發(fā)布時(shí)間：2018-10-21 12:55

【摘要】：[目的]研究高糖狀態(tài)下,體外培養(yǎng)的小鼠前成骨細(xì)胞系(MC3T3-E1)的細(xì)胞增殖、分化、細(xì)胞外礦物質(zhì)沉積的情況以及葡萄糖濃度對(duì)P38MAPK信號(hào)通路的影響,進(jìn)一步探討P38MAPK特異性抑制劑SB203580對(duì)高糖所導(dǎo)致的成骨細(xì)胞活性改變的影響,以闡明P38MAPK信號(hào)通路在高糖狀態(tài)下成骨細(xì)胞活性中的作用。[方法]將培養(yǎng)的小鼠前成骨細(xì)胞系(MC3T3-E1)分為8組：1)正常對(duì)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(xì)胞接種后,待細(xì)胞生長(zhǎng)至80%密度后,同步化24小時(shí)后,分別用以下各因素處理細(xì)胞并繼續(xù)培養(yǎng)。1、甲基噻唑基四唑(MTT)法檢測(cè)3、7、14d MC3T3-El細(xì)胞增殖情況；2、堿性磷酸酶(ATP)試劑盒檢測(cè)3d、7d細(xì)胞裂解液中ALP活性；3、茜素紅染色觀察細(xì)胞細(xì)胞礦化結(jié)節(jié)的大小和形態(tài)；4、熒光實(shí)時(shí)定量PCR檢測(cè)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(dá)。收集細(xì)胞,提取總RNA,逆轉(zhuǎn)錄cDNA,采用實(shí)時(shí)熒光定量PCR法檢測(cè)各組成骨細(xì)胞中成骨相關(guān)基因的表達(dá)。5、Western blot檢測(cè)P38MAPK和磷酸化P38MAPK在3d、7d的表達(dá)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(xì)胞的增殖。且經(jīng)過(guò)SB203580干預(yù)后,細(xì)胞增殖水平繼續(xù)下降,差異具有統(tǒng)計(jì)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(xì)胞堿性磷酸酶(ALP)活性、細(xì)胞礦化和成骨相關(guān)基因的表達(dá)。SB203580干預(yù)后,ALP活性、細(xì)胞礦化及成骨相關(guān)基因(Cbfal和OCN)表達(dá)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(duì)P38MAPK,總蛋白分泌無(wú)明顯的作用,但能夠促進(jìn)P-P38MAPK的表達(dá)且成劑量相關(guān)關(guān)系,經(jīng)過(guò)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(xì)胞明顯增殖異常和礦化功能下降,并且激活P38MAPK信號(hào)傳導(dǎo)。2、P38MAPK特異性抑制劑SB203580,可阻斷P38MAPK信號(hào)傳導(dǎo)通路,導(dǎo)致ALP活性和成骨相關(guān)基因的表達(dá)增加。3、高糖所導(dǎo)致的小鼠成骨細(xì)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(hào)：2285149