本文選題：17-β雌二醇 + MC3T3-E1細(xì)胞��； 參考：《山東大學(xué)》2014年碩士論文

【摘要】：背景： 雌激素在骨改建中發(fā)揮重要作用,雌激素缺乏可以引起全身性骨質(zhì)疏松。雌激素通過(guò)誘導(dǎo)成骨細(xì)胞增殖和抑制破骨細(xì)胞活性促進(jìn)骨形成和抑制骨吸收,以增加骨量。相鄰細(xì)胞間縫隙連接蛋白構(gòu)成的縫隙連接介導(dǎo)的細(xì)胞間信號(hào)通路,使骨骼細(xì)胞在接受胞外刺激后產(chǎn)生的信號(hào)分子在相關(guān)細(xì)胞中迅速傳遞,使相關(guān)細(xì)胞群形成一功能整體。前期實(shí)驗(yàn)對(duì)MC3T3-E1細(xì)胞施力10-8mol/L17-β雌二醇作用5d后,應(yīng)用全基因組基因芯片分析發(fā)現(xiàn)縫隙連接信號(hào)通路Gja1因子表達(dá)明顯增強(qiáng),提示縫隙連接可能在成骨細(xì)胞對(duì)雌激素反應(yīng)的信號(hào)調(diào)控中起重要作用。因此,探討雌激素作用下縫隙連接細(xì)胞間通訊對(duì)成骨細(xì)胞作用機(jī)制,可以為提高骨創(chuàng)傷修復(fù)質(zhì)量及牙槽骨改建塑形以及相關(guān)骨疾病的治療提供理論依據(jù)。 目的： 本實(shí)驗(yàn)在前期研究的基礎(chǔ)上通過(guò)18a-甘草次酸(18a-glycyrrhetinic acid,AGA)抑制縫隙連接信號(hào)通路中的關(guān)鍵因子縫隙連接蛋白43,結(jié)合MTT、ALP、礦化結(jié)節(jié)染色、鈣離子濃度測(cè)定、Runx2蛋白和mRNA測(cè)定,探討雌激素作用下縫隙連接細(xì)胞間通訊對(duì)成骨細(xì)胞作用機(jī)制,并針對(duì)這個(gè)機(jī)制和過(guò)程采取更有效的應(yīng)對(duì)策略,為提高骨創(chuàng)傷修復(fù)質(zhì)量及牙槽骨改建塑形以及相關(guān)骨疾病的治療開(kāi)辟新的更廣闊的前景。 方法： 1.體外培養(yǎng)小鼠MC3T3-E1細(xì)胞。實(shí)驗(yàn)分為四組即：空白對(duì)照組：AGA組(30μmol/L);17-βP雌二醇(10-8mol/L);17-βP雌二醇(10-8mol/L)+AGA組(30μmol/L)。 2.應(yīng)用MTT法和ALP法分別檢測(cè)四組細(xì)胞進(jìn)行相應(yīng)處理后的增殖能力及細(xì)胞分化活性。 3.茜素紅染色及鈣離子濃度測(cè)定四組細(xì)胞分化成骨能力。 4.運(yùn)用RT-PCR及免疫印記Western blot檢測(cè)四組細(xì)胞進(jìn)行相應(yīng)處理后的Runx2蛋白及mRNA表達(dá)水平。 5.采用SPSS17.0對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析。 結(jié)果： 1.MC3T3-E1成骨樣細(xì)胞生長(zhǎng)狀況良好。 2.細(xì)胞增殖情況：17-β雌二醇組與空白對(duì)照組相比,細(xì)胞增殖活性增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；加入AGA后,不論是17-β雌二醇組,還是非17-β雌二醇組,細(xì)胞增殖活性均明顯降低(P0.05)。 3.細(xì)胞堿性磷酸酶表達(dá)情況：3天組：與空白對(duì)照組相比,17-β雌二醇可增加MC3T3-E1細(xì)胞堿性磷酸酶活性(P0.05),而5天組,17-p雌二醇增加MC3T3-E1細(xì)胞堿性磷酸酶活性的作用不明顯(P0.05)；加入AGA后,不論是3天組,還是5天組,細(xì)胞增殖活性均明顯降低(P0.05)；5天組與3天組相比,隨時(shí)間延長(zhǎng),各組細(xì)胞堿性磷酸酶活性均明顯增高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 4.鈣化結(jié)節(jié)茜素紅染色：肉眼及倒置相差顯微鏡下可見(jiàn),礦化結(jié)節(jié)數(shù)量：17-β雌二醇組空白對(duì)照組17-β雌二醇+AGA組AGA組。 鈣離子濃度：17-β雌二醇組空白對(duì)照組,17-β雌二醇+AGA組AGA組,有統(tǒng)計(jì)學(xué)差異(P0.05)；加入AGA后,不論17-β雌二醇組,還是非17-β雌二醇組,鈣離子濃度均明顯降低(P0.05)。 5.實(shí)時(shí)定量RT-PCR檢測(cè)成骨分化相關(guān)基因Runx2mRNA表達(dá)水平：結(jié)果顯示,加入AGA后,不論是在17-β雌二醇下,還是空白對(duì)照組,Runx2mRNA表達(dá)水平均明顯降低(P0.05)；17-β雌二醇組與空白對(duì)照組相比,Runx2mRNA表達(dá)上調(diào)約1.2倍,有統(tǒng)計(jì)學(xué)意義(P0.05)；17-β雌二醇+AGA聯(lián)合刺激組與僅AGA刺激組相比,Runx2mRNA表達(dá)增加上調(diào)兩倍,有統(tǒng)計(jì)學(xué)意義(P0.05)。 