人BMP-4腺病毒表达载体对人退变腰椎间盘细胞的影响  

EFFECT OF ADENOVIRUS HUMAN BONE MORPHOGENETIC PROTEIN 4 ON HUMAN DEGENERATIVE LUMBAR INTERVERTEBRAL DISC CELLS

作　　者：曹德军[1] 权正学[1] 蒋电明[1] 罗小辑[1] 钟伟洋[1] 刘港[1] 

机构地区：[1]重庆医科大学附属第一医院骨科,重庆400016

出　　处：《中国修复重建外科杂志》2012年第12期1442-1447,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30901530);重庆市科学技术委员会自然科学基金资助项目(CSTC2009BB5074)~~

摘　　要：目的研究人BMP-4腺病毒表达载体(adenovirus human BMP-4,Ad-hBMP-4)转染体外培养人退变腰椎间盘细胞后,对细胞蛋白多糖、Ⅱ型胶原及Sox9表达的影响。方法取Ad-hBMP-4重组腺病毒进行扩增,并检测病毒滴度。取ModicⅢ级、27～50岁腰椎间盘突出症患者自愿捐赠的退变椎间盘体外分离、培养椎间盘细胞,激光共聚焦显微镜观察Ⅱ型胶原在细胞中的表达。取第1代椎间盘细胞转染Ad-hBMP-4重组腺病毒(实验组),应用RT-PCR和Western blot法分别检测病毒转染后3、6 d细胞蛋白多糖、Ⅱ型胶原、Sox9基因及蛋白多糖、Ⅱ型胶原蛋白的表达,与未转染细胞(对照组)进行比较。结果 Ad-hBMP-4重组腺病毒滴度为5×106PFU/mL。激光共聚焦显微镜观察示Ⅱ型胶原主要表达于椎间盘细胞质。Ad-hBMP-4重组腺病毒转染后细胞形态无明显变化。转染3、6 d后实验组蛋白多糖、Ⅱ型胶原、Sox9 mRNA表达以及蛋白多糖、Ⅱ型胶原蛋白表达均显著高于对照组,比较差异有统计学意义(P<0.05);实验组各指标3、6 d间比较差异亦有统计学意义(P<0.05)。结论 Ad-hBMP-4可有效转染体外培养人退变椎间盘细胞,促进细胞蛋白多糖、Ⅱ型胶原及Sox9表达,提示hBMP-4可能对早期退变的椎间盘具有修复功能。Objective To research the transfer ofadenovirus human bone morphogenetic protein 4 （Ad-hBMP-4） to human degenerative lumbar intervertebral disc cells in vitro and analyze its effect on the proteoglycan, collagen type II, and Sox9 of intervertebral disc cells. Methods Identified Ad-hBMP-4 was amplified and detected. Degenerative lumbar intervertebral disc cells were aspirated from the degenerative lumbar intervertebral disc of patients with Modic III level disc protrusion （aged, 27-50 years）. The expressing position of collagen type II was identified in the intervertebral disc cells through the laser confocal microscope. The intervertebral disc ceils at passage 1 were transfected with Ad-hBMP-4 as experimental group. After 3 and 6 days of transfection, RT-PCR was used to detect the mRNA expressions of proteoglycan, collagen type II, and Sox9, and Western blot to detect the expressions of proteoglycan and collagen type II proteins. Non-transfected cells at passage 1 served as control group. Results The virus titer of Ad-hBMP-4 was 5 x 106 PFU/mL. No morphological changes in the cells after transfection by Ad-hBMP-4. Collagen type II mainly expressed in the cell cytoplasm. The mRNA expressions of the proteoglycan, collagen type II, and Sox9 in experimental group at 3 and 6 days after transfection were significantly higher than those in control group by RT-PCR （P 〈 0.05）, and the expressions of proteoglycan and collagen type II proteins were significantly higher than those in contorl group by Western blot （P 〈 0.05）. There were significant differences between 3 days and 6 days in experimental group （P 〈 0.05）. Conclusion Ad-hBMP-4 could transfect human degenerative lumbar intervertebral cells with high efficiency and promote collagen type II, proteoglycan, and Sox9 expressions, hBMP-4 may play an important role in the repair process during early disc degeneration.

关 键 词：BMP-4 椎间盘细胞 腺病毒表达载体 基因转染 椎间盘退变 

分 类 号：R681.53[医药卫生—骨科学]