rhBMP-2基因转染小鼠间充质干细胞的成骨活性和BMP-2的释放规律分析

Analysis of Osteogenic Activity and BMP-2 Release Rules of C3H10T1/2 Cells Transfected with rhBMP-2 Gene

通过pcDNA3.1-rh BMP-2质粒转染小鼠间充质干细胞C3H10T1/2研究其成骨能力和增殖效应,探讨BMP-2的释放规律。将预先准备好的重组rhBMP-2质粒转染至C3H10T1/2细胞,利用G418抗药性筛查转染成功的细胞。将研究实验细胞分为转染组、空载组、空白组。通过ELISA检测各组细胞上清液中的BMP-2含量,CCK-8试剂盒检测各组细胞的增值活性,通过测定ALP活性和进行ALP、钙茜素红染色,检测各组细胞成骨活性。结果显示转染组BMP-2浓度随着时间推移逐渐达到高峰并维持一段时间,各检测点均明显高于空白组和空载组（p〈0.05）,转染组细胞较其余两组更具增殖活性、成骨活性（p〈0.05）。此结果表明C3H10T1/2细胞是pcDNA3.1/rhBMP-2重组质粒的良好宿主,转染后能在一定时间内平稳和持续地表达外源性BMP-2,有较强诱导细胞成骨分化能力。

In this study, we aimed to investigate osteogenic and proliferative effects and BMP-2 release profile in rat mesenchymal stem cells, C3H10T1/2, which transfected by cDNA3.1-rhBMP-2 plasmid. We used a pre-arranged recombinant BMP-2 plasmid DNA transfer into C3H10T1/2 cells and used G418 resistance to screen transfectd cells. The experimental cell divided into transfer group, blank group（empty plasmid） and blank group（no plasmid）. Subsequently, the concentration of medium supernatant liquor BMP-2 was detected by BMP-2 ELISA kit, the osteogenic activity was quantified by and ALP kit, ALP stain and Alizarin red stain. The results showed that with the passage of time, the concentration of BMP-2 increased in transfected group and were significantly higher than the blank group and control group（p〈0.05）, cell proliferation trend of transfection group was significantly higher than that of the blank group and control group, there were significant differences（p〈0.05）.The activity of ALP in transfection group was higher than that in blank control group and in blank group, and the difference was significant（p 〈0.05）. The quantitative analysis of ALP activity of transfer group obviously appear ALP-positive and intense positive ompared with other groups. These results indicated C3H10T1/2 cells is suited to been parasitifer for pcDNA3.1/rhBMP-2 recombinant plasmid, which can stably express exogenous BMP-2 in a certain period of time and has strongly ability to induce osteogenic differentiation