摘要
目的探讨hTGF-β1 转染骨髓间充质干细胞（BMSCs）复合藻酸钙凝胶三维培养构建组织工程化软骨的可行性。方法全骨髓法获取和培养大鼠BMSCs，取第3代细胞接种于细胞培养板。实验分成3组：Ad．hTGF-β1 转染组、Ad．EGFP转染组、空白对照组，前两组分别加入含Ad-hTGF-β1 与Ad．EGFP的无血清培养基，空白对照组添加不做任何处理的完全培养基。7d后，运用实时荧光定量PCR与Westernblot检测TGF-β1 的表达情况。将Ad．hTGF-β1 成功转染组的BMSCs继续大量扩增后。按照1．0×107／ml的细胞密度制成细胞-藻酸钙凝胶复合物，将复合物置于培养箱中继续体外培养。10d后，观察凝胶材料中的细胞形态与增殖情况，并将复合物包埋、切片。进行组织学及免疫组织化学染色分析软骨分化情况。结果转染后7d，实时荧光定量PCR结果显示TGF-β1 表达量的平均相对比值Ad．hTGF-β1 转染组（0．863）明显高于Ad．EGFP转染组（0．183）、空白对照组（0．180），差异有统计学意义（P〈0．05）。Westernblot检测发现Ad．hTGF．B1转染组的细胞内TGF-β1表达呈强阳性．而Ad-EGFP转染组及空白对照组细胞内的TGF．S1表达很弱。倒置显微镜观察到细胞在藻酸钙凝胶中能够很好的维持球形生长；MTr检测显示，不同时间点细胞在藻酸钙凝胶中的数量变化差异无统计学意义（P〉O．05）。HE染色可见有大量软骨陷窝形成，甲苯胺蓝、Masson染色可见有软骨基质分泌。CollagenⅡ免疫组织化学染色呈阳性表达。结论hTGF-β1 成功转染的BMSCs，在藻酸钙凝胶材料中培养在维持细胞形态的同时能够很好的向软骨细胞分化，此种培养方法更能模拟细胞在体内的生长方式。
To investigate the feasibility of hTGF-β1 transfected bone mrrow mesenchymal stem cell （BMSCs） combined with calcium alginate gel in three dimensional condition to construct tissue engineering cartilage. Methods Rats BMSCs were obtained and cultured by whole bone marrow method, and then the thirdgeneration cells were seeded into cell culture plate, and were divided into 3 groups： AdhTGF-β1 transfected group, Ad-EGFP transfected group and control group. The control group was added in common medium without any treatment while the other 2 groups were respectively added in serumfree medium containing Ad-hTGF-131 or that containing AdEGFP. Seven days later, realtime fluorescent quantitation PCR and Western blot were employed for detecting the expression of TGF-β1. Then, the BMSCs which suc- cessfully transfected by AdhTGF-β1, were continually cultured in vitro. And the confound of cells-calcium alginate gel, the cell density was 1.0 x 107 per ml, were prepared and cultuered in constant temperature incubator. Ten days later, examine the morphous and proliferation of cell. Last, paraffin slice of the cellgel confound was stained by HE, toluidine blue and Masson staining, while immunohistochemical for the secretion of collagen Ⅱ. Results Seven days after the transfection, realtime fluorescent quantitation PCR revealed that the average relative expression of TGF-β1 was： AdhTG-β1 group 0.863, and AdEGFP group0.183, the control group 0.180, and the expression difference of TGF-β1was statistically diffence （P 〈 0.05）. Western blot proved strong TGF-β1 expression in AdhTGF-β1group while it was detected a little in the other two groups. Globose cells were observed through inverted microscope in the calcium alginate gel. MTY proved the amount of cells were not statistically diffence （P 〉 0.05） at different time point. HE staining proved amount cartilage lacuna formation in the gel, while the secretion of cartilage matrix were proved by toluidine blue and Masson, and immunohistochemical proved the
出处
《中华显微外科杂志》
CSCD
北大核心
2012年第1期40-45,97,共7页
Chinese Journal of Microsurgery
基金
国家自然科学基金资助项目（30770574）
湖北省卫生厅科技基金资助项目（JX3821）
关键词
骨髓间充质干细胞
转化生长因子-Β1
基因转染
软骨
组织工程
三维培养
Bone marrow mesenchymal stem cells
TGF-β1
Gene transfection
Chondrocytes
Tissue engineering
Three dimensional culture
