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周栩,权正学,罗小辑,唐可,周强,钟伟洋,张圆,江维.Id1基因在BMP-2维持椎间盘细胞软骨特性中的作用[J].陆军军医大学学报(原第三军医大学学报),2015,37(20):2011-2016.
Zhou Xu,Quan Zhengxue,Luo Xiaoji,Tang Ke,Zhou Qiang,Zhong Weiyang,Zhang Yuan,Jiang Wei.Role of Id1 gene in maintenance of cartilage phenotype in BMP-2-induced intervertebral disc cells[J].J Amry Med Univ (J Third Mil Med Univ),2015,37(20):2011-2016.
周栩,权正学,罗小辑,唐可,周强,钟伟洋,张圆,江维.Id1基因在BMP-2维持椎间盘细胞软骨特性中的作用[J].陆军军医大学学报(原第三军医大学学报),2015,37(20):2011-2016. DOI:
Zhou Xu,Quan Zhengxue,Luo Xiaoji,Tang Ke,Zhou Qiang,Zhong Weiyang,Zhang Yuan,Jiang Wei.Role of Id1 gene in maintenance of cartilage phenotype in BMP-2-induced intervertebral disc cells[J].J Amry Med Univ (J Third Mil Med Univ),2015,37(20):2011-2016. DOI:
目的 构建Id1基因过表达和RNAi慢病毒载体,并以兔椎间盘髓核细胞为研究对象,观察Id1基因表达水平调整后,BMP-2诱导髓核细胞分泌软骨特性因子的变化。 方法 ①设计针对Id1基因过表达和RNA干扰的序列,应用基因重组技术将目的基因片段连接到慢病毒载体,转染293T细胞后测定病毒滴度。②将2种慢病毒分别感染髓核细胞,QRT-PCR和Western blot检测髓核细胞Id1 mRNA和蛋白表达。③重组腺病毒AdBMP2感染髓核细胞,利用已制备的2种慢病毒再次感染细胞,QRT-PCR检测细胞表达软骨特性分子的能力。 结果 ①慢病毒感染髓核细胞后,Id1基因的mRNA与蛋白的表达量与未感染病毒组(0.428±0.079)及空载病毒组(0.400±0.045)相比,过表达组(0.848±0.154)增加,RNAi组(0.115±0.014)下降,差异具有统计学意义(P0.01),符合预期结果。②细胞内Id1的表达增加可促进colⅡ(0.407±0.083)和ACAN(0.284±0.074)的mRNA表达增加,与GFP组比较,差异具有统计学意义(P0.01)。③ADBMP2处理细胞后,Id1的表达增加可使colⅡ(0.590±0.072)和ACAN(0.351±0.066)的mRNA表达进一步显著升高;Id1表达降低时,以上因子的表达被明显抑制[colⅡ:(0.244±0.060)
ACAN:(0.168±0.034)
差异具有统计学意义(P0.01)]。 结论 Id1能够促进椎间盘细胞软骨特性分子的表达,细胞内Id1基因水平变化能够显著影响BMP-2诱导细胞分泌软骨特性因子,推测Id1是BMP-2维持椎间盘细胞软骨特性作用的关键因子。
Objective To construct lentiviral vectors for overexpression and RNA interference of inhibitors of differentiation 1 (Id1) gene
and observe the changes of cartilage phenotype molecules secreting from bone morphogenetic proteins 2 (BMP-2)-induced nucleus pulposus cells after adjusting the expression level of Id1 gene. Methods The sequences for overexpression and RNA interference of human Id1 gene were designed and linked into lentiviral vectors by genetic recombination technology
and then the constructed lentiviral vectors were introduced into 293T cells to determine the titers. After the nucleus pulposus cells were transfected with the 2 kinds of recombinant lentiviruses
the expression of Id1 at mRNA and protein levels were assayed by real-time quantitative PCR and Western blotting respectively. Then
the nucleus pulposus cells were sequentially transfected with recombinant adenovirus AdBMP2 and the 2 kinds of lentiviruses
and the capacity of expressing cartilage phenotype molecules was detected by qRT-PCR. Results The expression of Id1 at mRNA and protein levels in the nucleus pulposus cells were changed after lentivirus transfection
and compared with the normal cell control group (0.428±0.079) and negative control group (0.400±0.045)
the Id1 gene expressions were increased in the overexpression group (0.848±0.154) and decreased in the RNAi group (0.115±0.014)
with statistically significant differences (P0.01). The Id1 gene overexpression could promote the mRNA expression of collagen Ⅱ (0.407±0.083) and Agreecan (0.284±0.074)
and there was statistically significant difference as compared with the GFP group (P0.01). After AdBMP2 transfection
the Id1 gene overexpression could further promote the mRNA expression of collagen Ⅱ (0.590±0.072) and Agreecan (0.351±0.066)
while the down-regulation expression of Id1 gene could inhibit the expressions of collagen Ⅱ (0.244±0.060) and Agreecan (0.168±0.034)
with statistically significant difference (P0.01). Conclusion Id1 can promote the expression of cartilage phenotype molecules
and its expression change can significantly affect the secretion of cartilage phenotype molecules in the BMP-2-induced nucleus pulposus cells
thus Id1 may be the key factor in maintaining cartilage phenotype of the BMP-2-induced intervertebral disc cells.
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