摘要
目的：通过研究兔骨髓基质干细胞（BMSC）的体外获取方法、生物学鉴定以及新型靶向性非病毒载体介导转化生长因子β1（TGF-β1）基因在BMSC中的瞬时转染和表达，为下一步的骨组织工程基因治疗研究奠定基础。方法：取4周龄新西兰大白兔骨髓，通过密度梯度离心法和全骨髓培养法获取BMSC，通过倒置相差显微镜、BrdU标记染色观察其形态学特征和生长状况，免疫组化观察其表面抗原表达情况。固相合成法合成含RGD肽K16GRGDSPC（K16-RGD）。以K16-RGD为载体介导TGF-β1基因转染兔BMSC，免疫组化检测其瞬时转染和表达·效率。结果：分离培养的细胞在第10-12代以前生长性状稳定，增殖能力强。免疫组化检测兔BMSC表达CD90、CD44，但不表达CD34、CD45、CD11b和层黏连蛋白Laminine。与全血培养法相比较，密度梯度离心法获取的BMSC纯度更高。K16-RGD介导TGF-β1基因瞬时转染效率可达（21．6±4．3）％。结论：分离培养的细胞成分较为单一，具有干细胞特性；TGF-β1基因能在BMSC中转染和表达，为下一步利用BMSC、K16．RGD和TGF-β1基因进行骨组织工程基因治疗研究奠定了基础。
Objective ： To establish the method of isolation and identification of rabbit-derived bone marrow stromal cells （BMSC） and to investigate the ability of BMSC to accept and express exogenous transforming growth factor （TGF）-β1 gene mediated with integrin-targeted nonviral gene delivery system-RGD containing peptide K16GRGDSPC （K16-RGD） Methods：The bone marrow tissues were collected from rabbits and BMSC were isolated, respectively, by the methods of density gradient centrifuge and full-marrow culture. Microscopy, BrdU label and immunohistochemistry stain were employed to observe the BMSC morphologic features, growth status and cell-surface antigens. K16-RGD was used to transfect exogenous TGF-β1 gene into BMSC and the gene expression was examined by a immunohistochemistry technique. Results： The cultured cells grew stably and proliferated rapidly before the 10th to 12th generation. The positive expressions of CD90 and CD44 were detectable while those of CD34, CIM5, CD11 b and laminin were not detectable. The purity of BMSC isolated by the method of density gradient centrifuge was higher than that by the method of full-marrow culture. The transient transfection efficiency of TGF-β1 gene mediated by K16-RGD was about 18.6%. Conclusion： The cultured cells were only composed of undifferentiated BMSC, They had the properties of stem cells. Exogenous TGF-151 could be transfected into BMSC by the mediation of K16-RGD peptide and expressed effectively. These provide experimental foundations for further gene modified bone tissue engineering studies.
出处
《医学研究生学报》
CAS
2006年第6期508-511,i0012,共5页
Journal of Medical Postgraduates
基金
国家自然科学基金资助项目(批准号:302000633047083)
关键词
骨髓基质干细胞
转化生长因子-Β1基因
含RGD肽
转染
Bone marrow stromal cells
Transforming growth factor β1 gene
RGD-containing pep-tide
Gene transfer
