CGRP在人骨髓间充质干细胞增殖中的作用研究

发布时间：2018-01-20 23:01

本文关键词： 降钙素基因相关肽间充质干细胞增殖骨形态发生蛋白-2 胰岛素样生长因子-1　出处：《第三军医大学》2008年博士论文　论文类型：学位论文

【摘要】： 降钙素基因相关肽(CGRP)是一种在骨组织中广泛分布的神经肽,已发现,CGRP与骨组织的增殖、塑形密切相关。通过组织学研究,在骨折愈合试验中观察到,骨组织中,含CGRP的神经纤维主要分布在骨生成和塑形活跃的区域,例如在长骨的干骺端分布就比骨干部位高约10倍,且神经分布及CGRP含量的变化与骨折痊愈的过程紧密相关。研究证实,CGRP能显著促进成骨细胞增殖,增强成骨活性。骨髓间充质干细胞(MSCs)是成骨细胞重要的来源干细胞,也是骨组织工程的主要种子细胞来源。MSCs的增值能力,直接影响骨的生长、塑形和修复,也对组织工程骨的构建效能和成骨能力起着决定性的作用。因此,对影响MSCs增殖的因素的研究,一直是骨科界和组织工程学界研究的重点。目前的研究已经发现,MSCs在骨组织的分布,主要集中在干骺端的红骨髓中,这与CGRP能的神经分布相符合,相关研究也发现,CGRP能促进骨髓有核细胞集落的形成。但是关于CGRP对MSCs的增殖的影响及其机制,仍有大量研究需要深入。第一部分人骨髓间充质干细胞表面CGRP受体存在证据目的:寻找人骨髓MSCs表面存在CGRP受体的确切证据(从基因及蛋白质表达水平) 方法:原材料采自健康志愿者骨髓,应用梯度离心法及贴壁培养筛选,获得MSCs细胞。体外培养扩增后,取对数生长期细胞,采用RT-PCR技术进行细胞CGRP受体mRNA表达检测;采用杂交瘤技术,获得兔抗人CGRP受体蛋白抗体,利用该抗体,使用Westernblot技术进行MSCs表达CGRP受体蛋白检测。结果:采用梯度离心及贴壁培养筛选,细胞纯度较高,表面标记稳定,且与文献报道相符;经RT-PCR检测结果,证实人骨髓MSCs表达CGRP受体mRNA;采用杂交瘤技术获得的兔抗人CGRP受体抗体,结构稳定,滴度较高,能较好的满足Westernblot试验需要;采用Westernblot的DAB显色法,检测出人MSCs表达CGRP受体蛋白,且表达量较大。结论:采用梯度离心及贴壁培养法获得MSCs纯度较高,增殖效果稳定;经过RT-PCR检测,证实MSCs表达CGRP受体mRNA;Western-blot检测证实,MSCs表达CGRP受体蛋白。第二部分CGRP对人骨髓间充质干细胞增殖的作用目的:观察在添加外源性CGRP的情况下,MSCs增殖的改变,并证实这种改变与添加CGRP间的关联性方法:MSCs获取仍采自健康志愿者骨髓,应用梯度离心法及贴壁培养筛选,获得MSCs细胞。分组采用对照组、CGRP组(根据CGRP添加的终浓度分为10-7mol/ L、10-8mol/ L和10-9mol/ L三组),以MTT法检测细胞增殖曲线变化;体外培养扩增后,取相同时相点,光镜下进行细胞形态观察;将各组细胞在第三代传代后72小时的对数生长期,进行细胞周期检测,观察各组细胞处在DNA合成期及细胞分裂前期的比例。结果:经过对照培养,细胞增殖同时相点,对照组细胞细胞密度低于各实验组,细胞形态各组均较为典型、规则;细胞周期检测,各实验组处于细胞分裂前期及DNA合成期细胞比例明显高于对照组,各实验组间该比例为,10-8mol/ L10-9mol/ L组10-7mol/ L,但各实验组间差异无统计学意义;MTT组检测,在对数生长期,各实验组增殖速率高于对照组,实验组间细胞增殖速率为10-8mol/ L10-9mol/ L组10-7mol/ L。10-8mol/ L速率与另外两实验组间差异有统计学意义,其余两组间无显著性差异结论:采用MTT法,证实添加外源性CGRP能促进对数增殖期MSCs细胞增殖速度;采用流失细胞法检测,证实,外源性CGRP能提高MSCs细胞处于DNA合成和有丝分裂前期的比例。第三部分: CGRP对人骨髓间充质干细胞细胞信号传导的变化的影响的研究目的:观察在添加外源性CGRP的情况下,MSCs胞间通讯连接的改变,并验证改变与CGRP的关联性方法:MSCs获取仍采自健康志愿者骨髓,应用梯度离心法及贴壁培养筛选,获得MSCs细胞。分组采用对照组、CGRP组和拮抗剂组,放射免疫法检测各组胞间信号分子cAMP含量改变;使用CFDA染料,应用激光共聚焦技术观察细胞间缝隙连接和胞间信号传导能力变化;采用荧光定量PCR技术检测缝隙连接分子Cx43mRNA表达变化。结果:放免法检测结果显示,各组间,以CGRP组胞间cAMP含量最高,与其余两组间存在统计学差异;CGRP结合拮抗剂组的含量高于对照组,后两者间无统计学差异。采用激光共聚焦技术,结合CFDA生物活性染料,显示CGRP组荧光信号恢复幅度较对照组及拮抗剂组大,差异有显著性(P0.05);拮抗剂组恢复幅度较对照组大,但两组间差异不具有显著性(P0.05)。三组细胞Cx43mRNA表达,CGRP组表达量高于抑制剂组及对照组,差异有显著性(P0.05);抑制剂组表达量高于对照组,两者间差异无显著性。结论:CGRP能促进MSCs胞间缝隙连接,促进缝隙连接蛋白的基因表达。第四部分:CGRP对人骨髓间充质干细胞细胞增殖相关因子的作用目的:研究在添加外源性CGRP的情况下,与MSCs增殖及分化相关的细胞因子IGF-1、BMP-2的受体mRNA表达变化;研究外源性CGRP是否造成MSCs对成骨诱导因子BMP-2的mRNA表达。方法:MSCs获取、分离及培养方法同前。实验分组为对照组、CGRP组和拮抗剂组,采用荧光定量PCR技术分别检测增殖相关因子IGF-1及其受体mRNA表达;以及成骨分化因子BMP-2及其受体mRNA表达。结果:采用相对定量技术,CGRP组MSCs表达IGF-1、IGF-1受体以及BMP-2受体mRNA量高于拮抗剂组及对照组,且差异有统计学意义。而三组在表达BMP-2 mRNA无显著差异,且Ct值≥35。结论:外源性CGRP能够提高MSCs表达IGF-1、IGF-1受体以及BMP-2受体mRNA,而不能诱导MSCs表达BMP-2mRNA。三组BMP-2的mRNA平均Ct值均在35左右,可以认为阴性表达。
