转染人骨形态发生蛋白-2基因的羊骨髓间充质干细胞的异位成骨研究

Ectopic bone formation of hBMP-2 gene transfected goat bone marrow-derived mesenchymal stem cells

目的评价腺病毒介导的人骨形态发生蛋白-2(hBMP-2)基因转染羊骨髓间充质干细胞(MSCs)后的成骨活性。方法实验分为3组:(1)hBMP-2转染细胞组,(2)半乳糖苷酶基因(βgal)转染细胞组,(3)未转染细胞组。从羊骨髓中分离培养MSCs,基因转染后利用免疫沉淀和Westernblot方法检测BMP-2的表达,并在体外进行碱性磷酸酶(ALP)和VonKossa染色、ALP定量测定、透射电镜观察。分别将细胞悬液注入裸鼠股后部肌内,分期进行X线和组织学检查。结果Westernblot检测发现只有hBMP-2转染细胞组的MSCs表达并分泌hBMP-2。该组于转染后第12d,ALP活性达高峰,与其它两组比较,差异有显著性意义;于16d后出现钙结节,而其它两组未见。透射电镜观察见hBMP-2转染细胞组的细胞内粗面内质网、线粒体和溶酶体增多。裸鼠肌内注射细胞悬液后3周,hBMP-2转染细胞组即有异位成骨,6周时明显增多,其它两组仅见纤维组织形成或微量成骨。结论hBMP-2基因转染能诱导羊MSCs分化为成骨细胞,并在裸鼠体内诱导成骨。

Objective When marrow-derived mesenchymal stem cells(MSCs)tranfected by adenovirus tranduced hBMP-2 were transplanted autograft, it could display the ability to produce BMP-2 and induce bone formation. In the present, MSCs obtained from goat were tranfected with BMP-2 gene in vitro and evaluate its osteogenic potential ectopically in nude mice. Methods BMP-2cDNA was inserted into the vector of PAC-CMV, and the recombinant plasmid of PAC-CMVBMP-2 was accomplished, furthermore, type V adenovirus gene by zymic cutting with Cla I enzyme and PAC-CMVBMP-2 zymic cutting with Xho I enzyme were reconstructed in 293 cell line to achieve recombinant adenovirus with hBMP-2 (Adv-hBMP-2). By above method, recombinant adenovirus with βgal (Adv-βgal) was also made as control gene. Goat MSCs were transfected by Adv-hBMP-2 gene (group 1 ), by Adv-βgal gene (group 2), and uninfected served as control groups (group 3). Western blot analysis, ALP staining, Von Kossa staining and transmission electron microscopy were used to determine the phenotype of MSCs. Then the MSCs were injected into the thigh muscles of nude mice. Radiographic and histological evaluations were performed on schedule to conform the condition of bone formation. Results By immunoprecipitation test and Western blot analysis, only the Adv-hBMP-2 transfected MSCs produced hBMP-2, and the cells were positive for ALP staining at the 12th day with the 2.417 of ALP activity significantly higher than that of βgal gene and uninfected MSCs. Whereas in Von Kossa staining at the 16th day, calcified nodules were demonstrated in the muscles transplanted with Adv-hBMP-2 gene, otherwise, the other two groups were negative. Eelectron microscopy observation showed that there were more rough endoplasmic reticulum, mitochondria and lysosomes in the Adv-hBMP-2 transfected MSCs comparing to the MSCs of other two groups. At the 3rd and 6th week after cells injection, ectopic bones were observed in muscles of nude mice of group 1, only fibrous tissue or little bone was found in other two groups. Conclusion BMP-2 gene transfected MSCs could differentiate into osteoblast in vitro and induce bone formation ectopically in vivo.