脂质体介导下重组pcDNA3┐hBMP3转染兔关节软骨细胞的条件 _医学论文.docVIP

脂质体介导下重组pcDNA3┐hBMP3转染兔关节软骨细胞的条件 _医学论文 脂质体介导下重组pcDNA3┐hBMP3转染兔关节软骨细胞的条件 _医学论文 作者：韩一生，胡蕴玉，金格乐，李松建 【关键词】 基因 　　关键词: 基因软骨细胞骨形成蛋白 　　摘 要:目的 探讨基因转移效率与pcDNA3-hBMP3质粒及阳离子脂质体的浓度、转染时机以及细胞的种殖密度等因素的关系，以寻求最佳的基因转染条件. 方法 细胞密度按2×104 #12539cm -2 接种后，对细胞接种后即时、孵化2，5和7d进行转染效率研究G418浓度每mL DMEM分别含有200，300，400，500和600μg，探讨G418对软骨细胞的杀伤作用选择不同细胞接种密度、不同脂质体浓度和pcDNA3-BMP3浓度，在细胞对数生长期且细胞融合至70%～80%时开始做转染，各组软骨细胞爬片的原位杂交结果通过计算机图像分析，以其灰度值判定基因转染的效率. 结果 最佳期的转染时机为软骨细胞接种后在孵箱内放置2d转染，即在软骨细胞对数生长期内做转染.G418对软骨细胞的最佳筛选浓度为400mg#12539L-1 .最佳的细胞种植密度、pcDNA3-BMP3浓度和脂质体的量应分别为2×10 4 #12539cm-2 ，5μL和10μL. 结论 通过本研究探索出脂质体介导下pcDNA3-BMP3转染兔关节软骨细胞的最佳条件. 　　Keywords:genechondrocyteBMP 　　Abstract:AIM To explore the relationship between the ef-ficiency of the gene transfection in vitro and the amount of re-combination pcDNA3-hBMP3，the transfection opportunity and cell density ect to find the best situation of gene trans-fection to chondrocyts.METHODS We studied the different time points of transfection to find its best opportunity and the action of G418in different densities of200/300/400/500/600μg in order to get its screen density.The different densities of chondrocytes of1×104 #12539cm -2 ，2×104 #12539cm-2 and1×105 #12539cm -2 ，and of profectamine adopt5，10and15μL，and5μg for amount of DNA were chosen.The transfection was done during cell logarithm growth period with the cell fusion of70%～80%.Furthermore，we analyzed the in situ hy-bridization results of chondrocytes in each group through computer image，according to which the transfection efficien-cy was determined，and cell planting density and lipisome density to transfection efficacy were made.Under such densi-ties of pcDNA3-BMP3as2，5and10μg，and with the pro-fectamine density of10μL，the transfection was done2d af-ter cell vaccination.RESULTS The best opportunity of cell transfection was2d in incubator after vaccination，and the optimum screen density of G418to chondrocytes was400mg#12539L-1 .The optimum cell planting density，optimum pcD-NA3-BMP3density and optimum li