6. Runx2蛋白表達(dá)情況：加入AGA后,不論是否在17-β雌二醇作用下,Runx2蛋白表達(dá)水平均明顯降低(P0.05)；17-β雌二醇組與空白對(duì)照組相比,Runx2蛋白表達(dá)上調(diào),有統(tǒng)計(jì)學(xué)意義(P0.05)；17-β雌二醇+AGA聯(lián)合刺激組與僅加入AGA刺激組相比,Runx2蛋白表達(dá)量上調(diào),有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論： 1.適宜濃度及作用時(shí)間的17-β雌二醇可以通過(guò)Cx43介導(dǎo)的縫隙連接信號(hào)通路調(diào)節(jié)MC3T3-E1成骨樣細(xì)胞的增殖及分化,促進(jìn)細(xì)胞外基質(zhì)礦化。 2.縫隙連接信號(hào)通路是介導(dǎo)適宜雌激素作用下調(diào)節(jié)成骨細(xì)胞活性的有效通路之一,其具體調(diào)控機(jī)制還有待于進(jìn)一步研究。
[Abstract]:Background:
Estrogen plays an important role in bone remodeling. Estrogen deficiency can cause systemic osteoporosis. Estrogen can increase bone formation and inhibit bone absorption by inducing osteoblast proliferation and inhibiting osteoclast activity, in order to increase bone mass. Intercellular signaling pathway mediated by gap junctions of connexin between adjacent cells makes bone The signal molecules produced by external stimulation of the iliac cells are transmitted rapidly in the related cells to make the related cell group form a functional whole. After the preliminary experiment on the effect of the action of 10-8mol/L17- beta estradiol on MC3T3-E1 cells, the expression of Gja1 factor in the gap junction signal pathway was obviously enhanced by the whole genome microarray analysis. Gap junctions may play an important role in the regulation of estrogen response signals in osteoblasts. Therefore, the study of the mechanism of gap junctional intercellular communication on osteoblasts under the action of estrogen can provide a theoretical basis for improving the quality of bone trauma repair, the remodeling of alveolar bone and the treatment of related bone diseases.
Objective:
In this experiment, the key factor of gap junction protein 43 in gap junction signaling pathway was inhibited by 18a- glycyrrhizic acid (18a-glycyrrhetinic acid, AGA), combined with MTT, ALP, mineralized nodules, determination of calcium ion concentration, Runx2 protein and mRNA, and the discussion of intercellular communication between gap junctions under estrogen action to bone formation. In order to improve the quality of bone repair and the remodeling of alveolar bone and the treatment of bone disease, the mechanism and process of the mechanism and process are more effective.
Method錛，

本文編號(hào)：1875098