【摘要】：[目的]研究高糖狀態(tài)下,體外培養(yǎng)的小鼠前成骨細胞系(MC3T3-E1)的細胞增殖、分化、細胞外礦物質(zhì)沉積的情況以及葡萄糖濃度對P38MAPK信號通路的影響,進一步探討P38MAPK特異性抑制劑SB203580對高糖所導(dǎo)致的成骨細胞活性改變的影響,以闡明P38MAPK信號通路在高糖狀態(tài)下成骨細胞活性中的作用。[方法]將培養(yǎng)的小鼠前成骨細胞系(MC3T3-E1)分為8組：1)正常對照組5.5mM葡萄糖；2)高糖組A：15.5mM葡萄糖；3)高糖組B：25.5mM葡萄糖；4)高糖組C：35.5mM葡萄糖；5)SB203580組A：5.5mM葡萄糖+10gM SB203580；6)SB203580組B：15.5mM葡萄糖+10μMSB203580;7)SB203580組C：25.5mM葡萄糖+10μMSB203580；8)SB203580組D：35.5mM葡萄糖+10μM SB203580。細胞接種后,待細胞生長至80%密度后,同步化24小時后,分別用以下各因素處理細胞并繼續(xù)培養(yǎng)。1、甲基噻唑基四唑(MTT)法檢測3、7、14d MC3T3-El細胞增殖情況；2、堿性磷酸酶(ATP)試劑盒檢測3d、7d細胞裂解液中ALP活性；3、茜素紅染色觀察細胞細胞礦化結(jié)節(jié)的大小和形態(tài)；4、熒光實時定量PCR檢測核心結(jié)合因子1(core binding factor-1, Cbfal)和骨鈣蛋白基因(osteocalcin, OCN)分別在培養(yǎng)3d、7d的表達。收集細胞,提取總RNA,逆轉(zhuǎn)錄cDNA,采用實時熒光定量PCR法檢測各組成骨細胞中成骨相關(guān)基因的表達。5、Western blot檢測P38MAPK和磷酸化P38MAPK在3d、7d的表達情況。[結(jié)果]1、與低濃度葡萄糖相比,高糖組(≥15.5mM)可抑制MC3T3-E1細胞的增殖。且經(jīng)過SB203580干預(yù)后,細胞增殖水平繼續(xù)下降,差異具有統(tǒng)計學(xué)意義。(P0.05)。2、與低濃度葡萄糖相比,高糖組(≥15.5mM)可抑制MC3T3-E1細胞堿性磷酸酶(ALP)活性、細胞礦化和成骨相關(guān)基因的表達。SB203580干預(yù)后,ALP活性、細胞礦化及成骨相關(guān)基因(Cbfal和OCN)表達水平較未干預(yù)組升高。3、與低濃度葡萄糖相比,高糖組(≥15.5mmM)對P38MAPK,總蛋白分泌無明顯的作用,但能夠促進P-P38MAPK的表達且成劑量相關(guān)關(guān)系,經(jīng)過SB203580干預(yù)后,可明顯抑制P38MAPK磷酸化。(P0.05)[結(jié)論]1、高糖可以導(dǎo)致MC3T3-E1細胞明顯增殖異常和礦化功能下降,并且激活P38MAPK信號傳導(dǎo)。2、P38MAPK特異性抑制劑SB203580,可阻斷P38MAPK信號傳導(dǎo)通路,導(dǎo)致ALP活性和成骨相關(guān)基因的表達增加。3、高糖所導(dǎo)致的小鼠成骨細胞功能抑制可能與P38MAPK信通路有關(guān)
[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，

本文編號：2285149