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目的:建立hBMP-4反转录病毒体系,为BMP-4基因治疗在骨组织工程中的应用提供研究平台。方法:应用亚克隆方法构建pLEGFP-hBMP-4真核表达载体,脂质体介导下转染PA317细胞,包装病毒颗粒。收集病毒上清,感染NIH3T3细胞系,测定滴度。以同样方法转染、包装获得了绿色荧光蛋白LEGFP反转录病毒,作为实验对照。将实验组和对照组病毒液分别注射至4只裸鼠肌内,8周后取材,组织学检查,鉴定成骨的效果。结果:成功构建了pLEGFP-hBMP-4载体。转染包装细胞后,获得了较高滴度的LEGFP-hBMP-4和LEGFP反转录病毒颗粒。裸鼠肌内注射后8周,hBMP-4病毒注射部位有新生骨块形成,而EGFP组则未见新生骨块。结论:成功建立了LEGFP-hBMP-4反转录病毒体系,为利用BMP-4基因治疗进行骨修复的研究提供了反转录病毒工具。
Objective: To establish hBMP-4 retroviral system and provide a research platform for the application of BMP-4 gene therapy in bone tissue engineering. Methods: The eukaryotic expression vector pLEGFP-hBMP-4 was constructed by subcloning method. PA317 cells were transfected by lipofectamine and the virus particles were packaged. Virus supernatants were collected and infected with NIH3T3 cell line for titration. The same method of transfection, packaging obtained green fluorescent protein LEGFP retrovirus, as an experimental control. The experimental group and the control group were injected into the virus fluid in nude mice, respectively. After 8 weeks, the samples were taken for histological examination to confirm the osteogenic effect. Results: The pLEGFP-hBMP-4 vector was successfully constructed. After transfection of the packaging cells, higher titers of LEGFP-hBMP-4 and LEGFP retroviral particles were obtained. Eight weeks after the intramuscular injection of nude mice, there was a new bone formation at the injection site of hBMP-4 virus, but no new bone mass was found in the EGFP group. CONCLUSION: The LEGFP-hBMP-4 retrovirus system was successfully established and provided a retroviral tool for the study of bone repair using BMP-4 gene therapy.