摘要
目的探究慢病毒介导shRNA质粒TRPV2和TRPV4基因双沉默模式在大鼠脊柱侧弯模型椎间盘退变中的作用。方法本研究采用经典的双足大鼠脊柱侧弯模型构建的方法。根据siRNA干扰原理,构建shRNA-TRPV2和shRNA-TRPV4干扰载体,根据实验目的将SD大鼠分成4组,即模型组,shRNA-TRPV2组,shRNA-TRPV4组和双基因沉默组。利用X射线对上述动物模型进行摄片,利用Masson染色检测成骨组织,利用抗酒石酸酸性磷酸酶(TRAP)染色检测破骨细胞活性,利用RT-PCR检测BMP,BGP和ALP的mRNA表达水平。结果 shRNA-TRPV2的慢病毒转染效率较高,为(92.3±2.7)%,shRNA-TRPV4的慢病毒转染效率较高,为(91.5±3.3)%,影像摄片结果显示,双基因沉默组大鼠模型Cobb角度相较于模型组具有明显改善(P<0.05)。Masson染色结果显示,双基因沉默组大鼠模型新生成骨组织明显多于模型组(P<0.05)。TRAP染色结果显示,双基因沉默组大鼠模型破骨细胞活性及数量明显多于模型组(P<0.05)。RT-PCR结果显示,双基因沉默组大鼠模型的BMP-2,BGP和ALP的mRNA相对表达量要明显高于模型组和单基因沉默组(P<0.05)。结论 shRNATRPV2和shRNA-TRPV4双基因沉默可以促进骨组织的生成,促进脊柱侧弯动物模型的修复。
Objective To investigate the role of lentivirus mediated double silencing of TRPV2 and TRPV4 gene in disc degeneration in scoliosis rats. Methods The classic scoliosis model of bipedal rats was used in this study. According to the principle of siRNA interference, shRNA-TRPV2 and shRNA-TRPV4 interference vectors were constructed. According to the experimental purpose, SD rats were divided into four groups: the model group, shRNA-TRPV2 group, shRNA-TRPV4 group and double gene silencing group. The above animal models were photographed by X-ray, the osteoblasts were detected by Masson staining, the osteoclast activity was detected by tartrate resistant acid phosphatase(TRAP) staining, and the mRNA expression levels of BMP, BGP and ALP were detected by RT-PCR. Results The efficiency of shRNA-TRPV2 and shRNA-TRPV4 was(92.1±2.7)% and(91.5±3.3)%, respectively. The imaging results showed that the Cobb angle of the model group was significantly improved compared with the model group(P<0.05). The results of Masson staining showed that the number of new osteoblasts in the model group was significantly higher than that in the model group(P<0.05). The results of TRAP staining showed that the osteoclast activity and quantity of the model group were significantly higher than that of the model group(P<0.05). The result of the RT-PCR showed that the relative expression of BMP-2, BGP and ALP mRNA in the model group was significantly higher than that in the model group and the single gene silencing group(P<0.05). Conclusion The double gene silencing of shRNA-TRPV2 and shRNA-TRPV4 can promote the formation of bone tissue and the repair of scoliosis animal model.
作者
李琰
孔建军
祝聪聪
LI Yan;KONG Jian-jun;ZHU Cong-cong(Department of Orthopaedics,General Hospital of Xingtai Mining Group of Central Hebei Energy,Xingtai 054000;Department of Pediatric Internal Medicine,Second Affiliated Hospital of Xingtai Medical College,Xingtai 054000,China)
出处
《解剖科学进展》
2020年第4期462-465,470,共5页
Progress of Anatomical Sciences
基金
邢台市科技计划项目(2019ZC340)。
关键词
基因沉默
青少年特发性脊柱侧弯
动物模型
成骨
gene silencing
adolescent idiopathic scoliosis
animal model
osteogenesis
作者简介
通信作者:祝聪聪。
