胰高血糖素样肽-1受体激动剂促进前成骨细胞向成骨分化及其机制研究

发布时间：2018-03-30 17:40

本文选题：骨质疏松　切入点：利拉鲁肽　出处：《河北医科大学》2017年博士论文

【摘要】：胰高血糖素样肽-1(glucagon-like peptide 1,GLP-1)是肠道L型细胞分泌的一种肠促胰岛素,主要的生理学作用包括进食后呈葡萄糖依赖性刺激胰岛素的分泌和释放,促进胰腺β细胞的增殖并抑制其凋亡,抑制胰高血糖素分泌,抑制胃排空,促进饱食感产生等。但GLP-1在体内容易被二肽基肽酶Ⅳ(dipeptidyl peptidase-Ⅳ,DPP-Ⅳ)降解,稳定性差,无法应用于临床,因此科研人员研发了多种GLP-1类似物。利拉鲁肽与人GLP-1有97%同源,半衰期约12~14小时;Exendin-4与哺乳动物GLP-1的氨基酸序列53%同源,为含有39个氨基酸的多肽,注射后2小时就可到达血药浓度峰值。由于利拉鲁肽和Exendin-4都不会被体内的DPP-Ⅳ降解,因此两者既克服了天然GLP-1易被降解的缺点,又保留了GLP-1的各种生理作用和治疗优势。利拉鲁肽和Exendin-4是目前临床上常用的降糖药,不仅可以控制血糖,还可通过多种途径影响骨代谢。Zhan等研究发现,GLP-1可以抑制Runt相关转录因子2(runt-related transcription factor2,Runx2)的产生进而抑制小鼠血管平滑肌细胞的成骨分化。然而,多数体外研究证实,GLP-1可以促进成骨分化的发生。因此,GLP-1及其类似物到底是抑制还是促进成骨分化尚未定论。Smads是转化生长因子-β(transforming growth factor-β,TGF-β)的信号转导和调节分子。已证实Wnt/β-catenin和磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)信号通路均可调节Smad2和Smad3的活性和核转移,并参与成骨分化过程。此外,Hedgehog也是影响细胞分化的重要信号通路,Hedgehog信号可使骨髓间充质干细胞选择性向成骨细胞分化。为阐述胰高血糖素样肽-1类似物促进前成骨细胞的成骨分化及其机制,本研究分四部分:第一部分利拉鲁肽促进前成骨细胞向成骨分化;第二部分利拉鲁肽对前成骨细胞向成骨分化影响的信号通路研究;第三部分Exendin-4促进前成骨细胞向成骨分化;第四部分Exendin-4对前成骨细胞向成骨分化影响的信号通路研究。第一部分利拉鲁肽促进前成骨细胞向成骨分化目的:明确不同浓度利拉鲁肽对小鼠成样骨细胞mc3t3-e1增殖和分化的影响。方法:1将体外培养的小鼠成样骨细胞mc3t3-e1分为对照组和干预组(10-9mol/l利拉鲁肽,10-8mol/l利拉鲁肽,10-7mol/l利拉鲁肽),接种细胞后第24、48和72h对细胞进行ckk-8检测并利用流式细胞术进行细胞周期检测。2将普通培养液中培养的mc3t3-e1细胞设置为空白对照组,成骨培养液中培养的mc3t3-e1细胞设置为成骨对照组,成骨干预组中利拉鲁肽浓度分别设为10-9mol/l、10-8mol/l和10-7mol/l。不同浓度利拉鲁肽干预细胞48h后进行免疫荧光染色定位细胞glp-1受体(glucagon-likepeptide1receptor,glp-1r)的表达部位;分别取第3、7、14和21天的细胞进行碱性磷酸酶(alkalinephosphatase,alp)活性测定和茜素红染色半定量测定;利用westernblot分析和real-timert-pcr检测检测细胞glp-1r、runx2和骨钙素(osteocalcin,ocn)的mrna水平和蛋白表达。结果:1利拉鲁肽对mc3t3-e1细胞的细胞活力和细胞周期均无显著影响。2mc3t3-e1细胞的细胞质与细胞核中均存在glp-1r,利拉鲁肽可以增加成骨培养基中mc3t3-e1细胞glp-1r的表达,并具有剂量依赖性;在成骨培养基中加入10-7mol/l利拉鲁肽后,14天时glp-1r表达增加最明显。3利拉鲁肽可剂量依赖性促进mc3t3-e1细胞向成骨细胞分化时的alp活性和钙沉积量。4利拉鲁肽上调runx2和ocn的mrna水平和蛋白表达,并具有剂量依赖性;runx2的mrna水平和蛋白表达在7天达到峰值,ocn的mrna水平和蛋白表达在21天达到峰值。第二部分利拉鲁肽对前成骨细胞向成骨分化影响的信号通路研究目的:探索smad2和smad3是否参与了利拉鲁肽促进小鼠成样骨细胞mc3t3-e1向成骨分化的过程,并进一步明确利拉鲁肽是否通过wnt/β-catenin信号通路和pi3k-akt信号通路调节smad2和smad3的转录和蛋白表达。方法:1将普通培养液中培养的mc3t3-e1细胞设置为空白对照组,成骨培养液中培养的mc3t3-e1细胞设置为成骨对照组,成骨干预组中利拉鲁肽浓度分别设为10-9mol/l、10-8mol/l和10-7mol/l。利用real-timert-pcr检测细胞smad2和smad3的基因表达水平,用westernblot分析技术检测p-smad2、smad2、p-smad3和smad3蛋白表达水平。2体外培养小鼠前成样骨细胞mc3t3-e1,经小干扰rna(smallinterferingrna,sirna)转染以沉默smad2和smad3基因表达。取成骨诱导培养条件下的mc3t3-e1细胞随机分为8组,分别为空白对照组、空载体组、空载体+利拉鲁肽组、smad2-sirna组、sismad2+利拉鲁肽组、smad3-sirna组和sismad3+利拉鲁肽组。利用real-timert-pcr检测细胞smad2、smad3和runx2的基因表达水平,用westernblot分析技术检测p-smad2、smad2、p-smad3、smad3和runx2的蛋白表达水平,并通过检测alp活性和茜素红染色及半定量法评价细胞的分化程度。2我们进一步应用了wnt/β-catenin信号通路抑制剂dkk-1和pi3k-akt信号通路抑制剂ly294002以确定wnt/β-catenin信号通路和pi3k-akt信号通路在利拉鲁肽对smad2和smad3影响中的作用。结果:1利拉鲁肽上调smad2和smad3的mrna水平,并具有剂量反应关系。2利拉鲁肽可以增加p-smad2、smad2、p-smad2/smad2、p-smad3、smad3和p-smad3/smad3蛋白的表达,并具有剂量反应关系。3通过sismad2或sismad3转染抵消了利拉鲁肽对mc3t3-e1细胞向成骨细胞分化的促进作用。4wnt/β-catenin信号通路阻断剂dkk-1和pi3k-akt信号通路阻断剂ly294002可以抑制利拉鲁肽诱导的smad2和smad3相关蛋白的表达增加。第三部分exendin-4促进前成骨细胞向成骨分化目的:明确不同浓度exendin-4对小鼠前成骨细胞mc3t3-e1向成骨分化的影响。方法:1将普通培养液中培养的mc3t3-e1细胞设置为空白对照组,成骨培养液中培养的mc3t3-e1细胞设置为成骨对照组,成骨干预组中exendin-4浓度分别设为10-9mol/l、10-8mol/l和10-7mol/l。