hBMP-2基因转染兔骨髓间充质干细胞体内移植后hBMP-2的活性检测  

Detection of hBMP-2 activity after transplantation of hBMP-2 gene-trasfected bone marrowmesenchymal stem cells in rabbits

作　　者：石正松[1] 李强[1] 宁寅宽[1] 蔡伟良[1] 李诗鹏 

机构地区：[1]桂林医学院附属医院急诊创伤外科,桂林541001

出　　处：《中国骨质疏松杂志》2015年第11期1333-1337,共5页Chinese Journal of Osteoporosis

基　　金：国家自然科学基金资助项目(31160199);广西区自然科学基金资助项目(2014jj AA40064)

摘　　要：目的探究人骨形态发生蛋白-2(human bone morphogenetic protein-2,h BMP-2)基因植入兔股骨头坏死模型体内后的活性,为后续实验奠定理论基础。方法 1.采用髓芯减压联合液氮冰冻法制备兔股骨头坏死模型;2.将造模成功的兔子随机分为A、B、C3组(n=12),A组不植入材料,为对照组,B、C组分别植入DBM/BMSCs、h BMP-2/BMSCs/DBM;3.术后第1、第2、第3周各组分别处死4只兔子,分别运用PCR、RTFQ-PCR、Western Blot检测股骨头内h BMP-2基因和蛋白的表达量,评估h BMP-2基因在兔体内的活性。结果植入后,在第1、第2、第3周,PCR结果示:A、B两组h BMP-2基因呈阴性表达,C组h BMP-2基因呈阳性表达;RTFQ-PCR示:C组相对表达量分别为1,0.947±0.025,0.895±0.059,P=0.081;Western blot示:A、B两组h BMP-2蛋白在兔体内呈阴性表达,C组h BMP-2蛋白呈阳性表达;凝胶成像系统分析显示:C组h BMP-2蛋白表达灰度值(目的/内参)分别为:0.530±0.056、0.507±0.066、0.475±0.086,且两两之间比较P>0.05。结论 h BMP-2基因转染BMSCs植入兔股骨头坏死模型体内后能够稳定表达,进而能够持续诱导BMSCs向骨细胞分化,达到修复股骨头坏死的目的。Objective To explore the activity of hBMP-2 after transfection to BMSCs in rabbit femoral neck necrosis model, and to lay the theoretical foundation for the subsequent experiments. Methods 1 ） Rabbit femoral head necrosis model was established by core decompression combined with liquid nitrogen frozen. 2） After the model establishment, animals were randomly divided into 3 groups （n = 12 per group）, Group A as control group, Group B with implant of DBM/BMSCs, Group C with implant of hBMP- 2/BMSCs/DBM- 3） Four rabbits of each group were killed at 1, 2, and 3 weeks after surgery, hBMP-2 gene and protein were detected using PCR, RTFQ-PCR, and Western blotting. Results After 1-, 2-, and 3-week implant, PCR results showed that the expression of hBMP-2 gene in group A and group B were negative, and the expression of hBMP-2 gene in C group was positive. RTFQ-PCR showed that the relative expression in group C was 1, 0. 947 ± 0. 025, and 0. 895 ±0. 059, P = 0. 081. Western blotting showed that the expression of hBMP-2 protein in group A and group B were negative, and the expression in group C was positive. Gel imaging system analysis showed that hBMP-.2 protein expression gray values （objective/reference） in group C was 0. 530 ± 0. 056, 0. 507 ± 0. 066, and 0. 475 ± 0. 086, respectively （ P 〉 0.05 ）. Conclusion The hBMP-2 can be stably expressed in transfected BMSCs after implant into the femoral head necrosis rabbit model and it can induce the differentiation of BMSCs to bone cells, and repair the necrosis of the femoral head.

关 键 词：骨组织工程 人骨形态发生蛋白2 股骨头坏死 PCR 荧光定量PCR 免疫印迹 

分 类 号：R687.34[医药卫生—骨科学]