P38MAPK信号通路介导高糖状态对MC3T3-E1细胞影响的作用机制研究
发布时间：2018-10-21 12:55
【摘要】：[目的]研究高糖状态下,体外培养的小鼠前成骨细胞系(MC3T3-E1)的细胞增殖、分化、细胞外矿物质沉积的情况以及葡萄糖浓度对P38MAPK信号通路的影响,进一步探讨P38MAPK特异性抑制剂SB203580对高糖所导致的成骨细胞活性改变的影响,以阐明P38MAPK信号通路在高糖状态下成骨细胞活性中的作用。[方法]将培养的小鼠前成骨细胞系(MC3T3-E1)分为8组：1)正常对照组5.5mM葡萄糖；2)高糖组A：15.5mM葡萄糖；3)高糖组B：25.5mM葡萄糖；4)高糖组C：35.5mM葡萄糖；5)SB203580组A：5.5mM葡萄糖+10gM SB203580；6)SB203580组B：15.5mM葡萄糖+10μMSB203580;7)SB203580组C：25.5mM葡萄糖+10μMSB203580；8)SB203580组D：35.5mM葡萄糖+10μM SB203580。细胞接种后,待细胞生长至80%密度后,同步化24小时后,分别用以下各因素处理细胞并继续培养。1、甲基噻唑基四唑(MTT)法检测3、7、14d MC3T3-El细胞增殖情况；2、碱性磷酸酶(ATP)试剂盒检测3d、7d细胞裂解液中ALP活性；3、茜素红染色观察细胞细胞矿化结节的大小和形态；4、荧光实时定量PCR检测核心结合因子1(core binding factor-1, Cbfal)和骨钙蛋白基因(osteocalcin, OCN)分别在培养3d、7d的表达。收集细胞,提取总RNA,逆转录cDNA,采用实时荧光定量PCR法检测各组成骨细胞中成骨相关基因的表达。5、Western blot检测P38MAPK和磷酸化P38MAPK在3d、7d的表达情况。[结果]1、与低浓度葡萄糖相比,高糖组(≥15.5mM)可抑制MC3T3-E1细胞的增殖。且经过SB203580干预后,细胞增殖水平继续下降,差异具有统计学意义。(P0.05)。2、与低浓度葡萄糖相比,高糖组(≥15.5mM)可抑制MC3T3-E1细胞碱性磷酸酶(ALP)活性、细胞矿化和成骨相关基因的表达。SB203580干预后,ALP活性、细胞矿化及成骨相关基因(Cbfal和OCN)表达水平较未干预组升高。3、与低浓度葡萄糖相比,高糖组(≥15.5mmM)对P38MAPK,总蛋白分泌无明显的作用,但能够促进P-P38MAPK的表达且成剂量相关关系,经过SB203580干预后,可明显抑制P38MAPK磷酸化。(P0.05)[结论]1、高糖可以导致MC3T3-E1细胞明显增殖异常和矿化功能下降,并且激活P38MAPK信号传导。2、P38MAPK特异性抑制剂SB203580,可阻断P38MAPK信号传导通路,导致ALP活性和成骨相关基因的表达增加。3、高糖所导致的小鼠成骨细胞功能抑制可能与P38MAPK信通路有关
[Abstract]:[objective] to study the proliferation, differentiation, extracellular mineral deposition and the effect of glucose concentration on P38MAPK signaling pathway of mouse proosteoblast cell line (MC3T3-E1) cultured in vitro under high glucose condition. To further investigate the effect of P38MAPK specific inhibitor SB203580 on the changes of osteoblast activity induced by high glucose in order to elucidate the role of P38MAPK signaling pathway in osteoblast activity under high glucose condition. [methods] Mouse proosteoblast cell line (MC3T3-E1) was divided into 8 groups: 1) 5.5mM glucose in normal control group, 2) A:15.5mM glucose in high glucose group, 3) B:25.5mM glucose in high glucose group, 4) C:35.5mM glucose in high glucose group. 5) SB203580 group A:5.5mM glucose 10gM SB203580;6) SB203580 group B:15.5mM glucose 10 渭 MSB203580;7) SB203580 group C:25.5mM glucose 10 渭 MSB203580;8) SB203580 group D:35.5mM 10 渭 M SB203580. After inoculation, the cells grew to 80% density, then synchronized for 24 hours, then the cells were treated with the following factors and cultured continuously. 1. The proliferation of MC3T3-El cells was detected by methylthiazolyl tetrazole (MTT) assay. 2Alkaline phosphatase (ATP) kit was used to detect the activity of ALP in the cell lysate for 3 days, and the size and morphology of the mineralized nodules were observed by alizarin red staining. 4. The expression of core binding factor 1 (core binding factor-1, Cbfal) and osteocalcin gene (osteocalcin, OCN) were detected by real-time fluorescence quantitative PCR. The expression of osteoblast-associated genes in osteoblasts was detected by real-time fluorescence quantitative PCR, and the expression of P38MAPK and phosphorylated P38MAPK was detected by Western blot. [results] 1. Compared with low glucose concentration, high glucose group (鈮，
本文编号：2285149
[Abstract]:[objective] to study the proliferation, differentiation, extracellular mineral deposition and the effect of glucose concentration on P38MAPK signaling pathway of mouse proosteoblast cell line (MC3T3-E1) cultured in vitro under high glucose condition. To further investigate the effect of P38MAPK specific inhibitor SB203580 on the changes of osteoblast activity induced by high glucose in order to elucidate the role of P38MAPK signaling pathway in osteoblast activity under high glucose condition. [methods] Mouse proosteoblast cell line (MC3T3-E1) was divided into 8 groups: 1) 5.5mM glucose in normal control group, 2) A:15.5mM glucose in high glucose group, 3) B:25.5mM glucose in high glucose group, 4) C:35.5mM glucose in high glucose group. 5) SB203580 group A:5.5mM glucose 10gM SB203580;6) SB203580 group B:15.5mM glucose 10 渭 MSB203580;7) SB203580 group C:25.5mM glucose 10 渭 MSB203580;8) SB203580 group D:35.5mM 10 渭 M SB203580. After inoculation, the cells grew to 80% density, then synchronized for 24 hours, then the cells were treated with the following factors and cultured continuously. 1. The proliferation of MC3T3-El cells was detected by methylthiazolyl tetrazole (MTT) assay. 2Alkaline phosphatase (ATP) kit was used to detect the activity of ALP in the cell lysate for 3 days, and the size and morphology of the mineralized nodules were observed by alizarin red staining. 4. The expression of core binding factor 1 (core binding factor-1, Cbfal) and osteocalcin gene (osteocalcin, OCN) were detected by real-time fluorescence quantitative PCR. The expression of osteoblast-associated genes in osteoblasts was detected by real-time fluorescence quantitative PCR, and the expression of P38MAPK and phosphorylated P38MAPK was detected by Western blot. [results] 1. Compared with low glucose concentration, high glucose group (鈮，
本文编号：2285149
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