摘要
[目的]从永生化骨骺干细胞中克隆Sox9基因并构建真核表达载体,并探讨Sox9诱导骨髓基质细胞向骨骺干细胞分化的可能性。[方法]以RT-PCR方法获得Sox9全长,插入pGEM-TEasy克隆载体中,测序正确后与pEG-FP-IRES2表达载体酶切后连接,复合质粒以脂质体法转染骨髓基质细胞,观察转染效率,Sox9和FGFR-3的表达。流式细胞术鉴定细胞表型,MTT法检测细胞增殖活性。[结果]成功的完成了Sox9的扩增和表达载体的构建,重组载体转染骨髓基质细胞后能检测到Sox9、FGFR-3的表达,增殖活性与骨骺干细胞无异。[结论]成功构建了Sox9真核表达载体,其能诱导骨髓基质细胞分化为骨骺干细胞并具有其特性。
[ Objective ] To construct eukaryotic expression vector containing Sox9 derived from immortalized precartilaginous stem cells and to probe its inductive effect on marrow stroma cells differentiating into precartilaginous stem cells. [ Method] Sox9 was obtained with RT - PCR and then inserted into pGEM - T Easy cloning vectors, pEGFP - IRES2 - Sox9 was established after sequencing. Then it＇ s transfected into marrow stroma cells. The expression of Sox9 and FGFR - 3 was detected by Western - blot and immunohistochemisty. Identification and proliferation was determined by flow cytometry and MTT. [ Result] Sox9 cloning and expression vector construction was achieved. Sox9 and FGFR - 3 expression was identified in marrow stroma cells after transfec- tion,whose proliferation activity was similar to precartilaginous stem cells. [ Conclusion ] Sox9 eukaryotic expression vector has been successfully established. Sox9 can induce marrow stroma cells to differentiate into precartilaginous stem cells.
出处
《中国矫形外科杂志》
CAS
CSCD
北大核心
2009年第12期931-934,共4页
Orthopedic Journal of China
基金
国家自然科学基金资助项目(编号:30650006)
教育部博士点基金资助项目[No.教技发中心函(2005)216]
关键词
骨骺干细胞
SOX9
骨髓基质细胞
转染
：precartilaginous stem cells
Sox9
marrow stroma cells
transfection
