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背景:骨形态发生蛋白(bonemorphogeneticprotein,BMP)是诱导和促进种子细胞向骨细胞方向转化的最重要的细胞因子之一。天然BMP难溶解于培养基,对培养的种子细胞无法有效发挥作用;可溶性重组BMP虽可溶解于培养基,但价格昂贵,而且难于适时、适量的作用于种子细胞。基因治疗为上述问题的解决提供了新的思路。目的:转染外源BMP-3基因入成纤维细胞,筛选获得稳定表达BMP-3的阳性成纤维细胞克隆。设计:单一样本研究。单位:解放军第四军医大学西京医院全军骨科研究所。材料:成纤维细胞(NIH3T3细胞)由第四军医大学口腔医学院司徒镇强教授提供。方法:2000-04/2003-11在全军重点实验室第四军医大学全军骨科研究所实验室完成。采用脂质体转染的方法将外源BMP-3基因转染入NIH3T3细胞,G418筛选后寻找细胞集落,分离、培养,免疫组织化学鉴定,BMP-3染色阳性者为阳性BMP-3表达细胞克隆。主要观察指标:①NIH3T3细胞筛选浓度。②转染阳性细胞克隆的筛选。③筛选的细胞克隆内BMP-3的表达。结果:成功将BMP-3基因转染入NIH3T3细胞,经免疫组化筛选出阳性BMP-3表达成纤维细胞的克隆,建立了BMP-3稳定表达成纤维细胞株。结论:将BMP-3基因真核表达载体转染入成纤维细胞,并经筛选获得了可稳定表达BMP-3的成纤维细胞株,为将来骨组织工?
BACKGROUND Bone morphogenetic protein (BMP) is one of the most important cytokines that induce and promote the transformation of seed cells into osteoblasts. Natural BMP difficult to dissolve in the medium, the seed cells can not effectively play a role; soluble recombinant BMP although soluble in culture medium, but expensive, and difficult to timely and appropriate role in the seed cells. Gene therapy provides a new way of solving the above problems. OBJECTIVE: To transfect exogenous BMP-3 gene into fibroblasts and screen positive clones of BMP-3-positive fibroblasts. Design: Single Sample Study. Unit: Xijing Hospital, Fourth Military Medical University PLA Orthopedic Institute. MATERIALS: Fibroblasts (NIH3T3 cells) were provided by Prof. Zhenzhu Zhen from Stomatology School, Fourth Military Medical University. METHODS: The laboratory of the PLA General Orthopedic Research Institute of the Fourth Military Medical University from April 2000 to November 2003 was completed. The transfection of exogenous BMP-3 gene into NIH3T3 cells was carried out by lipofectamine transfection. The cells were screened by G418 for cell colonies, isolated, cultured and identified by immunohistochemistry. Positive BMP-3 cells were positive for BMP-3 expressing cells clone. MAIN OUTCOME MEASURES: ①NiH3T3 cell screening concentration. ② transfection of positive cell clones screening. ③ screening of cell clones BMP-3 expression. Results: The BMP-3 gene was successfully transfected into NIH3T3 cells. The positive clones of BMP-3-expressing fibroblasts were screened by immunohistochemistry and the stable expression of BMP-3 fibroblasts was established. CONCLUSION: The eukaryotic expression vector of BMP-3 gene is transfected into fibroblasts, and the fibroblast cell line stably expressing BMP-3 is screened out for future bone tissue engineering.