人骨形成蛋白2腺病毒表达载体转染体外培养兔腰椎间盘细胞的研究

ADENOVIRUS-MEDIATED HUMAN BONE MORPHOGENETIC PROTEIN 2 GENE TRANSFERRED TO RABBIT INTERVERTEBRAL DISC CELLS IN VITRO

目的研究人骨形成蛋白2腺病毒表达载体(adenovirus-mediatedhumanbonemorphogeneticprotein2gene,Ad-hBMP-2)转染体外培养兔腰椎间盘细胞,分析其对腰椎间盘细胞的影响。方法成年健康新西兰大白兔4只,体重4～5kg,取L2、L3到L6、L7节段椎间盘细胞体外培养。利用免疫荧光和蛋白印迹法(Westernblot)检测空白对照组(未转染)、Ad-LacZ组[感染复数(multiplicityofinfection,MOI)150MOI]和不同剂量Ad-hBMP-2(50、100、150MOI)组转染后细胞hBMP-2的表达水平。采用RT-PCR检测转染后细胞型胶原和aggrecanmRNA表达水平。结果转染Ad-hBMP-2后通过免疫荧光和蛋白印迹法可见随转染剂量增加hBMP-2表达量逐渐增加,与50MOI组比较100MOI组升高102%,150MOI组升高183%。在转染后第6天RT-PCR结果显示转染组aggrecan和型胶原mRNA的表达高于对照组,并且随转染剂量增高mRNA的表达量均逐渐增加。aggrecanmRNA从50MOI组的61.7%增加到150MOI组的167.3%。型胶原mRNA从50MOI组的77.3%增加到150MOI组的169.0%。结论Ad-hBMP-2可高效转染体外培养兔腰椎间盘细胞,随转染剂量增加hBMP-2表达量逐渐增加,同时上调aggrecan和型胶原mRNA的表达,并存在剂量依赖关系。

Objective To study the adenovirus-mediated human bone morphogenetic protein-2 gene （AdhBMP-2）transferred to the intervertebral disc cells of the New Zealand rabbit in vitro. Methods The cells of New Zealand white rabbits were isolated from their lumbar discs. The cells were grown in the monolayer and treated with an adenovirus encoding the LacZ gene （Ad-LacZ） and Ad-hBMP-2 （50, 100, 150 MOI,multiplicity of infection） in the Dulbecco＇s Modified Eagle Medium and the Ham＇s F-12 Medium in vitro. Three days after the Ad-hBMP-2 treatment, the expression of hBMP-2 in the cells that had been infected by different doses of MOI was determined by immunofluorescence and the Western blot analysis, and the expression was determined in the cells with the Ad-LacZ treatment in a dose of 150 MOI. Six days after the Ad-hBMP-2 treatment, mRNA was extracted for the reverse transcription polymerase chain reaction （RT-PCR） and the difference was detected between the control group and the culture group that was treated with Ad-hBMP-2 in doses of 50, 100 and 150 MOI so that the expressions of aggrecan and collagen R mRNA could be observed. Results The expression of hBMP-2 in the cells was gradually increased after the transfection in an increasing dose, which was observed by immunofluorescence and the Western blot analysis. At 6 days the aggrecan and collagen type Ⅱ mRNA expressions were up-regulated by Ad-hBMP-2 after the transfection at an increasing viral concentration in the dose-dependent manner. Conclusion The results show that Ad-hBMP-2 can transfect the rabbit intervertebral disc cells in vitro with a high efficiency rate and the expression of hBMP-2 after the infection is dose-dependent in the manner. Ad-hBMP-2 after transfection can up-regulate the expression of aggreean and collagen Ⅱ mRNA at an increasing viral concentration.