在诱导后3、7、14和21天,利用real-timert-pcr和westernblot分析检测细胞glp-1r的mrna和蛋白表达水平。2检测alp活性和茜素红染色及半定量法评价细胞的分化程度,利用real-timert-pcr和westernblot分析检测细胞runx2和ocn的mrna和蛋白表达水平。结果:1exendin-4可使mc3t3-e1细胞glp-1r的mrna和蛋白表达水平表达升高,且呈剂量依赖性。2mc3t3-e1细胞alp活性和茜素红染色半定量的od值随exendin-4浓度升高而增加。3exendin-4可使mc3t3-e1细胞runx2和ocn的mrna和蛋白表达水平表达升高,且呈剂量依赖性。第四部分exendin-4对前成骨细胞向成骨分化影响的信号通路研究目的:明确hedgehog/gli1信号通路是否参与调控了exendin-4促进mc3t3-e1细胞向成骨分化过程。方法:1利用小干扰rna(smallinterferingrna,sirna)转染技术以沉默gli1的基因表达。取成骨诱导培养条件下的mc3t3-e1细胞分为6组,空白对照组、空载体组、gli1-sirna组、空载体+exendin-4组、gli1-sirna+exendin-4组。利用real-timert-pcr和westernblot分析方法检测空白对照组、空载体组和gli1-sirna组中gli1的表达水平以确认转染效率,并在第7天检测所有组的alp活性和runx2组的表达水平,在第21天进行茜素红染色半定量分析。2应用hedgehog信号通路抑制剂环杷明(cyclopamine)以检测gli1的干扰效率以及阻断效率,并确定hedgehog/gli1信号通路在exendin-4对MC3T3-E1细胞促成骨分化影响中的作用。结果:1 Exendin-4可使MC3T3-E1细胞Hedgehog和Gli1的mRNA和蛋白表达水平表达升高,且呈剂量依赖性。2 SiGli1转染降低了细胞中Gli1的mRNA和蛋白表达水平,并抵消了Exendin-4对MC3T3-E1细胞向成骨细胞分化的促进作用。3 Hedgehog信号通路抑制剂环杷明抵消了Exendin-4对MC3T3-E1细胞向成骨细胞分化的促进作用。结论:1利拉鲁肽对MC3T3-E1细胞增殖无显著影响。2 MC3T3-E1细胞细胞质与细胞核中存在GLP-1R,利拉鲁肽可以增加成骨培养基中MC3T3-E1细胞GLP-1R的表达,并具有剂量依赖性。3利拉鲁肽通过Wnt/β-catenin和PI3K-AKT信号通路上调Smad2和Smad3相关蛋白的表达,呈剂量依赖性促进MC3T3-E1细胞向成骨分化。4 Exendin-4可以增加成骨培养基中MC3T3-E1细胞GLP-1R的表达,并通过Hedgehog/Gli1信号通路剂量依赖性促进其向成骨分化。
[Abstract]:Glucagon like peptide -1 (glucagon-like peptide 1, GLP-1) is a kind of intestinal secretion of intestinal tract L cell insulin, the main physiological role include eating after a Glucose dependent stimulation of insulin secretion and release, promote pancreatic beta cell proliferation and apoptosis, inhibition of glucagon secretion. Inhibition of gastric emptying, promote satiety produced. But GLP-1 in vivo to two dipeptidyl peptidase IV (dipeptidyl IV peptidase-, DPP- IV) degradation, poor stability, not in clinical application, so researchers developed a variety of GLP-1 analogues. Liraglutide and 97% identity with GLP-1, the half-life of about 12~14 hours; homologous amino acid sequences of 53% Exendin-4 and mammalian GLP-1, polypeptide containing 39 amino acids, 2 hours after injection can reach the peak plasma concentration. Because liraglutide and Exendin-4 will be DPP- IV degradation in vivo, because This not only overcomes the disadvantages of natural GLP-1 susceptible to degradation, and retain a variety of physiological effects and treatment GLP-1 advantage. Liraglutide and Exendin-4 are commonly used in clinical drug, can not only control blood sugar, but also through a variety of ways to influence on bone metabolism in.Zhan found that GLP-1 can inhibit Runt related transcription factor 2 (runt-related transcription FACTOR2, Runx2) produced thereby inhibiting vascular smooth muscle cells of mouse osteoblast differentiation in vitro. However, most studies have confirmed that GLP-1 can promote the osteogenic differentiation. Therefore, GLP-1 and its analogues to promote or inhibit the osteogenic differentiation of undetermined.Smads transforming growth factor beta (transforming growth factor- beta, beta TGF-) signal transduction and regulatory molecules. It has been confirmed that Wnt/ beta -catenin and phosphatidylinositol 3- kinase (phosphatidylinositol 3-kinase PI3K) signal transduction pathway Adjustable Smad2 and Smad3 activation and nuclear transfer, and participate in the osteogenic differentiation process. In addition, Hedgehog is also an important pathway of cell differentiation, Hedgehog signal can make the choice of bone marrow mesenchymal stem cells to differentiate into osteoblasts. For this glucagon like peptide analogues of -1 can promote the osteogenic differentiation and the mechanism of osteoblast cells, this study includes four parts: the first part liraglutide promote pre osteoblast osteogenic differentiation; the second part of liraglutide on bone cells to the signal transduction pathway in osteogenic differentiation of the third part of the Exendin-4; promote the osteoblast osteogenic differentiation; the fourth part Exendin-4 of the to study the signaling pathway of bone cells osteogenic differentiation effect. The first part of liraglutide promote pre osteoblast osteogenic differentiation Objective: to determine the different concentrations of liraglutide on mouse MC3T3-E1 cells proliferation and differentiation of bone The effect. Methods: 1 in vitro mouse bone cells MC3T3-E1 divided into control group and intervention group (10-9mol/l 10-8mol/l, liraglutide, liraglutide 10-7mol/l, liraglutide), inoculated cells after 24,48 and 72h on cell ckk-8 detection and.2 detection of cell cycle by flow cytometry to normal culture in the culture medium of MC3T3-E1 cells is set to the blank control group, osteoblasts cultured in MC3T3-E1 cells arranged into bone control group, a group of backbone pre liraglutide concentrations were set to 10-9mol/l, 10-8mol/l and 10-7mol/l. of different concentrations of liraglutide 48h cells by immunofluorescence localization of cell GLP-1 receptor (glucagon-likepeptide1receptor, GLP-1R the expression site; respectively) on days 3,7,14 and 21 cells were alkaline phosphatase (alkalinephosphatase, ALP) activity assay and alizarin red staining and semi quantitative measurement ; using Westernblot analysis and real-timert-pcr detection of GLP-1R cells, Runx2 and Osteocalcin (osteocalcin, OCN) expression levels of mRNA and protein. Results: there were no significant effects of GLP-1R are cytoplasmic and nuclear.2mc3t3-e1 cells in 1 liraglutide cell viability and cell cycle of MC3T3-E1 cells, liraglutide can increase osteoblast culture the expression of MC3T3-E1 GLP-1R cells in the medium in a dose-dependent manner; in osteoblast culture medium added 10-7mol/l liraglutide after 14 days the expression of GLP-1R increased obviously.3 liraglutide dose dependently stimulated MC3T3-E1 cells to express mRNA protein level and osteogenic differentiation of the ALP activity and calcium deposition with.4 peptide upregulation of Runx2 and OCN, in a dose-dependent manner; the expression level of mRNA protein and Runx2 reached the peak on the 7 day, the expression level of mRNA protein and OCN was reached in 21 days Peak. The second part of liraglutide on pre