促红细胞生成素对大鼠磨牙牙髓血运重建术后SDF-1和VEGF表达水平及疼痛的影响

Effects of EPO(Erythropoietin)on the Expression of SDF-1 and VEGF and Pain After Pulp Revascularization in Molar Rats

目的:探讨EPO(促红细胞生成素)对大鼠磨牙牙髓血运重建术后SDF-1、VEGF表达水平及疼痛的影响。方法:48只大鼠随机分为正常组、模型组(磨牙牙髓血运重建大鼠)、EPO-A组(磨牙牙髓血运重建大鼠腹腔注射250IU/Kg EPO)及EPO-B组(磨牙牙髓血运重建大鼠腹腔注射500IU/Kg EPO),采用ELISA法检测各组大鼠血清中TNF-α、IL-6及IL-1β表达,HE染色观察根管组织形态,免疫印迹检测根管组织内SDF-1、VEGF蛋白水平。将人牙髓细胞分为正常组(人牙髓细胞)、EPO组(人牙髓细胞给予EPO干预)、VEGF组(人牙髓细胞给予VEGF干预)及SDF-1组(人牙髓细胞给予SDF-1干预),采用CCK-8检测24h、48h及72h细胞相对增值率,qRT-PCR检测SDF-1、VEGF mRNA相对表达。结果:与和正常组相比,模型组大鼠血清中TNF-α、IL-6及IL-1β水平升高(P<0.05),与模型组相比,EPO-A组及EPO-B组大鼠血清中炎性指标均降低(P<0.05),且EPO-B组低于EPO-A组(P<0.05);模型组大鼠根管组织中SDF-1及VEGF蛋白表达水平低于正常组(P<0.05),EPO-A组及EPO-B组SDF-1及VEGF蛋白表达水平高于模型组(P<0.05),EPO-B组与EPO-A组比较差异显著(P<0.05);人牙髓细胞克隆至第二代时,倒置显微镜下观察细胞形态,镜下可见,细胞生长状况良好,呈梭形或星形,细胞间排列紧密,胞浆丰富,胞浆的突起互相连接,细胞核呈卵圆形,位于细胞中央,有明显的核仁;EPO组、SDF-1组及VEGF组人牙髓细胞的在24h、48h及72h的相对增殖率均高于正常组(P<0.05);与正常组相比,EPO组、SDF-1组及VEGF组细胞中SDF-1及VEGF mRNA表达升高(P<0.05),EPO组、SDF-1组及VEGF组SDF-1及VEGF mRNA水平无意义(P>0.05),SDF-1mRNA与VEGF mRNA呈现正向相关性(r=0.576,P=0.003)。结论:促红细胞生成素能够降低大鼠磨牙牙髓血运重建术后炎症水平,缓解疼痛,并通过提高SDF-1、VEGF表达促进血管新生,参与血运重建。

Objective:To investigate the effects of EPO(erythropoietin)on the expression of SDF-1 and VEGF and pain after pulp revascularization in molar rats.Methods:Totally 48 rats were randomly divided into normal group,model group(molar pulp revascularization rats),EPO-A group(molar pulp revascularization rats intraperitoneal injection of 250IU/Kg EPO)and EPO-B group(molar pulp revascularization rats intraperitoneal injection of 500IU/Kg EPO).ELISA method was used to detect the expression of TNF-α,IL-6 and IL-1βin the serum of each group of rats.HE staining was used to observe the morphology of root canal tissue.Western blotting was used to detect the protein levels of SDF-1 and VEGF in the root canal tissue.Human dental pulp cells were divided into normal group(human dental pulp cells),EPO group(human dental pulp cells given EPO intervention),VEGF group(human dental pulp cells given VEGF intervention)and SDF-1 group(human dental pulp cells given SDF-1 intervention).CCK-8 was used to detect the relative proliferation rate of 24h,48h and 72h cells.qRT-PCR detects the relative expression of SDF-1 and VEGF mRNA.Results:Compared with the normal group,the serum levels of TNF-α,IL-6 and IL-1βin the model group increased(P<0.05).Compared with the model group,the inflammatory indexes in the serum of rats in the EPO-A group and EPO-B group were reduced(P<0.05),and the EPO-B group was lower than the EPO-A group(P<0.05);the expression level of SDF-1 and VEGF protein in the root canal tissue of the model group was lower than that of the normal group(P<0.05).The protein expression levels of SDF-1 and VEGF in the EPO-A and EPO-B groups were higher than those in the model group(P<0.05),and there was a significant difference between the EPO-B group and the EPO-A group(P<0.05).When the human dental pulp cells were cloned to the second generation,the morphology of the cells was observed under inverted microscope.It was found that the cells were in good growth condition,showing spindle shape or star shape.The cells were closely arranged,the cytoplasm was abundant,the processes of the cytoplasm were interconnected,and the nuclei were oval and located in the center of the cells,with obvious nucleoli.The relative proliferation rate of human dental pulp cells in EPO group,SDF-1 group and VEGF group at 24h,48h and 72h was higher than that in normal group(P<0.05).Compared with the normal group,the mRNA expressions of SDF-1 and VEGF in EPO,SDF-1 and VEGF groups were increased(P<0.05),but the mRNA levels of SDF-1 and VEGF in EPO,SDF-1 and VEGF groups were not significant(P>0.05).Sdf-1 mRNA was positively correlated with VEGF mRNA(r=0.576,P=0.003).Conclusion:Eerythropoietin can reduce IL-1Βlevel and relieve pain after molar pulp revascularization in rats,and promote angiogenesis to participate in revascularization by up-regulating the expression of SDF-1 and VEGF.