【作者】 宋檀婧；

【导师】 汤其群； 宋后燕； 马端； 黄海艳；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2008， 博士

【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。更多还原

【Abstract】 The increase of adipose tissue mass associated with obesity is due in part to an increase in the number of adipocytes (hyperplasia). This hyperplasia results from the recruitment of pluripotent stem cells present in the vascular stroma of adipose tissue. These stem cells have the capacity to undergo commitment to adipose, muscle, bone or cartilage lineages.The development of mesenchymal stem cells into terminally differentiated adipocytes can be divided into four stages: lineage commitment, preadipocyte proliferation, growth arrest and terminal differentiation. Many of the key players in the latter phases of the adipocyte development program, i.e., mitotic clonal expansion and terminal differentiation, have been well characterized. However, little is known about the factors/genes that trigger the commitment process. The goal of our research is to define the role of BMP signaling pathway during the commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage.In this article, we firstly report that bone morphogenetic protein 2 (BMP-2), as well as BMP-4, can induce the commitment of C3H10T1/2 cells to preadipocytes that, when subjected to an adipocyte differentiation protocol (MDI), develop into cells of the adipocyte phenotype.BMPs exhibit their biological effects through the sequential activation of two types of transmembrane receptors, namely BMPR-I and BMPR-II, which possess intrinsic serine/threonine kinase activity. BMPR-I is composed of many subtypes, of which BMPR-IA and BMPR-IB are downstream of BMP-2 and BMP-4. In this article, we examined the expression of BMP receptors in C3H10T1/2 stem cells by RT-PCR. Data show that C3H10T1/2 cells express BMPR-IA and BMPR-II but not BMPR-IB.A variety of studies indicate that BMPR-IA and BMPR-IB may transmit different signals during the specification and differentiation of mesenchymal lineages. To investigate the role of BMPR-IA and BMPR-IB in the BMP-2 or BMP-4 signaling pathways in C3H10T1/2, we constructed constitutively active BMPR-IA (Ca-BMPR-IA), constitutively active BMPR-IB (Ca-BMPR-IB), dominant-negative BMPR-IA (DN-BMPR-IA) plasmids and then transferred them into C3H10T1/2 by using a retroviral system. The results showed that C3H10T1/2 cells that overexpressed Ca-BMPR-IA and Ca-BMPR-IB underwent differentiation into adipocyes after MDI induction, while overexpression of DN-BMPR-IA decreased the proportion of adipocyte induced by BMP-2 or BMP-4 and subsequent MDI. Since BMPR-IB is absent in this cell line, above data show that BMP-2 or BMP-4 induces the commitment of C3H10T1/2 stem cells to the adipocyte lineage through BMPR-IA.To define which signal pathway in the downstream of BMP-2 or BMP-4 is responsible for the commitment process, we blocked Smad, p38 MAPK and ERK pathway by specific chemical inhibtors and/or RNA interference. We found SB203580, a chemical inhibitor of p38 MAPK, inhibited the commitment of C3H10T1/2 stem cells to the adipocyte lineage induced by BMP-2 or BMP-4, and p38 Stealth ? RNAi also decreased the proportion of adipocyte induced by BMP and MDI. PD98059, a chemical inhibitor of MEK1, kinase of ERK, did not have influence on the commitment process. We also found that Smad4 Stealth ? RNAi reduced the proportion of adipocyte induced by BMP and MDI. And the inhibitory effect could be enhanced by co-transfection with Smad4 and p38 RNAi remarkably. So, we conclude that both Smad and p38 MAPK pathways are required for the commitment of C3H10T1/2 stem cells to the adipocyte lineage induced by BMP-2 or BMP-4, while ERK is not involved in the process.Taken together, our study identified BMP-2 and BMP-4 induced the commitment of C3H10T1/2 stem cells to the adipocyte lineage through BMPR-IA and its downstream signal molecules—Smad, p38 were responsible for this process.更多还原