Ghrelin通过MAPK通路诱导兔BMSCs增殖及成骨分化的机制研究

发布时间：2018-05-28 11:16

本文选题：骨髓基质干细胞 + 内源性脑肠肽　；参考：《重庆医科大学》2016年博士论文

【摘要】：骨髓基质干细胞(bone marrow mesenchymal stem cells,BMSCs)是干细胞家族的主要成员,其最初在骨髓被发现,它具有多项功能诸如,分化潜能,造血支持,免疫调节等。其增殖和分化为现如今的热点。在1999年,Ghrelin被日本学者Kojima等发现。经研究发现,它是由28个氨基酸残基组成,分子量为3.3KD。Ghrelin是生长激素促分泌素受体(Growth hormone secretagogue receptor,GHS-R)的内源性配体,当它与其受体GHS-R结合后会产生具有很强的促进生长激素(Growth hormone,GH)释放的作用。对MAPK途径的研究主要表现在哺乳动物细胞中的研究,其主要方法是过表达、失活、基因沉默等方法。MAPK信号通路主要有4种类型,用它们的核心命名为MAPK。多种生理活动的调节均由其参与。每条通路都可以和其它通路相整合交叉,细胞的命运主要由他们的信号强弱及活化程度决定。目的:本研究就ghrelin对兔BMSCs(rBMSCs)的增殖与分化的作用,及ghrelin通过哪种途径来实现对兔BMSCs的增殖与分化的作用机制进行研究。方法:从健康清洁新西兰大白兔体内分离纯化得到rBMSCs。传代后免疫荧光检测rMSCs细胞标记物CD44、CD34的表达,用MTT检测不同代的rmscs在10天内的生长情况,从而得到最佳的生长时间。利用mtt法检测不同浓度的ghrelin对rmscs的增殖作用,从而找到ghrelin对rmscs的增殖作用的最佳的作用浓度与作用时间。用westernblot的方法来检测ghrelin对rmscs不同时间点的mapk通路的关键蛋白(erk1/2,p38和jnk)的磷酸化情况,来研究ghrelin发挥其作用的主要通路。为了进一步研究ghrelin对rmscs增殖的作用,将mapk通路的关键蛋白进行抑制,用westernblot来检测其下游蛋白的磷酸化情况,及细胞增殖情况。同时用ghrelin受体抑制剂作用于细胞,用westernblot方法检mapk通路的关键蛋白(erk1/2,p38和jnk)的磷酸化情况,用mtt法检测细胞增殖情况。同时用ghrelin对rbmcs向成骨细胞分化的影响进行研究。用westernblot检测加入ghrelin后成骨细胞标志因子alp,runx2和osterix的表达情况,同时检测mapk通路的关键蛋白(erk1/2,p38和jnk)的磷酸化情况,来确定ghrelin对rbmcs向成骨细胞分化影响的作用机制。用mapk通路的关键蛋白的抑制剂作用于细胞,及ghrelin受体抑制剂作用于rbmcs向成骨细胞分化的过程,在分别检测alp,runx2和osterix的表达情况。结果:由新西兰大白兔提rmscs,经培养得到较纯的细胞,其在第四代的生长最佳,且cd44有强烈表达,而cd34则没有表达。ghrelin对rmscs有明显的促进生长的作用。ghrelin对rmscs的最佳作用浓度为600ng/ml,作用时间为第三天。用600ng/ml的ghrelin作用于rbmcs并于0,20,40和60min后,磷酸化的erk1/2的表达量在ghrelin作用后随着作用时间的增加而增加(p0.05)。且在40min时达到量高而后保持不变(p0.05)。说明ghrelin对rbmcs的作用是通过erk1/2通路起作用的。为了进一步证明erk1/2通路的做用,用erk1/2磷酸化抑制剂u0126作用细胞,发现ghrelin对细胞的促进生长作用消失。当细胞在600ng/mlghrelin的作用下,加入ghrelin受体抑制剂,抑制了细胞的生长。从而说明在ghrelin促进rmscs生长增殖时也是ghrelin首先与其受体结合然后才发挥的促进作用的。在本研究中将alp,runx2和osterix作为rbmscs向成骨细胞分化的检测指标,结果表明在ghrelin存在的情况下,alp,runx2和osterix的表达量升高,更有利于rbmscs向成骨细胞分化。为了进一步研究ghrelin促进rbmscs向成骨细胞分化的作用机制,本研究对mapk通路的主要蛋白erk1/2,jnk和p38磷酸化情况进行了检测。结果表明ghrelin促进rbmscs向成骨细胞分化主要是通过erk1/2通路来实现的,为了进一步证明此机制,我们用erk1/2磷酸化抑制剂u0126作用于rbmscs,结果表明erk1/2磷酸化被抑制,而alp,runx2和osterix这三种蛋白的表达量均显著下降,且低于只加地塞米松组的表达量,说明rbmscs向成骨细胞分化过程减慢。进一步证明了ghrelin促进rbmscs向成骨细胞分化主要是通过erk1/2通路来实现的。为了证明ghrelin促进rbmscs向成骨细胞分化的实现是ghrelin先与其受体结合然后才发挥其作用的,本研究还用ghrelin受体抑制剂作用于rbmscs后磷酸化的erk1/2,jnk,p38的表达量在不同的时间段均没有发生明显的变化。同时检测了细胞中alp,runx2和osterix的表达量也没有变化。表明ghrelin的促进作用受到抑制。结论:本研究证明了ghrelin促进rbmscs向成骨细胞分化的机制是ghrelin先与其受体结合然后通过erk1/2通路促进rbmscs的增殖及向成骨细胞分化的。
[Abstract]:Bone marrow stromal cells (bone marrow mesenchymal stem cells, BMSCs) are the main members of the stem cell family. Originally found in the bone marrow, it has a number of functions such as differentiation potential, hematopoiesis support and immunoregulation. Its proliferation and differentiation are now hot spots. In 1999, Ghrelin was found by Japanese scholar Kojima, and so on. The study found that the bone marrow stromal stem cells were found in 1999. It is composed of 28 amino acid residues, and the molecular weight of 3.3KD.Ghrelin is an endogenous ligand for the Growth hormone secretagogue receptor (GHS-R) receptor. When it is combined with its receptor GHS-R, it produces a strong role in promoting the release of growth hormone (Growth hormone, GH). The main table for the MAPK pathway is studied. Now the main methods in mammalian cells are over expression, inactivation, and gene silencing. The.MAPK signaling pathway is mainly 4 types, and the regulation of various physiological activities, named MAPK., is involved. Each pathway can be intersecting with other pathways, and the fate of the cells is mainly due to their strong signals. The purpose of this study is to study the effect of ghrelin on the proliferation and differentiation of rabbit BMSCs (rBMSCs), and the mechanism of the action mechanism of ghrelin on the proliferation and differentiation of rabbit BMSCs. Method: to isolate and purify New Zealand white rabbits from healthy and clean New Zealand white rabbits, and to detect rMSCs finely after rBMSCs. passages. The expression of cellular marker CD44, CD34, the growth of rMSCs in different generations in 10 days was detected by MTT, and the optimum growth time was obtained. The MTT method was used to detect the proliferation of ghrelin at different concentrations to rMSCs, and the optimum concentration and time of action of ghrelin to rMSCs proliferation was found. A Westernblot method was used to detect the effect