本文關(guān)鍵詞：BMP信號通路在多潛能干細(xì)胞向前脂肪細(xì)胞定向中作用及機制研究　出處：《復(fù)旦大學(xué)》2008年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨形成蛋白 多潛能干細(xì)胞 前脂肪細(xì)胞 定向 分化 Smad p38 ERK

【摘要】：肥胖的產(chǎn)生是由于脂肪細(xì)胞體積增大和數(shù)目增多。其中,脂肪細(xì)胞數(shù)目增多則是由于脂肪組織中多潛能干細(xì)胞向前脂肪細(xì)胞定向的增多,后者在一定的環(huán)境因素作用下分化為成熟的脂肪細(xì)胞。從多潛能干細(xì)胞發(fā)育成為脂肪細(xì)胞是一個多步驟的過程,包括多潛能干細(xì)胞向前脂肪細(xì)胞定向、前脂肪細(xì)胞增殖、生長抑制及終末分化。以前的研究主要集中于從前脂肪細(xì)胞到成熟脂肪細(xì)胞的過程,對于如何從干細(xì)胞以及多潛能干細(xì)胞向前脂肪細(xì)胞定向研究甚少。 Tang等研究表明BMP-4促進C3H10T1/2多潛能干細(xì)胞向前脂肪細(xì)胞定向,但是其下游機制并不明確,并且對于其它在多潛能干細(xì)胞向前脂肪細(xì)胞定向過程發(fā)揮作用的細(xì)胞因子了解很少。在本課題中,我們觀察了BMP家族另一成員BMP-2對多潛能干細(xì)胞向前脂肪細(xì)胞定向過程的影響,并且重點研究了BMP-2和BMP-4共同參與的信號轉(zhuǎn)導(dǎo)通路在促進多潛能干細(xì)胞向前脂肪細(xì)胞定向的作用機制,彌補了這一部分研究的空白。 在研究中,我們發(fā)現(xiàn)BMP-2可促進C3H10T1/2多潛能干細(xì)胞向前脂肪細(xì)胞定向,并且按照標(biāo)準(zhǔn)脂肪細(xì)胞分化方案誘導(dǎo)后,已定向的前脂肪細(xì)胞可分化為成熟脂肪細(xì)胞,細(xì)胞由成纖維形態(tài)變?yōu)閳A形,胞漿內(nèi)聚集甘油三酯,脂肪細(xì)胞特異性蛋白質(zhì)脂肪酸結(jié)合蛋白(422/aP2)表達。BMP-2因此成為被發(fā)現(xiàn)的除BMP-4以外能夠促進多潛能干細(xì)胞向前脂肪細(xì)胞定向的又一重要細(xì)胞因子。 為了進一步研究BMP信號通路在多潛能干細(xì)胞向前脂肪細(xì)胞定向過程中作用機制,我們觀察了不同類型BMP受體對多潛能干細(xì)胞向前脂肪細(xì)胞定向的作用。首先,我們檢測了C3H10T1/2多潛能干細(xì)胞內(nèi)各種類型BMP受體mRNA表達水平。結(jié)果表明C3H10T1/2細(xì)胞表達BMPR-ⅠA和BMPR-Ⅱ,而BMPR-ⅠB不表達。這一結(jié)果暗示了BMP-2或BMP-4N能是通過BMPR-ⅠA發(fā)揮功能。然后,我們構(gòu)建了含有持續(xù)激活BMPR-ⅠA、BMPR-ⅠB和激酶部位缺失的BMPR-ⅠA(可競爭抑制BMPR-ⅠA活性)基因的質(zhì)粒,利用逆轉(zhuǎn)錄病毒表達系統(tǒng)將持續(xù)激活的BMPR-ⅠA、BMPR-ⅠB和激酶部位缺失的BMPR-ⅠA基因?qū)隒3H10T1/2多潛能干細(xì)胞,結(jié)果表明持續(xù)激活BMPR-ⅠA和BMPR-ⅠB的細(xì)胞不需BMP誘導(dǎo),直接經(jīng)MDI誘導(dǎo)后可分化為脂肪細(xì)胞,而激酶部位缺失的BMPR-ⅠA顯著抑制了BMP-2或BMP-4引起的多潛能干細(xì)胞向前脂肪細(xì)胞定向。這一系列結(jié)果證實了BMP-2和BMP-4促進多潛能干細(xì)胞向前脂肪細(xì)胞定向是通過BMPR-ⅠA實現(xiàn)的。 BMP受體主要可激活兩條重要的信號通路,即Smad和p38 MAPK信號通路。我們檢測了BMP-2和BMP-4處理分裂期的C3H10T1/2多潛能干細(xì)胞后下游信號通路激活情況,Smad和p38 MAPK信號通路均可被BMP-2和BMP-4激活。目前少量文獻報導(dǎo),BMP還可激活Ras/ERK信號通路。 為了進一步明確BMP在多潛能干細(xì)胞向前脂肪細(xì)胞定向中作用機制,我們使用特異性化學(xué)抑制劑抑制某些信號分子活性和/或利用RNA干擾方法下調(diào)特定蛋白質(zhì),觀察了Smad、p38 MAPK和ERK信號通路對BMP引起的多潛能干細(xì)胞向前脂肪細(xì)胞定向的影響。我們利用RNA干擾手段下調(diào)了C3H10T1/2多潛能干細(xì)胞內(nèi)共介導(dǎo)Smad即Smad4的蛋白水平,從而阻斷磷酸化的調(diào)節(jié)性Smad(R-Smads,Smad1/Smad5/Smad8)與之形成復(fù)合物進入細(xì)胞核內(nèi)發(fā)揮功能,達到阻斷BMP/Smad信號通路的目的。