短发夹RNA TRPV4基因对脊柱侧弯大鼠模型椎间盘退变的抑制作用

doi:10.3969/j.issn.1674-5817.2019.05.004

摘要/Abstract

摘要： 目的探究短发夹RNA(shRNA)瞬时感受器电位香草素4(transient receptor potential vanilloid, TRPV4)基因对抑制SD大鼠脊柱侧弯动物模型椎间盘退变的机制。方法采用经典的双足大鼠脊柱侧弯模型构建动物模型,根据小干扰RNA(siRNA)干扰原理,构建短发夹RNA(shRNA)-TRPV4干扰载体,根据实验目的大鼠分为对照组、模型组和shRNA组。采用影像学摄片观察大鼠脊柱侧弯的程度和cobb角度; HE染色观察退变椎间盘组织; 免疫组织化学染色检测髓核组织中II型胶原和多聚蛋白多糖(Aggrecan)的表达; 实时荧光定量PCR(RT-PCR)和Western blotting从基因和蛋白两个层面检测诱导型一氧化氮合成酶(iNOS); 炎症相关因子环氧合酶2(COX-2)和金属蛋白酶9 (MMP-9); 以及凋亡相关因子B淋巴细胞瘤-2基因(Bcl-2),Bcl-2家族抗凋亡因子(Bad),Bcl-2家族相关X蛋白(Bax)以及含半胱氨酸的天冬氨酸蛋白水解酶-3(Caspase-3), Caspase-6和Caspase-9的表达水平。结果 0.011 μL shRNA-TRPV4慢病毒量转染效率较高,为90.3%; 对照组SD大鼠未发生侧弯; 模型组SD大鼠脊柱向左侧弯,cobb角为35.22°±2.18°; shRNA组SD大鼠脊柱向左侧弯,cobb角为24.07°±3.09°; RT-PCR结果显示, 模型组髓核组织中iNOS, COX-2,MMP-9,Bad,Bax和凋亡相关因子Caspase-3,Caspase-6,Caspase-9的mRNA表达水平均高于对照组(P<0.05); 而抗凋亡因子Bcl-2的表达水平要低于对照组(P<0.05）; Western blotting结果显示, 模型组髓核组织中iNOS和MMP-9的蛋白表达水平均高于对照组(P<0.05); 而低于shRNA组(P<0.05); 免疫组织化学染色结果显示, shRNA组髓核组织中II型胶原蛋白和多聚蛋白多糖(Aggrecan)蛋白的蛋白表达水平均低于对照组(P<0.05); 而高于模型组(P<0.05)。结论 shRNA-TRPV4可以抑制脊柱侧弯鼠模型中髓核细胞的凋亡, 降低髓核组织中iNOS以及炎性因子COX-2和MMP-9的表达, 对髓核组织的退变起到保护作用。

关键词: 髓核细胞, 脊柱侧弯, 凋亡, 机械敏感性离子通道, 瞬时感受器电位香草素4(TRPV4)

Abstract: Objective To explore the mechanism of short hairpin (shRNA) transient receptor potential vanilloid 4 (TRPV4) gene on intervertebral disc degeneration intervention in SD rat scoliosis model. Methods The classical scoliosis model of bipedal rats was used to construct the animal model. Based on the principle of siRNA interference, shRNA-TRPV4 interference vector was constructed. According to the purpose of the experiment, animals were divided into three groups, control group, model group and shRNA group. The degree of scoliosis and cobb angle were observed by radiography, degenerative intervertebral disc tissue was stained by pathological HE, expression of collagen II and Aggrecan in nucleus pulposus tissue was detected by immunohistochemical staining, inducible nitric oxide synthase (iNOS) was detected by RT-PCR and Western Blotting at gene and protein levels as well as inflammatory related factors cyclooxygenase 2 (COX-2) and matrix metalloproteinase-9 (MMP-9) and the expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 antagonist of cell death(Bad), Bcl-2 associated X protein(Bax), cysteinyl aspartate specific proteinase-3(Caspase-3), Caspase-6 and Caspase-9. Results The transfection efficiency of 0.011 μL shRNA-TRPV4 lentivirus was 90.3%. The control group had no scoliosis, the model group had left scoliosis, and the cobb angle was 35.22°±2.18°, the shRNA group had left scoliosis, and the Cobb angle was 24.07°±3.09°. RT-PCR results showed that iNOS, COX-2, MMP-9, Bad, Bax and apoptosis-related factors such as Caspase-3, Caspase-6, Caspase-9 in the model group was higher than that of control group (P<0.05), while the expression of anti-apoptotic factor Bcl-2 was lower than that of control group (P<0.05). Western blotting showed that the expression of iNOS and MMP-9 in nucleus pulposus of model group was higher than that of control group (P<0.05), but lower than that of shRNA group (P<0.05). Immunohistochemical staining showed that the expression level of Type II collagen and Agrecan protein was lower than that of control group (P<0.05), but higher than that of model group (P<0.05). Conclusion ShRNA-TRPV4 can inhibit the apoptosis of nucleus pulposus cells in scoliosis rat model, reduce the expression of iNOS and inflammatory factors COX-2 and MMP-9 in nucleus pulposus tissue, and protect the degeneration of nucleus pulposus tissue.

Key words: Nucleus pulposus cells, Scoliosis, Apoptosis, Mechanically sensitive ion channels, Transient receptor potential vanilloid 4 (TRPV4)

中图分类号:

Q95-33

李琰, 孔建军, 陈子奇, 祝聪聪. 短发夹RNA TRPV4基因对脊柱侧弯大鼠模型椎间盘退变的抑制作用[J]. 实验动物与比较医学, 2019, 39(5): 356-363.

LI Yan, KONG Jian-jun, CHEN Zi-qi, ZHU Cong-cong. Inhibitory Effect of shRNA TRPV4 Gene on Intervertebral Disc Degeneration in Scoliosis Rat Model[J]. Laboratory Animal and Comparative Medicine, 2019, 39(5): 356-363.

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