of ghrelin on the proliferation of rMSCs. Ghrelin studies the phosphorylation of key proteins (erk1/2, p38 and JNK) of the MAPK pathway at different time points of rMSCs to study the main pathway of ghrelin to play its role. In order to further study the effect of ghrelin on rMSCs proliferation, the key proteins of the MAPK pathway are suppressed, and the phosphorylation of the downstream proteins in the MAPK pathway is detected by Westernblot. At the same time, the cell proliferation was detected by the ghrelin receptor inhibitor, and the phosphorylation of the key proteins (erk1/2, p38 and JNK) in the MAPK pathway was detected by the Westernblot method. The cell proliferation was detected by MTT method. The effect of ghrelin on the differentiation of rbmcs into osteoblasts was studied with ghrelin. Westernblot was used to detect the osteoblasts after ghrelin. The expression of marker factors ALP, Runx2 and osterix, and the phosphorylation of key proteins (erk1/2, p38 and JNK) of the MAPK pathway to determine the mechanism of ghrelin's effect on the differentiation of rbmcs into osteoblast. The inhibitor of the key protein of the MAPK pathway acts on the cells, and the ghrelin receptor inhibitor acts on the rbmcs to osteoblasts. The process of differentiation, the expression of ALP, Runx2 and osterix were detected respectively. Results: a New Zealand white rabbit was given rMSCs and cultured to obtain more pure cells. The growth of the fourth generation was the best, and CD44 had a strong expression, while CD34 did not express the effect of.Ghrelin on rMSCs. The best concentration of.Ghrelin for rMSCs was the concentration of CD34. 600ng/ml, the action time is third days. The expression of phosphorylated erk1/2 is increased after ghrelin action with the action of 600ng/ml ghrelin on rbmcs and after 0,20,40 and 60min (P0.05). And it reaches a high quantity at 40min and then remains unchanged (P0.05). In order to further prove the use of the erk1/2 pathway and use the erk1/2 phosphorylation inhibitor U0126 to act on the cells, it is found that ghrelin can promote the growth of the cells. When the cells are under the action of 600ng/mlghrelin, the ghrelin receptor inhibitors are added to inhibit the growth of the cells. Thus, it is said that ghrelin promotes rMSCs proliferation and ghrelin in ghrelin. In this study, ALP, Runx2 and osterix were used as an indicator of rBMSCs differentiation to osteoblasts in this study. The results showed that, in the presence of ghrelin, the expression of ALP, Runx2 and osterix increased, which was more conducive to the differentiation of rBMSCs into osteoblasts. In order to further study ghrelin to promote RB. The mechanism of MSCs to osteoblast differentiation, this study examines the major protein erk1/2, JNK and p38 phosphorylation of the MAPK pathway. The results show that ghrelin promotes the differentiation of rBMSCs into osteoblasts mainly through erk1/2 pathway. In order to further prove this mechanism, we use erk1/2 phosphorylation inhibitor U0126 to act on RBMs. CS, the results showed that the phosphorylation of erk1/2 was inhibited, and the expression of the three proteins of ALP, Runx2 and osterix were significantly decreased, and lower than that of only dexamethasone group, which indicated that rBMSCs slowed down the differentiation process of osteoblasts. It further proved that ghrelin promotes rBMSCs to osteogenic cell differentiation mainly through erk1/2 pathway. It is demonstrated that ghrelin promotes the differentiation of rBMSCs into osteoblasts by combining ghrelin with its receptor before playing its role. This study also uses the ghrelin receptor inhibitor to phosphorylate erk1/2 after rBMSCs, JNK, and p38 has no significant changes at different time periods. At the same time, ALP, Runx2 and OSTE in cells are detected. The expression of Rix has not changed. It shows that the promoting effect of ghrelin is inhibited. Conclusion: This study demonstrated that the mechanism of ghrelin to promote the differentiation of rBMSCs into osteoblasts is that ghrelin combines with its receptor first and promotes the proliferation of rBMSCs through the erk1/2 pathway and differentiation into osteoblasts.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R329.2

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