整合素α6对hDPSCs增殖和成牙本质向分化的影响

Effect of Integrin Alpha 6 on Proliferation and Odontoblast Differentiation of Human Dental Pulp Stem Cells

目的:探讨整合素α6(integrin alpha 6)对人牙髓干细胞(human dental pulp stem cells,hDPSCs)增殖和成牙本质向分化的影响。方法:采用酶消化法,体外分离并培养hDPSCs,构建整合素α6沉默慢病毒,感染hDPSCs,分为阴性对照组(shMock)和整合素α6沉默组(shITGA6)。采用噻唑蓝(MTT)法及对增殖标志物Ki-67进行免疫荧光染色,检测整合素α6对hDPSCs增殖的影响;采用碱性磷酸酶(alkaline phosphatase,ALP)染色、Western blot检测矿化相关蛋白牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)和牙本质基质蛋白1(dentin matrix protein-1,DMP-1)的表达以及茜素红染色检测整合素α6对hDPSCs成牙本质向分化和矿化的影响。结果:MTT结果显示,沉默组hDPSCs的增殖能力明显降低(P<0.01);Ki-67免疫荧光染色结果显示,沉默组Ki-67阳性细胞率明显低于阴性对照组(P<0.001)。碱性磷酸酶染色结果显示,沉默组ALP染色明显加深;Western blot结果显示,沉默组DSPP和DMP-1的蛋白表达量明显增加,高于阴性对照组(P<0.05);茜素红染色结果显示,沉默组染色矿化结节明显增多。结论:整合素α6可以促进hDPSCs的增殖,抑制其成牙本质向分化。

Objective:To investigate the effect of integrin alpha 6 on proliferation and odontoblast differentiation of human dental pulp stem cells(hDPSCs).Methods:HDPSCs were isolated and cultured by enzyme digestion in vitro,and the integrinα6 silencing lentivirus was constructed to infect hDPSCs.The hDPSCs were divided into negative control group(shMock group)and integrinα6 silencing group(shITGA6 group).3-(4,5-dimethylthithioazole-2)-2,5-diphenyltetrazole bromide(MTT)and Ki-67 immunofluorescence staining were used to detect the effect of integrinα6 on the proliferation of hDPSCs.Alkaline phosphatase(ALP)staining and Western blot were used to detect the expression of dentin sialophosphoprotein(DSPP)and dentin matrix protein-1(DMP-1),and alizarin red staining was used to detect the effect of integrinα6 on the odontoblast differentiation and mineralization of hDPSCs.Results:MTT results showed that the proliferation capacity of hDPSCs in shITGA6 group was significantly reduced(P<0.01).The immunofluorescence staining of Ki-67 showed that the positive cell rate of Ki-67 in shITGA6 group was significantly lower than that in shMock group(P<0.001).Alkaline phosphatase staining showed that ALP staining in shITGA6 group was obviously deeper.Western blot showed that the expression of DSPP and DMP-1 in shITGA6 group was significantly up-regulated than that of shMock group(P<0.05).Alizarin red staining showed that the mineralized nodules increased significantly in shITGA6 group.Conclusion:Integrinα6 can promote the proliferation of hDPSCs and inhibit the odontoblast differentiation of hDPSCs.