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目的:构建骨形态发生蛋白4(BMP4)重组腺病毒,探讨其对NIH3T3成纤维细胞向成骨方向分化的影响。方法:将BMP4基因克隆连接到载体pAdTrack-CMV中,在细菌BJ5183中与pAdEasy腺病毒基因组进行同源重组,得到BMP4重组腺病毒基因组,通过转染HEK293细胞包装出重组腺病毒。利用BMP4重组腺病毒转染NIH3T3细胞,采用逆转录-多聚酶链反应(RT-PCR)和western-blot检测BMP4在细胞中的mRNA和蛋白表达。Gomori改良钙钴法检测BMP4重组腺病毒转染后的NIH3T3细胞碱性磷酸酶表达;von kossa染色检测BMP4重组腺病毒转染后NIH3T3细胞的成骨分化情况。结果:获得了BMP4转移质粒pAdTrack-BMP4和BMP4重组腺病毒基因组,并包装出重组腺病毒。BMP4在转染后的NIH3T3细胞中得到表达,表达的BMP4蛋白具有生物学活性。转染后的NIH3T3细胞碱性磷酸酶表达增加。BMP4促进了NIH3T3细胞向成骨方向分化,形成钙结节。结论:本实验成功构建了BMP4重组腺病毒;BMP4重组腺病毒可促进NIH3T3细胞向成骨方向分化。
OBJECTIVE: To construct bone morphogenetic protein 4 (BMP4) recombinant adenovirus and investigate its effect on the osteogenic differentiation of NIH3T3 fibroblasts. Methods: The BMP4 gene was cloned into the vector pAdTrack-CMV and homologously recombined with the pAdEasy adenoviral genome in the bacterium BJ5183 to obtain the BMP4 recombinant adenovirus genome. The recombinant adenovirus was packaged and transfected into HEK293 cells. NIH3T3 cells were transfected with BMP4 recombinant adenovirus and the mRNA and protein expression of BMP4 in the cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western-blot. The expression of alkaline phosphatase in NIH3T3 cells transfected with BMP4 recombinant adenovirus was detected by Gomori method. The osteogenic differentiation of NIH3T3 cells transfected with BMP4 recombinant adenovirus was detected by von kossa staining. Results: BMP4 transfer plasmid pAdTrack-BMP4 and BMP4 recombinant adenovirus genome was obtained and packaged recombinant adenovirus. BMP4 was expressed in transfected NIH3T3 cells, and the expressed BMP4 protein was biologically active. After transfection NIH3T3 cells increased alkaline phosphatase expression. BMP4 promoted NIH3T3 cells to differentiate into osteoblasts, forming calcium nodules. Conclusion: BMP4 recombinant adenovirus was successfully constructed in this experiment. BMP4 recombinant adenovirus can promote NIH3T3 cells to differentiate into osteoblasts.