Toll样受体3处理的脐带间充质干细胞与人牙髓细胞共培养对细胞矿化与抗炎修复能力的影响

Effect of human umbilical cord mesenchymal stem cells treated with Toll-like receptor 3 and human dental pulp cells co-cultured on cell mineralization and anti-inflammatory repair ability

目的:探究Toll样受体3(TLR3)处理的人脐带间充质干细胞(hUCMSCs)与人牙髓细胞(hDPCs)共培养后,对细胞矿化能力及抗炎修复能力的影响。方法:从健康新生儿的脐带组织中分离培养hUCMSCs,采用流式细胞术和免疫荧光染色检测细胞表面标记物CD34、CD45、CD90及CD105的表达,以对hUCMSCs进行鉴定;以TLR3激动剂Poly(I:C)(10μg/mL)处理hUCMSCs,建立hUCMSCs与h DPCs的共培养体系,测定各组细胞碱性磷酸酶(ALP)水平,茜素红染色和Von Kossa染色观察各组细胞矿化情况;利用脂多糖(LPS,10μg/mL)刺激hDPCs模拟牙髓炎症反应,MTT法检测各组细胞的增殖活性,RT-qPCR法检测各组细胞中肿瘤坏死因子-α(TNF-α)、白细胞介素6(IL-6)、白细胞介素10(IL-10)、牙本质涎磷蛋白(DSPP)、牙本质基质蛋白-1(DMP-1)、骨钙素(OCN)及骨桥蛋白(OPN)的mRNA表达情况。结果:分离的hUCMSCs中CD34和CD45呈阴性表达,CD90和CD105呈阳性表达,表明成功分离出hUCMSCs;与单独培养的hUCMSCs和hDPCs比较,hUCMSCs与hDPCs共培养体系内细胞ALP染色加深,ALP活性升高,有较多的矿化结节形成(P<0.05);与hUCMSCs与hDPCs共培养比较,经Poly(I:C)处理的hUCMSCs与h DPCs共培养中细胞ALP活性进一步升高,矿化结节形成更多(P<0.05)。相较于未经LPS刺激的细胞,经LPS刺激后的各组细胞增殖活性降低,细胞中TNF-α、IL-6及IL-10的mRNA相对表达量均升高,DSPP、DMP-1、OCN及OPN的mRNA相对表达量均下降(P<0.05);在LPS刺激下,相较于hUCMSCs与hDPCs共培养下,经Poly(I:C)处理的hUCMSCs与h DPCs共培养中的细胞增殖活性较高,TNF-α、IL-6的mRNA相对表达量均下降,IL-10 mRNA相对表达量进一步升高,同时,DSPP、DMP-1、OCN及OPN的mRNA相对表达量也均升高(P<0.05)。结论:TLR3激动剂Poly(I:C)处理的hUCMSCs与hDPCs共培养,可增强细胞的矿化能力,抑制LPS刺激下炎症因子水平,并促进炎症状态下牙本质形成相关基因的表达,这可能有利于牙髓炎症下的抗炎修复。

Objective:To explore the effect of human umbilical cord mesenchymal stem cells(hUCMSCs)treated with Toll-like receptor 3(TLR3)and human dental pulp cells(hDPCs)co-cultured on cell mineralization and anti-inflammatory repair ability.Methods:hUCMSCs were isolated and cultured from the umbilical cord tissues of healthy newborns,and the expressions of cell surface markers CD34,CD45,CD90 and CD105 were detected by flow cytometry and immunofluorescence staining so as to identify hUCMSCs.hUCMSCs were treated with TLR3 agonist Poly(I:C)(10μg/mL)and co-cultured with hDPCs.The alkaline phosphatase(ALP)level of each group was determined and the cell mineralization in each group was observed by alizarin red staining and Von Kossa staining.LPS(10μg/mL)was used to stimulate hDPCs to simulate dental pulp inflammation.MTT assay was used to detect the proliferative activity of cells in each group and the mRNA expressions of tumor necrosis factor-α(TNF-α),interleukin 6(IL-6),interleukin 10(IL-10),dentin sialophosphate protein(DSPP),dentin matrix protein-1(DMP-1),osteocalcin(OCN)and osteopontin(OPN)in each group were detected by RT-qPCR.Results:The isolated hUCMSCs showed negative expressions of CD34 and CD45,and positive expressions of CD90 and CD105,indicating hUCMSCs were successfully isolated.Compared with hUCMSCs and hDPCs cultured separately,the ALP staining of cells in the co-cultured system of hUCMSCs and hDPCs was deepened,ALP activity increased,and many mineralized nodules were formed(P<0.05).Compared with co-cultured hUCMSCs and hDPCs,the ALP activity of co-cultured hUCMSCs treated with Poly(I:C)and hDPCs further increased and more mineralized nodules were formed(P<0.05).Compared with cells without LPS stimulation,the proliferative activity of cells in LPS stimulated groups decreased,the mRNA relative expressions of TNF-α,IL-6 and IL-10 increased,and the mRNA relative expressions of DSPP,DMP-1,OCN and OPN decreased(all P<0.05).Under LPS stimulation,compared with co-cultured hUCMSCs and hDPCs,the cell proliferation activity of co-cultured hUCMSCs treated with Poly(I:C)and hDPCs was higher,the mRNA relative expressions of TNF-αand IL-6 decreased,the mRNA relative expressions of IL-10 further increased and meanwhile the mRNA relative expressions of DSPP,DMP-1,OCN and OPN also increased(all P<0.05).Conclusion:hUCMSCs treated with TLR3 agonist Poly(I:C)co-cultured with hDPCs can enhance the mineralization ability of cells,inhibit the levels of inflammatory factors under LPS stimulation,and promote the expression of genes related to dentin formation under inflammation,which may be beneficial to the anti-inflammatory repair in pulpitis.