本文選題：神經(jīng)肽P物質(zhì) + 骨髓間充質(zhì)干細(xì)胞　； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：第一部分神經(jīng)肽P物質(zhì)對(duì)ST2細(xì)胞增殖分化的影響目的:初步探討神經(jīng)肽P物質(zhì)(Neuropeptide substance,SP)對(duì)ST2細(xì)胞成骨分化過(guò)程中增殖和分化的影響,并篩選出合適的促增殖分化的SP作用濃度和時(shí)間。方法:將第三代(P3)ST2以5×103/孔鋪板于96孔板,用含有不同濃度SP(10-10Mol/L、10-8Mol/L、10-6Mol/L、10-5Mol/L)的成骨條件培養(yǎng)基(Osteoblast Medium,OBM)進(jìn)行刺激,以僅含2%胎牛血清(FBS)的OBM作為對(duì)照組(control),每組設(shè)5個(gè)復(fù)孔和1個(gè)調(diào)零孔。分別以SP刺激ST2細(xì)胞24、48、72h,CCK-8檢測(cè)細(xì)胞增殖活性,篩選出合適的藥物濃度。然后取第三代(P3)的ST2按8×104/孔密度鋪板于6孔板,加入含有10-6Mol/L濃度SP 的 OBM,培養(yǎng) 1,3,5,7 天,酶聯(lián)免疫吸附實(shí)驗(yàn)(enzyme-linked immune sorbent assay,ELISA)檢測(cè)細(xì)胞培養(yǎng)上清液中堿性磷酸酶(alkaline phosphatase,ALP)、Ⅰ型膠原蛋白(CollaⅠ)和骨鈣素(osteocalcinv,OCN)的表達(dá),實(shí)驗(yàn)重復(fù)3次。并采用免疫熒光染色檢測(cè)細(xì)胞ALP的活性。結(jié)果:(1)神經(jīng)肽 P 物質(zhì)(Neuropeptide substance,SP)可促進(jìn)小鼠 BMSCs(ST2細(xì)胞)的增殖活性,且與SP的濃度和作用時(shí)間有一定的相關(guān)關(guān)系,當(dāng)SP濃度為10-6Mol/L時(shí),細(xì)胞增殖效應(yīng)最明顯。(2)神經(jīng)肽 P 物質(zhì)(Neuropeptide substance,SP)可促進(jìn)小鼠 BMSCs(ST2細(xì)胞)的成骨分化,早期成骨分化標(biāo)記物ALP的表達(dá)在第5天時(shí)達(dá)峰值,中晚期標(biāo)志物Colla Ⅰ、OCN的表達(dá)在第7天時(shí)最高。SP可增強(qiáng)ST2細(xì)胞ALP的活性。結(jié)論:yL經(jīng)肽 P 物質(zhì)(Neuropeptide substance,SP)可促進(jìn)小鼠 BMSCs(ST2 細(xì)胞)的增殖和成骨分化,該效應(yīng)具有劑量依賴性和時(shí)間依賴性。第二部分BMP信號(hào)通路對(duì)SP促ST2細(xì)胞成骨分化過(guò)程的調(diào)控作用研究目的:探討B(tài)MP信號(hào)通路在SP促ST2細(xì)胞成骨分化過(guò)程的作用。方法:取P3的ST2以8×104/孔的密度鋪板于6孔板,設(shè)3個(gè)復(fù)孔。實(shí)驗(yàn)分組為SP 組:10-6Mol/L SP;SP+BMP 通路抑制劑(Noggin)組:10-6Mol/L SP 和 100ng/ml Noggin;抑制劑組:100ng/mlNoggin;對(duì)照組:等體積2%FBS,培養(yǎng)5天或7天后采用ELISA法檢測(cè)細(xì)胞上清液中ALP,ColIa Ⅰ和OCN的表達(dá)量,并采用免疫熒光染色檢測(cè)細(xì)胞的ALP活性。結(jié)果:(1)SP可明顯促進(jìn)ST2細(xì)胞成骨分化標(biāo)記物ALP、ColIa Ⅰ和OCN的表達(dá),加入BMP信號(hào)通路抑制劑Noggin后,標(biāo)記物的表達(dá)顯著減少,且僅加入Noggin也可抑制ALP、ColIa Ⅰ和OCN的表達(dá)。(2)免疫熒光染色結(jié)果顯示SP可促進(jìn)ST2細(xì)胞ALP的表達(dá)活性,加入BMP信號(hào)通路抑制劑Noggin后,ALP活性受到抑制,單純加入Noggin也可抑制細(xì)胞ALP的活性表達(dá)。結(jié)論:SP可促進(jìn)ST2的成骨分化和ALP活性表達(dá),而B(niǎo)MP信號(hào)通路抑制劑Noggin可抑制其效應(yīng)的發(fā)揮,表明BMP信號(hào)通路可能在SP促ST2細(xì)胞成骨分化中產(chǎn)生了一定的調(diào)節(jié)作用。
[Abstract]:Part I effects of neuropeptide substance P on proliferation and differentiation of ST2 cells objective: to investigate the effects of neuropeptide substance P on proliferation and differentiation of ST2 cells during osteogenic differentiation. The appropriate SP concentration and time for proliferation and differentiation were selected. Methods: the third generation P3CST2 was incubated with 5 脳 10 3 / hole plate on 96 well plate. The osteogenic condition medium containing 10 ~ (-10) mol / L ~ (-10 ~ (-8) mol / L ~ (10 ~ (-6) mol / L) was used to stimulate the osteoblast medium. The OBM containing only 2% fetal bovine serum (FBS) was used as the control. Each group was given five multiple holes and one zero adjusting hole. The proliferation activity of ST2 cells stimulated by SP was determined by CCK-8 at 24 ~ 48 ~ 72 h, and the appropriate concentration of CCK-8 was screened out. Then the ST2 of the third generation of P3 was added to the 6-hole plate at the density of 8 脳 10 4 / pore, and the 10-6Mol/L containing SP was added to the plate, and cultured for 5 days. Enzyme linked immune sorbent Assay (Elisa) assay was used to detect the expression of alkaline phosphatase (ALP) and osteocalcinvan (OCNs) in the supernatant of cell culture. The activity of ALP was detected by immunofluorescence staining. Results (1) Neuropeptide substance P (Neuropeptide substance1) could promote the proliferation of mouse BMSCs(ST2 cells, which was related to the concentration of SP and the time of action. When SP concentration was 10-6Mol/L, The proliferation of BMSCs(ST2 cells was enhanced by neuropeptide substance P (NPP), and the expression of ALP, the marker of early osteogenic differentiation, reached its peak on the 5th day. The expression of Colla 鈪，

本文編號(hào)：1841210