【作者】 杨琳；

【导师】 王燕；

【作者基本信息】 山东大学 ， 口腔临床医学， 2014， 硕士

【摘要】 目的丝裂原活化蛋白激酶(Mitogen-activated protein kinases,MAPKs)信号通路是真核细胞将胞外信号转至胞内引起细胞反应的转导系统,在细胞生长、增殖、分化和凋亡等细胞反应的传导中起重要作用。人根尖乳头细胞(human apical papilla cells, HAPCs)具有多向分化潜能,是组织工程和生物牙根构建的种子细胞。无机三氧化物聚合体(mineral trioxide aggregate, MTA)已广泛应用于根尖诱导成形术,并且取得了良好的效果。然而,MTA在HAPCs分化中的作用和分子机制目前还不清楚,本文拟探讨MAPKs信号通路在MTA诱导HAPCs分化过程中的作用。方法第一部分：人根尖乳头细胞的分离培养以及体外诱导分化酶消化法分离培养人根尖乳头细胞,传代后获得稳定生长的细胞。矿化诱导液培养三周后,茜素红染色显示诱导组形成大量矿化结节,对照组仅有少量散在矿化结节形成。第二部分：MTA对人根尖乳头细胞增殖和分化的影响应用不同浓度MTA培养细胞,通过CCK-8检测MTA对HAPCs增殖的影响,结果显示10mg/ml和20mg/ml MTA对细胞增殖有明显的抑制作用(p<0.05),低浓度MTA对细胞增殖无明显影响。使用MTA条件培养基(0、0.02mg/ml、0.2mg/ml、2mg/ml)诱导HAPCs的分化,检测碱性磷酸酶(alkaline phosphatase, ALP)活性,茜素红染色检测矿化结节的形成；通过real-time PCR检测牙本质特异性标记物牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)以及成骨相关性转录因子(runt-related transcription factor2、骨涎蛋白(bone sialoprotein, BSP)、骨钙素(osteocalcin,OCN)mRNA的表达；Western Blot检测Runx2蛋白的表达。结果显示MTA诱导后细胞ALP活性增强；矿化结节增多；DSPP、Runx2、BSP、OCN mRNA表达升高；Runx2的蛋白表达量升高。以上结果充分说明MTA促进人根尖乳头细胞的成牙本质／成骨向分化。MTA浓度为0.2mg/ml时促进作用最显著,后续实验选用此浓度为诱导浓度。第三部分：MAPKs信号通路在MTA诱导的HAPCs分化过程中的作用应用Western Blot检测MTA刺激HAPCs后MAPKs信号通路蛋白磷酸化水平的变化,结果显示,MTA促进ERK和p38的磷酸化,表明MTA可以激活ERK和p38信号通路。在MTA诱导HAPCs分化的同时,分别用20uM浓度的ERK抑制剂U0126和p38MAPK抑制剂SB203580处理细胞,检测ALP活性,通过real-time PCR和Western Blot分别检测DSPP及其他矿化相关基因(Runx2、BSP和OCN) mRNA的表达和Runx2蛋白的表达。结果显示,抑制ERK和p38的活性后,人根尖乳头细胞ALP活性降低；DSPP、Runx2、BSP和OCN mRNA表达水平下降；Runx2蛋白的表达量也相应降低。以上结果表明阻断ERK和p38MAPK信号通路可降低MTA诱导的HAPCs成牙本质／成骨向分化。结论MTA促进人根尖乳头细胞的成牙本质／成骨向分化；MTA能够激活HAPCs中ERK和p38信号通路；阻断ERK和p38MAPK信号通路能够抑制MTA诱导的HAPCs成牙本质／成骨向分化。更多还原

【Abstract】 AimMAPKs (mitogen-activated protein kinases) are serine/threonine-specific protein kinases that respond to extracellular stimuli (mitogens, osmotic stress, heat-shock and proinflammatory cytokines) and regulate various cellular activities, such as gene expression, mitosis, differentiation, proliferation, and cell survival/apoptosis. Human apical papilla cells (HAPCs) have the capacity of multiple differentiation, they are used for tissue engineering and biological root construction. Mineral trioxide aggregate(MTA), as a new bioactive material, has been widely used in the clinical apexification and achieved good results. However the cellular and molecular mechanisms of MTA induced differentiation of HAPCs remain unclear. The purpose of this study was to investigate the effects of MAPKs signal pathway on the differentiation of HAPCs induced by MTA.MethodsPart I Culture and differentiation of human apical papilla cellsHuman apical papilla cells were cultured by using enzyme digestion, cells were in good condition after passaged. HAPCs were cultured in osteogenic induction medium for21days. The alizarin red staining showed that calcium deposition were increased after induction compared with the control group.Part II The effects of MTA on the proliferation and differentiation of HAPCsCCK-8assay was performed to evaluate the proliferation activity of HAPCs induced by MTA of different concentrations. The results showed that MTA with10mg/ml and20mg/ml inhibited the cell proliferation significantly (p<0.05). HAPCs were cultured in a-MEM medium supplemented with MTA of different concentrations (0、0.02mg/ml、0.2mg/ml、2mg/ml).Then alkaline phosphatase (ALP) activity assay and the alizarin red staining assay were performed to detect the early and later osteogenic differentiation. The expression of odonto/osteoblastic genes such as dentin sialophosphoprotein (DSPP), runt-related transcription factor2(Runx2), bone sialoprotein (BSP), osteocalcin (OCN) and Runx2protein were assessed by real-time PCR and Western blot respectively. The results showed that the ALP activity, calcium deposition, the expression of DSPP、Runx2、BSP and OCN mRNA, the expression of Runx2protein were increased after induction by MTA compared with the control group.In summary, MTA can promote odonto/osteoblastic differentiation of HAPCs. MTA at0.2mg/ml as the optimal concentration promoted odonto/osteoblastic differentiation of HAPCs, thus MTA of0.2mg/ml was used in the later experiments.Part Ⅲ The effects of MAPKs signaling pathway on MTA induced odonto/osteoblastic differentiation of HAPCsThe phosphorylation of ERK, p38MAPK and JNK were detected by Western blot in cells treated by MTA. The results showed that MTA can activate the phosphorylation of ERK and p38MAPK.The specific ERK inhibitor U0126and the specific p38MAPK inhibitor SB203580were used to inhibit the activity of ERK and p38MAPK during the induction of MTA on HAPCs. Then the alkaline phosphatase (ALP) activity, the expression of DSPP、Runx2、BSP and OCN mRNA, the expression of Runx2protein were detected on odonto/osteoblastic differentiation of HAPCs induced by MTA combined with treatment of p38MAPK inhibitor and ERK inhibitor. The results demonstrated that the ALP activity, the expression of DSPP、Runx2、BSP and OCN mRNA, the expression of Runx2protein were decreased after inhibition of ERK and p38MAPK. In summary, inhibition of ERK and p38MAPK can reduce odonto/osteoblastic differentiation of HAPCs induced by MTA.ConclusionMTA promote odonto/osteoblastic differentiation of HAPCs. MTA can activate the phosphorylation of ERK and p38MAPK. Inhibition of ERK and p38MAPK reduce odonto/osteoblastic differentiation of HAPCs induced by MTA.更多还原