osteoblasts to signal pathway of osteogenic differentiation effect: To explore Smad2 and Smad3 are involved in the promotion of liraglutide mouse like bone cell MC3T3-E1 to osteogenic differentiation, and further clarify the transcription and protein expression of liraglutide through wnt/ beta -catenin signal pathway and PI3K-Akt signal Smad2 pathway and Smad3. Methods: 1 normal cultured MC3T3-E1 cells were set to blank control group, osteoblasts cultured in MC3T3-E1 cells arranged into bone control group, a group of backbone pre liraglutide concentrations were set to 10-9mol/l, 10-8mol/l and 10-7mol/l. using real-timert-pcr to detect Smad2 and Smad3 the level of gene expression analysis techniques to detect p-Smad2, Smad2 with Westernblot, the expression of p-smad3 and Smad3 protein in cultured mouse bone cells into the level of MC.2 in vitro 3T3-E1, by small interfering RNA (smallinterferingrna, siRNA) expression to silence Smad2 gene and Smad3 Gene. The osteogenic induction in cultured MC3T3-E1 cells were divided into 8 groups under the conditions, namely control group, vector group, plasmid + liraglutide group, smad2-sirna group, sismad2+ group, smad3-sirna group and liraglutide. Sismad3+ liraglutide group detected by real-timert-pcr cells. Smad2, Smad3 and Runx2 gene expression level analysis to detect p-Smad2, Smad2, p-smad3 by Westernblot, the expression level of Smad3 and Runx2 protein were tested by ALP activity and alizarin red staining and semi quantitative evaluation method of cell differentiation.2 we further applied the wnt/ beta -catenin signal pathway inhibitor DKK-1 and PI3K-Akt signaling pathway inhibitor LY294002 to determine the effects of wnt/ beta -catenin and PI3K-Akt signal pathways of Smad2 and Smad3 in liraglutide in Results: 1. The role of liraglutide upregulation of Smad2 and Smad3 mRNA level, and there was a dose-response relationship between.2 liraglutide can increase p-Smad2, Smad2, p-smad2/smad2, p-smad3, Smad3 and p-smad3/smad3 protein expression, and there was a dose-response relationship between.3 with sismad2 or sismad3 was offset by liraglutide on MC3T3-E1 cells to differentiate into osteoblasts the increased expression of.4wnt/ beta -catenin signaling pathway related protein Smad3 and Smad2 blocking agent DKK-1 and PI3K-Akt signal pathway inhibitor LY294002 can inhibit liraglutide induced by exendin-4. In the third part, promote the osteoblast osteogenic differentiation Objective: to determine the different concentrations of exendin-4 on mouse osteoblastic MC3T3-E1 cells to osteogenic differentiation in vitro methods: 1 normal cultured MC3T3-E1 cells were set to blank control group, osteoblasts cultured in MC3T3-E1 cell set Osteogenic control group, into the backbone of the concentration of exendin-4 in the pre group were divided into 10-9mol/l, 10-8mol/l and 10-7mol/l. in 3,7,14 and 21 days after induction, analysis by real-timert-pcr and Westernblot mRNA and protein were detected the expression of GLP-1R differentiation level.2 ALP activity detection and evaluation of