本文選題：骨髓基質(zhì)干細胞 + 內(nèi)源性腦腸肽　； 參考：《重慶醫(yī)科大學》2016年博士論文

【摘要】：骨髓基質(zhì)干細胞(bone marrow mesenchymal stem cells,BMSCs)是干細胞家族的主要成員,其最初在骨髓被發(fā)現(xiàn),它具有多項功能諸如,分化潛能,造血支持,免疫調(diào)節(jié)等。其增殖和分化為現(xiàn)如今的熱點。在1999年,Ghrelin被日本學者Kojima等發(fā)現(xiàn)。經(jīng)研究發(fā)現(xiàn),它是由28個氨基酸殘基組成,分子量為3.3KD。Ghrelin是生長激素促分泌素受體(Growth hormone secretagogue receptor,GHS-R)的內(nèi)源性配體,當它與其受體GHS-R結(jié)合后會產(chǎn)生具有很強的促進生長激素(Growth hormone,GH)釋放的作用。對MAPK途徑的研究主要表現(xiàn)在哺乳動物細胞中的研究,其主要方法是過表達、失活、基因沉默等方法。MAPK信號通路主要有4種類型,用它們的核心命名為MAPK。多種生理活動的調(diào)節(jié)均由其參與。每條通路都可以和其它通路相整合交叉,細胞的命運主要由他們的信號強弱及活化程度決定。目的:本研究就ghrelin對兔BMSCs(rBMSCs)的增殖與分化的作用,及ghrelin通過哪種途徑來實現(xiàn)對兔BMSCs的增殖與分化的作用機制進行研究。方法:從健康清潔新西蘭大白兔體內(nèi)分離純化得到rBMSCs。傳代后免疫熒光檢測rMSCs細胞標記物CD44、CD34的表達,用MTT檢測不同代的rmscs在10天內(nèi)的生長情況,從而得到最佳的生長時間。利用mtt法檢測不同濃度的ghrelin對rmscs的增殖作用,從而找到ghrelin對rmscs的增殖作用的最佳的作用濃度與作用時間。用westernblot的方法來檢測ghrelin對rmscs不同時間點的mapk通路的關(guān)鍵蛋白(erk1/2,p38和jnk)的磷酸化情況,來研究ghrelin發(fā)揮其作用的主要通路。為了進一步研究ghrelin對rmscs增殖的作用,將mapk通路的關(guān)鍵蛋白進行抑制,用westernblot來檢測其下游蛋白的磷酸化情況,及細胞增殖情況。同時用ghrelin受體抑制劑作用于細胞,用westernblot方法檢mapk通路的關(guān)鍵蛋白(erk1/2,p38和jnk)的磷酸化情況,用mtt法檢測細胞增殖情況。同時用ghrelin對rbmcs向成骨細胞分化的影響進行研究。用westernblot檢測加入ghrelin后成骨細胞標志因子alp,runx2和osterix的表達情況,同時檢測mapk通路的關(guān)鍵蛋白(erk1/2,p38和jnk)的磷酸化情況,來確定ghrelin對rbmcs向成骨細胞分化影響的作用機制。用mapk通路的關(guān)鍵蛋白的抑制劑作用于細胞,及ghrelin受體抑制劑作用于rbmcs向成骨細胞分化的過程,在分別檢測alp,runx2和osterix的表達情況。結(jié)果:由新西蘭大白兔提rmscs,經(jīng)培養(yǎng)得到較純的細胞,其在第四代的生長最佳,且cd44有強烈表達,而cd34則沒有表達。ghrelin對rmscs有明顯的促進生長的作用。ghrelin對rmscs的最佳作用濃度為600ng/ml,作用時間為第三天。用600ng/ml的ghrelin作用于rbmcs并于0,20,40和60min后,磷酸化的erk1/2的表達量在ghrelin作用后隨著作用時間的增加而增加(p0.05)。且在40min時達到量高而后保持不變(p0.05)。說明ghrelin對rbmcs的作用是通過erk1/2通路起作用的。為了進一步證明erk1/2通路的做用,用erk1/2磷酸化抑制劑u0126作用細胞,發(fā)現(xiàn)ghrelin對細胞的促進生長作用消失。當細胞在600ng/mlghrelin的作用下,加入ghrelin受體抑制劑,抑制了細胞的生長。從而說明在ghrelin促進rmscs生長增殖時也是ghrelin首先與其受體結(jié)合然后才發(fā)揮的促進作用的。在本研究中將alp,runx2和osterix作為rbmscs向成骨細胞分化的檢測指標,結(jié)果表明在ghrelin存在的情況下,alp,runx2和osterix的表達量升高,更有利于rbmscs向成骨細胞分化。為了進一步研究ghrelin促進rbmscs向成骨細胞分化的作用機制,本研究對mapk通路的主要蛋白erk1/2,jnk和p38磷酸化情況進行了檢測。結(jié)果表明ghrelin促進rbmscs向成骨細胞分化主要是通過erk1/2通路來實現(xiàn)的,為了進一步證明此機制,我們用erk1/2磷酸化抑制劑u0126作用于rbmscs,結(jié)果表明erk1/2磷酸化被抑制,而alp,runx2和osterix這三種蛋白的表達量均顯著下降,且低于只加地塞米松組的表達量,說明rbmscs向成骨細胞分化過程減慢。進一步證明了ghrelin促進rbmscs向成骨細胞分化主要是通過erk1/2通路來實現(xiàn)的。為了證明ghrelin促進rbmscs向成骨細胞分化的實現(xiàn)是ghrelin先與其受體結(jié)合然后才發(fā)揮其作用的,本研究還用ghrelin受體抑制劑作用于rbmscs后磷酸化的erk1/2,jnk,p38的表達量在不同的時間段均沒有發(fā)生明顯的變化。同時檢測了細胞中alp,runx2和osterix的表達量也沒有變化。表明ghrelin的促進作用受到抑制。結(jié)論:本研究證明了ghrelin促進rbmscs向成骨細胞分化的機制是ghrelin先與其受體結(jié)合然后通過erk1/2通路促進rbmscs的增殖及向成骨細胞分化的。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R329.2

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