腺相关病毒介导hBMP-2体内转染对兔退变椎间盘髓核细胞凋亡的影响

The effects of AAV-hBMP-2 transfection to the rabbit degenerated intervertebal disces in vivo on the nucleus pulposus of apoptosis

目的:了解腺相关病毒介导骨形态发生蛋白-2(adeo-associated virus mediated human bone morpho-genetic protein-2,AAV-hBMP-2)基因体内转染兔退变椎间盘后对髓核细胞Fas与Caspase-3蛋白表达及细胞凋亡的影响。方法:36只普通级新西兰大白兔,采用针刺兔椎间盘的方法制作L2/3、L3/4、L4/5椎间盘退变模型。造模4周后,在MRI影像学中,髄核的信号比正常减弱,证明造模成功,再随机分为注射20μlAAV-hBMP-2组(6×106pfu,A组)、AAV组(6×106pfu,B组)和生理盐水组(C组),分别于注射2周、4周及8周后对椎间盘进行MRI检查,然后取材,石蜡包埋,组织切片应用SP免疫组织化学法和TUNEL法分别观察Fas与Caspase-3蛋白表达及髓核细胞凋亡情况,IPP6.0图像分析系统测定各指标的平均光密度。结果:注射后2周、4周及8周时A组MRI髓核信号结果与B组、C组比较有统计学意义(P<0.05)。各组Fas,Caspase-3及TUNEL染色结果随着时间推移逐渐增强,A组Fas、Caspase-3及TUNEL的平均光密度明显低于B组和C组(P<0.01),B组与C组间比较无显著性差异(P>0.05)。结论:体内转染AAV-hBMP-2能抑制兔腰椎间盘髓核细胞凋亡。

Objective：To study the effect of rabbit degenerated intervertebral disc pulpiform nucleus cells transfected with adeo-associated virus mediated human bone morphogenetic protein-2（AAV-hBMP-2） in vivo on apoptosis of Fas and Caspase-3 in the nucleus pulposus.Method：A total of 36 New Zealand white rabbits in ordinary grade were established a rabbit model of intervertebral discs degeneration in L2/3、L3/4、L4/5 induced by puncturing the anulus fibrosus with defined needle.At four weeks postsurgery,the nucleus signal is lower than the normal in magnetic resonance imaging that proved the model of intervertebral disc degeneration to be successful.Then the rabbits of intervedebral disc degeneration were divided into three groups in random,named A,B and C.Then the groups A,B,C were injected AAV-hBMP-2（20μl,the virus titer is 6×106 pfu）,AAV（20μl,the virus titer is 6×106 pfu） and normal saline（20μl） respectively.The analyses of radiology,TUNEL,immunohistochemistry with SP methods and IPP6.0 system were performed to examine the average optical of the apoptotic disc cells at two,four and eight weeks respectively after injection.Result：In the magnetic resonance imaging,within the group A,a remarkably highest signal intensity in T2-weighted images of the nucleus pulposus were detected in the three groups（P〈0.05）.The group A of the average optical of the expression of Fas and Caspase-3 and the apoptotic cells were significantly lowest in the three groups（P〈0.01）,while there were no differences between the group B and C（P〉0.05）.Conclusion：The AAV-hBMP-2 transfection to the rabbit degenerated intervertebal disces in vivo could suppress apoptosis of degenerated intervertebal disces.