結(jié)果顯示下調(diào)Smad4蛋白水平能明顯抑制BMP-2或BMP-4引起的多潛能干細(xì)胞向前脂肪細(xì)胞定向。同時,我們還研究了p38 MAPK和ERK信號通路在其中發(fā)揮的作用。我們分別使用p38 MAPK化學(xué)抑制劑SB203580和ERK激酶MEK1的化學(xué)抑制劑PD98059預(yù)先處理C3H10T1/2多潛能干細(xì)胞,再給予細(xì)胞BMP處理,經(jīng)MDI誘導(dǎo)后,我們發(fā)現(xiàn)SB203580明顯抑制多潛能干細(xì)胞向脂肪細(xì)胞分化,而PD98059對此沒有影響,表明p38 MAPK信號通路參與了BMP引起的多潛能干細(xì)胞向前脂肪細(xì)胞定向,而ERK信號通路在其中發(fā)揮的作用不是很明顯。為排除藥物副作用,我們合成了p38 RNAi,結(jié)果表明p38下調(diào)以后BMP引起的多潛能干細(xì)胞向前脂肪細(xì)胞定向被部分抑制。另外,當(dāng)我們在C3H10T1/2多潛能干細(xì)胞內(nèi)共同轉(zhuǎn)染了Smad4和p38 RNAi時,BMP引起的多潛能干細(xì)胞向前脂肪細(xì)胞定向幾乎被完全抑制。綜合這些數(shù)據(jù),我們得出了以下結(jié)論:BMP-2和BMP-4促進C3H10T1/2多潛能干細(xì)胞向前脂肪細(xì)胞定向依賴Smad和p38 MAPK兩條信號通路,與ERK信號通路無關(guān)。 Smad和p38 MAPK信號通路激活后究竟調(diào)節(jié)什么基因的變化而導(dǎo)致多潛能干細(xì)胞向前脂肪細(xì)胞定向呢?為了更深入的研究,我們對BMP處理前后的細(xì)胞進行了基因芯片檢測,發(fā)現(xiàn)了一系列基因的改變,其中最為顯著的是bHLH家族成員Id3的變化,至于這一系列變化基因的作用有待進一步研究。
[Abstract]:The emergence of obesity is due to increased fat cell size and number increase. The increase in the number of fat cells is due to the increase in adipose tissue stem cells to the adipocyte determination, the effect of certain environmental factors to differentiate into mature fat cells. The development of mesenchymal stem cells into fat cells is a process multiple steps, including stem cells to the adipocyte determination, preadipocyte proliferation, growth arrest and terminal differentiation. Previous studies have focused on the former fat cells to mature adipocytes, how from stem cells and pluripotent stem cells to adipocytes oriented research is little.
Tang study showed that BMP-4 promotes C3H10T1/2 stem cells to the adipocyte determination, but its downstream mechanism is not clear, and for other pluripotent stem cells in fat cells plays forward directional cytokines understand very little. In this study, we observed the effect of another member of the BMP family BMP-2 of pluripotent stem cells forward fat cell orientation process, and focus on the BMP-2 and BMP-4 are involved in the signal transduction pathway in the mechanism of promoting stem cells to the adipocyte determination, filling the gap in this part of the study.
