circRNA_079813抑制牙髓细胞损伤的机制研究

Mechanism of circRNA_079813 inhibiting dental pulp cells injury

目的探讨circRNA_079813对脂多糖(LPS)诱导的人牙髓细胞(hDPCs)损伤的作用和机制。方法原代分离培养脱落乳牙牙髓细胞,用免疫荧光化学法鉴定原代分离培养的hDPCs。将培养的hDPCs分为vector组、过表达circRNA_079813的重组载体组(circRNA_079813组)、腺相关病毒(AAV)空载体组(AAV-Null组)、Smad1过表达重组AAV载体组(AAV-Smad1组)、小干扰RNA(siRNA)沉默Smad1的重组载体组(si-Smad1组)以及siRNA阴性对照组(si-NC组)。另外,将hDPCs分为空白对照组(Control组)、LPS组、LPS+vector组、LPS+circRNA_079813组、LPS+circRNA_079813+si-NC组、LPS+circRNA_079813+si-Smad1组。采用CCK-8法和流式细胞术分别检测hDPCs的增殖活力和凋亡水平。ELISA法检测白细胞介素(IL)-1β和肿瘤坏死因子-α(TNF-α)水平。检测碱性磷酸酶(ALP)活性、ALP蛋白表达水平以及茜素红S(ARS)染色程度以评估成骨分化能力。Western blot检测runt相关转录因子2(RUNX2)以及Smad1的蛋白水平。结果在LPS诱导的hDPCs中过表达circRNA_079813可增加hDPCs的增殖活性,提高成骨分化能力,抑制hDPCs的凋亡和炎症反应,提高Smad1和RUNX2的蛋白表达,差异均有统计学意义(P<0.05)。在LPS基础上过表达Smad1与过表达circRNA_079813对hDPCs的影响一致(P<0.05),而沉默Smad1逆转了过表达circRNA_079813的上述效果(P<0.05)。结论过表达circRNA_079813通过Smad/RUNX2信号通路改善LPS诱导的hDPCs损伤。

Objective To investigate the effect and mechanism of circRNA_079813 on lipopolysaccharide(LPS)-induced injury of human dental pulp cells(hDPCs).Methods Primary pulp cells of deciduous teeth were isolated and cultured,and the hDPCs were identified by immunofluorescence chemistry.The cultured hDPCs were divided into the vector group,the overexpressed circRNA_079813 recombinant vector group(the circRNA_079813 group),the adeno-associated virus(AAV)empty vector group(the AAV-Null group),the Smad1-overexpressed recombinant AAV vector group(the AAV-Smad1 group)and the small interference RNA(siRNA)silencing Smad1 recombinant vector group(the si-Smad1 group)and the siRNA negative control group(the si-NC group).In addition,hDPCs were divided into the blank control group(the Control group),the LPS group,the LPS+vector group,the LPS+circRNA_079813 group,the LPS+circRNA_079813+si-NC group,and the LPS+circRNA_079813+si-Smad1 group.CCK-8 method and flow cytometry were used to detect the proliferative activity and apoptosis level of hDPCs.ELISA was used to detect the levels of interleukin(IL)-1β,tumor necrosis factor-α(TNF-α).Alkaline phosphatase(ALP)activity,ALP protein expression level,and alizarin red S(ARS)staining degree were detected to evaluate the osteogenic differentiation ability.The protein levels of runt-associated transcription factor 2(RUNX2)and Smad1 were detected by Western blot.Results Overexpression of circRNA_079813 in LPS-induced hDPCs might increase the proliferative activity of hDPCs,improve the osteogenic differentiation ability,inhibit the apoptosis and inflammation response of hDPCs,and increase the protein expression of Smad1 and RUNX2,with statistically significant differences(P<0.05).Overexpression of Smad1 on the basis of LPS had the same effect on hDPCs as overexpression of circRNA_079813(P<0.05),while silencing Smad1 reversed the above effect of overexpression of circRNA_079813(P<0.05).Conclusion Overexpression of circRNA_079813 improves LPS-induced hDPCs injury through the Smad/RUNX2 signaling pathway.