: group B: induced to 11 days: group C: induced to 14 days: group D: induced to 17d:E group: induced to 20 days. By means of cell morphology, TB staining, type 鈪，

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