摘要
目的观察人转化生长因子（hTGF）β2基因转染诱导脂肪间充质干细胞向软骨细胞的定向分化能力，探讨脂肪间充质干细胞作为种子细胞和在基因增强的软骨组织工程中应用的可行性。方法取3周龄Lewis大鼠的脂肪组织，消化法获得脂肪间充质干细胞，pcDNA3，1（＋）／hTGFβ2通过脂质体介导转染脂肪间充质干细胞，用免疫化学染色、逆转录-聚合酶链反应（RT-PCR）和Western blot检测筛选的阳性克隆细胞中hTGFβ2基因与软骨特异性蛋白-Ⅱ型胶原和蛋白多糖表达的情况；然后将基因转染的脂肪间充质干细胞与PLGA支架体外构建细胞-载体复合物，再将其植入裸鼠体内，12周后观察基因增强的组织工程软骨的形成情况。结果从成体大鼠脂肪组织中培养出脂肪间充质干细胞，能大量稳定增殖传代。hTGFβ2基因在脂肪间充质干细胞内能瞬时及稳定表达，并促使Ⅱ型胶原和蛋白多糖合成；细胞-载体复合物在裸鼠体内经12周的培养，可以形成形态、结构接近正常软骨的组织工程软骨。结论pcDNA3.1（＋）／hTGFβ2成功转染脂肪间充质干细胞，诱导其向软骨细胞分化，脂肪间充质干细胞可作为基因增强的软骨组织工程较理想的种子细胞。
Objective To investigate the effect of hTGFβ2 gene transfection on the differentiation of adipose mesenchymal stem cells into chondrocytes, and to discuss the feasibility of adipose mesenchymal stem cells as seeding cells of gene-enhanced cartilage tissue engineering. Methods Adipose mesenchymal stem cells were obtained from inguinal fat pads of Lewis rats with digestion. Adipose mesenchymal stem cells were induced into chondrocytes by hTGFβ2 gene transfection, and then the differentiations were examined by immuncytochemistry, RT-PCR and Western blotting. Cell-carrier composites were formed with the transfected adipose mesenchymal stem cells and PLGA scaffold, and then the composites were implanted into the subcutaneous tissue of nude mice. After 12 weeks, the tissue-engineering cartilage was examined by histochemistry. Results A population of adipose mesenchymal stem cells were successfully isolated from rat adipose tissue, and could stably proliferated and passaged in vitro. After transfected with pcDNA3.1 （ ＋ ）/hTGFβ2 ,the differentiation of adipose mesenchymal stem cells towards chondrocytes was verified by the positive results of type Ⅱ collagen and aggrecan through immunocytochemistry, RT-PCR and Western blot respectively. After the culture in nude mice for 12 weeks, H-E, alcian blue, toluidine blue and immunochemistry stain showed that cell-carrier composites developed to tissue engineering cartilage which to be similar to normal cartilage. Conclusion Mesenchymal stem ceils obtained from rat adipose tissue can differentiate into chondrocytes when transfected with hTGFβ2 gene in vitro and in vivo, and adipose mesenchymal stem cells can likely serve as optimal seeding cell source for gene-enhanced cartilage tissue engineering.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2008年第8期1033-1035,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（30500254）
关键词
脂肪
干细胞
基因转染
软骨
Adipose
Stem cell
Gene transfection
Cartilage