In the study, we found that BMP-2 can promote C3H10T1/2 stem cells to the adipocyte determination, and in accordance with the standard induction of adipocyte differentiation scheme, preadipocytes has been directed to differentiate into mature fat cells, cells by fibroblast morphology became round, intracellular triglyceride accumulation, adipocyte specific protein fatty acid the expression of.BMP-2 binding protein (422/aP2) as another important cell factor was found except BMP-4 can promote pluripotent stem cells to adipocytes oriented.
In order to further study the BMP signaling pathway in pluripotent stem cells forward mechanism of adipocyte determination process, we observed the different types of BMP receptors on pluripotent stem cells to adipocytes directional role. First, we examined the C3H10T1/2 expression of various types of pluripotent stem BMP receptor mRNA level in cells. The results showed that the expression of C3H10T1/2 cells A BMPR- I and BMPR- II, and BMPR- I B expression. This result suggests that BMP-2 or BMP-4N can function by BMPR- I A. Then, we have constructed a sustained activation of BMPR- I A, BMPR- I A I B and BMPR- kinase deletion (site can be competitive inhibition activity of A gene BMPR- 1) the plasmid, BMPR- I A by retroviral expression system will be continuously activated, BMPR- I and B I A BMPR- kinase deletion site C3H10T1/2 gene into pluripotent stem cells, results show that the activation of BMPR- I A and B MPR- of B cells could be induced by BMP, directly induced by MDI can differentiate into fat cells, and BMPR- I A kinase site deletion significantly inhibited BMP-2 or BMP-4 induced pluripotent stem cells directed forward fat cells. These results confirmed that BMP-2 and BMP-4 promote pluripotent stem cells to fat cells I was achieved by BMPR- A.
BMP receptor mainly activates two pathways important, namely Smad and p38 MAPK signaling pathways. We detected the BMP-2 and BMP-4 processing division phase C3H10T1/2 pluripotent stem cells after activation of downstream signaling pathways of p38, Smad and MAPK signaling pathway can be activated. BMP-2 and BMP-4 present some reports, BMP can also activation of Ras/ERK signaling pathway.
In order to further clarify the mechanism of BMP in pluripotent stem cells, fat cells in the forward orientation, we use a specific chemical inhibitor activity of some signal molecules and / or by using the RNA interference method for down-regulation of specific proteins, the effects of Smad, p38 MAPK and ERK signaling pathway on BMP induced pluripotent stem cells to adipocytes directional effects. We use RNA interference means cut C3H10T1/2 pluripotent stem cells were mediated by Smad protein level of Smad4, thereby blocking the regulatory phosphorylation of Smad (R-Smads, Smad1/Smad5/Smad8) formed a complex into the nucleus and achieve the function, blocking the BMP/Smad signaling pathway to pluripotency. The results showed that the down-regulation of Smad4 protein level significantly inhibited BMP-2 induced or BMP-4 stem cells to adipocytes orientation. At the same time, we also studied the p38 MAPK and ERK signal pathway in the hair The essential role. We use the p38 MAPK chemical inhibitors of PD98059 chemical inhibitors of SB203580 and ERK kinase MEK1 respectively pretreatment C3H10T1/2 pluripotent stem cells, and then given BMP cells, induced by MDI, we found that SB203580 significantly inhibited the pluripotent stem cells to differentiate into fat cells, while PD98059 had no effect, showed that the p38 MAPK signal pathway involved in BMP induced pluripotent stem cells on fat cell orientation, and ERK signal pathway in which the role is not obvious. In order to eliminate the side effects of the drug, we synthesized p38 RNAi, results show that the potential of p38 cut after BMP induced stem cells to adipocytes orientation was partially inhibited. In addition, when we are in C3H10T1/2 are multipotent stem cells in CO transfected Smad4 and p38 RNAi, BMP induced pluripotent stem cells to adipocytes was almost completely inhibited. The comprehensive orientation of this These data suggest that BMP-2 and BMP-4 promote C3H10T1/2 pluripotent stem cells to be adipocytes dependent on Smad and p38 MAPK two pathways, and are independent of ERK signaling pathway.
The changes of Smad and p38 MAPK signaling pathway gene regulation after what what caused pluripotent stem cells to adipocytes directed? In order to further research on BMP, we treated the cells of gene chip detection, found a series of genes, the most obvious change is a member of the bHLH family Id3, as a series of changes in gene function needs further study.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R329

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相關(guān)期刊論文 前1條

1 王茸影,易靜;骨形成蛋白調(diào)控成骨分化的信號機制[J];生命科學(xué);2005年01期

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本文編號：1377024