alizarin red staining and semi quantitative method using analysis of real-timert-pcr cells. The expression of Westernblot and mRNA and protein levels of OCN and Runx2 were detected. Results: 1exendin-4 mRNA and protein expression level of GLP-1R expression in MC3T3-E1 cells increased in a dose-dependent manner in.2mc3t3-e1 cells ALP activity and alizarin red staining and semi quantitative OD value can be increased.3exendin-4 mRNA and protein in MC3T3-E1 cells and the expression of OCN Runx2 the expression level increased with the concentration of exendin-4 increased in a dose-dependent manner. The fourth part exendin-4 of pre osteoblast osteogenic differentiation effect Objective to study the signal pathway: clear hedgehog/gli1 signaling pathway is involved in the regulation of exendin-4 promotes MC3T3-E1 cell osteogenic differentiation process. Methods: 1 using small interfering RNA (smallinterferingrna, siRNA) transfection technique to silence Gli1 gene expression. The osteogenic induction culture conditions of MC3T3-E1 cells were divided into 6 groups, control group, empty group, gli1-sirna group, empty vector group +exendin-4, gli1-sirna+exendin-4 group. Using real-timert-pcr and Westernblot analysis method to detect the expression of Gli1 water control group, empty vector group and gli1-sirna group in the flat to confirm the transfection efficiency and expression level of ALP activity and Runx2 group of all groups were detected in seventh days, semi quantitative alizarin red.2 staining analysis application of hedgehog signaling pathway inhibitor cyclopamine in twenty-first days (cyclopamine) to interfere with the efficiency of Gli1 detection and blocking efficiency, and to determine the hedgehog/ The Gli1 signaling pathway in the differentiation of osteogenic effect on MC3T3-E1 cells in exendin-4. Results: 1 Exendin-4 can make the mRNA and Hedgehog proteins in MC3T3-E1 cells and the expression level of Gli1 expression increased in a dose-dependent manner in.2 cells transfected with SiGli1 reduced Gli1 mRNA and protein expression level, and the offset of Exendin-4 on MC3T3-E1 cells osteogenic differentiation promoting effect of.3 Hedgehog signaling pathway inhibitor cyclopamine offset Exendin-4 of MC3T3-E1 cells to differentiate into osteoblasts to promote the role of 1. Conclusion: liraglutide has no significant effect on GLP-1R.2 MC3T3-E1 cell cytoplasm and nucleus of MC3T3-E1 cells proliferation, liraglutide can increase osteoblast cultured MC3T3-E1 cells express GLP-1R in the medium, and had dose dependent.3 liraglutide by Wnt/ beta -catenin and PI3K-AKT signaling pathway in the up regulation of Smad2 and Smad3 related protein table Da promoted the osteogenic differentiation of MC3T3-E1 cells in a dose-dependent manner..4 Exendin-4 increased the expression of GLP-1R in MC3T3-E1 cells and promoted the osteogenic differentiation through Hedgehog/Gli1 signaling pathway in a dose-dependent manner.

【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.1

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