BMP信号通路在多潜能干细胞向前脂肪细胞定向中作用及机制研究

发布时间：2018-01-04 04:25

本文关键词：BMP信号通路在多潜能干细胞向前脂肪细胞定向中作用及机制研究　出处：《复旦大学》2008年博士论文　论文类型：学位论文

【摘要】：肥胖的产生是由于脂肪细胞体积增大和数目增多。其中,脂肪细胞数目增多则是由于脂肪组织中多潜能干细胞向前脂肪细胞定向的增多,后者在一定的环境因素作用下分化为成熟的脂肪细胞。从多潜能干细胞发育成为脂肪细胞是一个多步骤的过程,包括多潜能干细胞向前脂肪细胞定向、前脂肪细胞增殖、生长抑制及终末分化。以前的研究主要集中于从前脂肪细胞到成熟脂肪细胞的过程,对于如何从干细胞以及多潜能干细胞向前脂肪细胞定向研究甚少。 Tang等研究表明BMP-4促进C3H10T1/2多潜能干细胞向前脂肪细胞定向,但是其下游机制并不明确,并且对于其它在多潜能干细胞向前脂肪细胞定向过程发挥作用的细胞因子了解很少。在本课题中,我们观察了BMP家族另一成员BMP-2对多潜能干细胞向前脂肪细胞定向过程的影响,并且重点研究了BMP-2和BMP-4共同参与的信号转导通路在促进多潜能干细胞向前脂肪细胞定向的作用机制,弥补了这一部分研究的空白。在研究中,我们发现BMP-2可促进C3H10T1/2多潜能干细胞向前脂肪细胞定向,并且按照标准脂肪细胞分化方案诱导后,已定向的前脂肪细胞可分化为成熟脂肪细胞,细胞由成纤维形态变为圆形,胞浆内聚集甘油三酯,脂肪细胞特异性蛋白质脂肪酸结合蛋白(422/aP2)表达。BMP-2因此成为被发现的除BMP-4以外能够促进多潜能干细胞向前脂肪细胞定向的又一重要细胞因子。为了进一步研究BMP信号通路在多潜能干细胞向前脂肪细胞定向过程中作用机制,我们观察了不同类型BMP受体对多潜能干细胞向前脂肪细胞定向的作用。首先,我们检测了C3H10T1/2多潜能干细胞内各种类型BMP受体mRNA表达水平。结果表明C3H10T1/2细胞表达BMPR-ⅠA和BMPR-Ⅱ,而BMPR-ⅠB不表达。这一结果暗示了BMP-2或BMP-4N能是通过BMPR-ⅠA发挥功能。然后,我们构建了含有持续激活BMPR-ⅠA、BMPR-ⅠB和激酶部位缺失的BMPR-ⅠA(可竞争抑制BMPR-ⅠA活性)基因的质粒,利用逆转录病毒表达系统将持续激活的BMPR-ⅠA、BMPR-ⅠB和激酶部位缺失的BMPR-ⅠA基因导入C3H10T1/2多潜能干细胞,结果表明持续激活BMPR-ⅠA和BMPR-ⅠB的细胞不需BMP诱导,直接经MDI诱导后可分化为脂肪细胞,而激酶部位缺失的BMPR-ⅠA显著抑制了BMP-2或BMP-4引起的多潜能干细胞向前脂肪细胞定向。这一系列结果证实了BMP-2和BMP-4促进多潜能干细胞向前脂肪细胞定向是通过BMPR-ⅠA实现的。 BMP受体主要可激活两条重要的信号通路,即Smad和p38 MAPK信号通路。我们检测了BMP-2和BMP-4处理分裂期的C3H10T1/2多潜能干细胞后下游信号通路激活情况,Smad和p38 MAPK信号通路均可被BMP-2和BMP-4激活。目前少量文献报导,BMP还可激活Ras/ERK信号通路。为了进一步明确BMP在多潜能干细胞向前脂肪细胞定向中作用机制,我们使用特异性化学抑制剂抑制某些信号分子活性和/或利用RNA干扰方法下调特定蛋白质,观察了Smad、p38 MAPK和ERK信号通路对BMP引起的多潜能干细胞向前脂肪细胞定向的影响。我们利用RNA干扰手段下调了C3H10T1/2多潜能干细胞内共介导Smad即Smad4的蛋白水平,从而阻断磷酸化的调节性Smad(R-Smads,Smad1/Smad5/Smad8)与之形成复合物进入细胞核内发挥功能,达到阻断BMP/Smad信号通路的目的。结果显示下调Smad4蛋白水平能明显抑制BMP-2或BMP-4引起的多潜能干细胞向前脂肪细胞定向。同时,我们还研究了p38 MAPK和ERK信号通路在其中发挥的作用。我们分别使用p38 MAPK化学抑制剂SB203580和ERK激酶MEK1的化学抑制剂PD98059预先处理C3H10T1/2多潜能干细胞,再给予细胞BMP处理,经MDI诱导后,我们发现SB203580明显抑制多潜能干细胞向脂肪细胞分化,而PD98059对此没有影响,表明p38 MAPK信号通路参与了BMP引起的多潜能干细胞向前脂肪细胞定向,而ERK信号通路在其中发挥的作用不是很明显。为排除药物副作用,我们合成了p38 RNAi,结果表明p38下调以后BMP引起的多潜能干细胞向前脂肪细胞定向被部分抑制。另外,当我们在C3H10T1/2多潜能干细胞内共同转染了Smad4和p38 RNAi时,BMP引起的多潜能干细胞向前脂肪细胞定向几乎被完全抑制。综合这些数据,我们得出了以下结论:BMP-2和BMP-4促进C3H10T1/2多潜能干细胞向前脂肪细胞定向依赖Smad和p38 MAPK两条信号通路,与ERK信号通路无关。 Smad和p38 MAPK信号通路激活后究竟调节什么基因的变化而导致多潜能干细胞向前脂肪细胞定向呢?为了更深入的研究,我们对BMP处理前后的细胞进行了基因芯片检测,发现了一系列基因的改变,其中最为显著的是bHLH家族成员Id3的变化,至于这一系列变化基因的作用有待进一步研究。
[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.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

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