shRNA-Piezo1对异常机械牵张应力作用下髓核细胞凋亡的影响及相关机制

Effects and mechanism of shRNA－Piezo1 on apoptosis of nucleus pulposus cells under abnormal mechanical stretch stress

中文关键词:  髓核细胞  Piezo1蛋白  shRNA干扰  牵张应力  凋亡

英文关键词:Nucleus pulposus cells  Piezo1 protein  shRNA interference  Ｍechanical stretch stress  Apotosis

中文摘要:

  【摘要】 目的：探讨短发夹RNA沉默压电离子蛋白1编码基因（shRNA-Piezo1）对异常机械牵张应力作用下髓核细胞凋亡的影响及相关机制。方法：取8例腰椎间突出症患者术中摘取的椎间盘组织（改良Pfirrmann分级为Ⅱ级或Ⅲ级），分别分离培养髓核细胞，取第2代髓核细胞构建体外机械牵张应力模型；利用293T细胞作为慢病毒感染靶细胞，检测最适慢病毒滴度，用最适滴度慢病毒感染髓核细胞；应用RT-PCR和Western-Blot法筛选出有效干扰序列，利用shRNA干扰技术构建shRNA-Piezo1干扰质粒；根据预实验处理结果，将细胞分成4组：空白对照组（取第2代髓核细胞不做机械牵张应力处理），牵张应力组（取第2代髓核细胞机械牵张应力处理24h），shRNA阴性对照组（空白载体质粒+第2代髓核细胞机械牵张应力处理24h），shRNA干扰组（shRNA-Piezo1干扰质粒+第2代髓核细胞机械牵张应力处理24h），应用Fluo3-AM试剂盒检测4组细胞的细胞质Ca2+水平；Cell Meter检测试剂盒检测4组细胞中线粒体膜电位的变化；AnnexinV-FITC试剂盒检测4组细胞的凋亡率。结果：（1）分离培养的细胞Ⅱ型胶原蛋白和Aggrecan蛋白表达阳性，符合髓核细胞特征。（2）当髓核细胞转染复数（MOI）=50时，慢病毒滴度为1×108TU/ml转染效率最高。（3）homo-3201序列为shRNA-Piezo1的有效序列。（4）4组髓核细胞细胞质Ca2+含量、线粒体膜电位翻转比例和细胞凋亡率有显著性差异（P<0.05），空白组与shRNA阴性对照组比较无显著性差异（P>0.05）；shRNA干扰组与牵张应力组和shRNA阴性对照组比较均显著性减少（P<0.05），与空白对照组比较无显著性差异（P＞0.05）。结论：shRNA－Piezo1可以抑制异常机械牵张应力作用下髓核细胞的过度凋亡，而且是通过抑制细胞质Ca2+水平和线粒体膜电位翻转来实现的。

英文摘要:

  【Abstract】 Objectives:　To investigate the effects and mechanism of shRNA-Piezo1 coding gene on apoptosis of nucleus pulposus cells induced by abnormal mechanical stretching stress.　Methods:　The nucleus pulposus cells from 8 cases of intervertebral disc herniation(Pfirrmann Ⅱ or Ⅲ) were isolated and cultured, and the second generation nucleus pulposus cells were used to construct the mechanical stretch stress model in vitro. The shRNA-Piezo1 interfering plasmid was constructed by shRNA interfering technique. The cells were divided into four groups according to the results of pretreatment: blank control group(the second generation of nucleus pulposus cells without mechanical stretching stress treatment), stretching stress group(the second generation of nucleus pulposus cells treated with mechanical stretching stress for 24 hours), shRNA negative control group(blank vector plasmid + 2nd generation nucleus pulposus cells with mechanical stretch stress treatment for 24 hours), shRNA interference group(shRNA-Piezo1 plasmid + 2nd generation nucleus pulposus cells with mechanical stretch stress treatment for 24 hours). Fluo 3-AM kit was used to detect the cytoplasmic Ca2+ level of the cells. Cell Meter kit was used to detect the mitochondrial membrane of the cells. Annexin　V-FITC kit was used to detect the apoptosis rate of the cells. Results:　(1)Collagen type Ⅱ and agrecan protein expression were positive in the cultured cells, which accorded with the characteristics of　nucleus pulposus cells.　(2)Lentiviral titer of 1×108TU/ml was the highest when the multiple transfection(MOI) of nucleus pulposus cells was 50. (3)The homo-3201 sequence was an effective sequence of shRNA-Piezo1. (4)There were significant differences in cytoplasmic Ca2+ content, mitochondrial membrane potential inversion ratio and apoptosis rate among the four groups(P<0.05); there was significant difference between the mechanical stress group and the blank control group(P<0.05), but there was no significant difference between the shRNA interference group and the shRNA negative control group(P>0.05); the shRNA interference group decreased significantly when compared with the stretch stress group and the shRNA negative control group(P<0.05), but there was no significant difference between the shRNA interference group and the blank control group(P>0.05). Conclusions: ShRNA Piezo1 can inhibit excessive apoptosis of nucleus pulposus cells under abnormal mechanical stretching stress by inhibiting cytoplasmic Ca2+ level and mitochondrial membrane potential inversion.