摘要
目的：构建双顺反子真核表达载体PIRES2-EGFP-hBMP-2,检测其表达。
方法：利用RT-PCR方法从人骨肉瘤组织中提取人骨形态发生蛋白hBMP-2基因,通过序列分析正确后,使之与PMD18-T载体连接,转化大肠杆菌JM109,选择阳性克隆载体,进行扩增,提取质粒。经酶切回收后,与目的载体pIRES2-EGFP连接。用PCR技术及基因序列分析技术鉴定构建结果。然后将构建好的载体转染HEK293,通过荧光显微镜及Western blot技术观测其表达。
结果：成功扩增了hBMP-2基因,基因片段长度为1102bp; pIRES2-EGFP-hBMP-2质粒构建成功,转染HEK293细胞后可在荧光显微镜下观察到绿色荧光；提取的总蛋白行Western blot检测,可观察到hBMP-2基因所表达蛋白的显影条带。
结论：重组HEK293细胞能成功表达EGFP及hBMP-2基因,并为pIRES2-EGFP-hBMP-2载体转染骨髓基质干细胞及应用于软骨及骨缺损治疗提供了前提基础。
Objective:To construct the eukaryotic vector pIRES2-EGFP-hBMP-2 and to observe its expression in HEK 293 cells.
Methods:hMBP-2 gene was cloned from human osteosarcoma by RT-PCR and inserted into PMD18-T vector. Following the DNA sequence verification, it was then sub-cloned into the eukaryotic vector pIRES2-EGFP. After restriction enzyme analysis, the pIRES2-EGFP-BMP-2 was transfected into HEK 293 cells. Then its expression was observed with fluorescence microscope and Western Blot.
Results:hBMP-2 gene was successfully cloned and identified by restriction enzyme analysis. Construction of pIRES2-EGFP-BMP-2 was verified, then the expression of EGFP and BMP-2 in HEK 293 cells was proved by fluorescence microscope and Western Blot.
Conclusion:It validated that HEK 293cells could be transduced by Lipofectamime 2000 efficiency and the eukaryotic vector pIRES2-EGFP-hBMP-2 could successfully express target protein in it. The present study demonstrated that the two gene sequences encoding BMP-2 and EGFP could be translated in protein respectively. These HEK239 cells with pIRES2-EGFP-BMP-2 provides a promizing tool for the further investigating the repairing mechanism of BMP-2 in bony and cartilaginous diseases.
引文
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1. Herrero-Beaumont G, Marcos ME, Sanchez-Pernaute O. et al. Effect of chondroitin sulphate in a rabbit model of atherosclerosis aggravated by chronic arthritis. Br J Pharmacol 2008;154(4):843-51
2. Zvi Schwartz, Maya Fisher, Christoph H. Lohmann, et al. Osteoprotegerin (OPG) Production by Cells in the Osteoblast Lineage is Regulated by Pulsed Electromagnetic Fields in Cultures Grown on Calcium Phosphate Substrates. Annals of Biomedical Engineering 2009; 37(3):437-444
3. Gelse K, Miihle C, Franke O, Park J, et al. Cell-based resurfacing of large cartilage defects: long-term evaluation of grafts from autologous transgene-activated periosteal cells in a porcine model of osteoarthritis. Arthritis Rheum 2008; 58(2):475-88
4. Kubo S, Cooper GM, Matsumoto T, et al. Blocking vascular endothelial growth factor with soluble FIt-1 improves the chondrogenic potential of mouse skeletal muscle-derived stem cells. Arthritis Rheum 2009;60(1):155-65
5. 许澍洽,许扬滨.BMSCs构建组织工程软骨的研究进展[J].中国修复重建外科杂志,2008；22(2)：163-166.
6. Matsumoto T, Cooper GM, Gharaibeh B, et al. Blocking VEGF as a potential approach to improve cartilage healing after osteoarthritis. J Musculoskelet Neuronal Interact 2008; 8(4):316-7
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43.邹德波,周东升,人骨形态发生蛋白-2基因转染兔骨髓基质干细胞及其表达[J].中华创伤骨科杂志,2006；8(2)：148-152