【作者】 张敏；

【导师】 吴东；

【作者基本信息】 福建医科大学 ， 口腔临床医学， 2020， 硕士

【摘要】 目的通过ERK1/2信号通路水平上调或下调以及mTOR抑制的细胞模型的建立,研究ERK1/2信号通路与自噬在细胞成骨之间的联系。方法U0126和Icariin诱导MSMSCs,抑制或激动ERK1/2信号通路,ELISA法检测磷酸化细胞外信号调节激酶(phosphorylated extracellular signal-regulated kinase,p ERK)蛋白;通过100 nM的RAPA抑制mTOR,RT-qPCR检测mTOR基因的表达,单丹磺酰尸胺(monodansylcadaverine,MDC)染色定性检测MSMSCs自噬水平;对硝基苯磷酸二钠(disodium4-nitrophenyl phosphate,pNPP)法定量检测MSMSCs中的ALP表达量;反转录-实时定量聚合酶链反应(reverse transcription-quantitative real time polymerase chain reaction,RT-qPCR)检测MSMSCs成骨相关基因RUNX2的表达与自噬基因LC3、beclin-1的表达。结果1.使MSMSCs的p ERK蛋白表达水平降低最明显的U0126的浓度是10μM,使MSMSCs的p ERK蛋白表达水平升高最明显的Icariin的浓度是800μg/L;100 nM RAPA诱导7天,成功抑制MSMSCs的mTOR的表达(p<0.05),且促进MSMSCs自噬。2.经分组处理的MSMSCs,成骨诱导后7天,根据统计学结果显示,使用激动剂Icariin组(B组)的ALP活性高于对照组(A组)(p<0.05),使用抑制剂U0126组(C组)的ALP活性低于对照组(p<0.05);使用激动剂Icariin组(B组)的自噬相关蛋白LC3基因、Beclin1基因表达高于对照组(A组)(p<0.05),使用抑制剂U0126组(C组)LC3基因、Beclin1基因表达低于对照组(p<0.05);使用激动剂Icariin组(B组)的成骨相关基因RUNX2表达高于对照组(A组)(p<0.05),使用抑制剂U0126组(C组)成骨相关基因RUNX2表达低于对照组(p<0.05)。结论上调ERK1/2信号通路水平,可激活MSMSCS自噬,促进MSMSCS成骨分化;下调ERK1/2信号通路水平,则抑制MSMSCS自噬,抑制MSMSCS成骨分化。更多还原

【Abstract】 ObjectiveTo establish a cellular model of up-regulation or down-regulation of ERK1/2signaling pathway and a cellular model of mTOR inhibition and to study the relationship between ERK1/2 signaling pathway and autophagy in cell osteogenesis.MethodU0126 and Icariin induced MSMSCs,inhibited or stimulated ERK1/2signaling pathway.ELISA was used to detect phosphorylated extracellular signal-regulated kinase(p ERK)protein;Use 100 nM RAPA to inhibit mTOR,RT-qPCR was used to detect the expression of mTOR gene,monodansylcadaverine(MDC)staining was used to detect the autophagic level of MSMSCs qualitatively;Disodium 4-nitrophenyl phosphate(pNPP)method were used to Quantitative test ALP expression level of MSMSCs;Reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR)was used to detect the expression of osteogenesis-related genes RUNX2 and autophagy genes LC3 and beclin1 inMSMSCs.Result1.The concentration of U0126 which reduced the expression level of p ERK protein of MSMSCs was 10μM,and the concentration of Icariin which increased the expression level of p ERK protein of MSMSCs was 800 ug / L;100 nM RAPA induced MSMSCSs for 7 days,successfully inhibiting the expression of mTOR inMSMSCs(p <0.05),and promote autophagy of MSMSCs.2.The MSMSCs treated by grouping showed 7 days after osteogenic induction.According to statistical results,the ALP activity in the group using the agonist Icariin(group B)was higher than that in the control group(group A)(p<0.05),the ALP activity in the group using the inhibitor U0126(group C)was lower than the control group(p<0.05);The expression of autophagy-related protein LC3 gene and Beclin1 gene in the group using the agonist Icariin(group B)was higher than that in the control group(group A)(p<0.05),the expression of LC3 gene and Beclin1 gene in the group using the inhibitor U0126(group C)was lower than the control group(p<0.05);The expression of osteogenesis-related genes RUNX2 in the group using the agonist Icariin(group B)was higher than that in the control group(group A)(p <0.05),the expression of osteogenesis-related genes RUNX2 in the group using the inhibitor U0126(group C)was lower than the control group(p <0.05).ConclusionRaise the level of ERK1/2signaling pathway can activate MSMSCS autophagy and promote osteogenic differentiation of MSMSCS;Decrease the level of ERK1/2 signaling pathway can inhibits MSMSCS autophagy and the osteogenic differentiation of MSMSCS.更多还原