[Abstract]:Calcitonin gene related peptide (CGRP) is a widely distributed in bone tissue has been found, neuropeptide CGRP and bone tissue proliferation, remodeling is closely related to the histological investigations, in fracture healing were observed, bone tissue, CGRP containing nerve fibers mainly distributed in bone formation and remodeling active region, for example in the metaphysis of a long bone distribution is about 10 times higher than the backbone of parts, and the changes of nerve distribution and the content of CGRP and the process of fracture recovery is closely related. The research shows that, CGRP can significantly promote the proliferation of osteoblasts, enhanced osteogenic activity.
Bone marrow mesenchymal stem cells (MSCs) is an important source of bone cells into stem cells,.MSCs main source of seed cells for bone tissue engineering is the ability to add value, directly affect the bone growth, remodeling and repair, but also plays a decisive role in the construction of tissue engineering bone efficiency and osteogenic ability. Therefore, study on the influence factors on the proliferation of MSCs, has been a research focus in the Department of orthopedics field and tissue engineering field.
The present research has found that the distribution of MSCs in bone tissue, bone marrow mainly concentrated in the metaphysis, and the CGRP nerve distribution is consistent with that of related studies have also found that CGRP can promote bone marrow cell colony formation. But the effect of CGRP on proliferation of MSCs and its mechanism. There are still a lot of research need further.
Part 1 evidence of CGRP receptor on the surface of human bone marrow mesenchymal stem cells
Objective: to find out the exact evidence of the existence of CGRP receptor on the surface of human bone marrow MSCs (from gene and protein expression level)
Methods: raw materials collected from healthy bone marrow donors, using gradient centrifugation and adherent culture screening, MSCs cells cultured in vitro. After the cells in the logarithmic growth phase of CGRP cell receptor mRNA expression was detected by RT-PCR technology; using hybridoma techniques, to obtain Rabbit anti human CGRP receptor protein antibody, the antibody. Detect the MSCs expression of CGRP receptor protein using Westernblot technology.
Results: by gradient centrifugation and adherent culture screening, cell surface markers of high purity, stability, and consistent with the literature; the RT-PCR results confirmed that human bone marrow MSCs expression of CGRP receptor mRNA was obtained by hybridoma technique; Rabbit anti human CGRP receptor antibody, stable structure, high titer, can better meet Westernblot test; using Westernblot DAB colorimetric method, detect the expression of CGRP receptor protein MSCs, and the expression of a large amount.
Conclusion: the MSCs obtained by gradient centrifugation and adherent culture is highly purified, and the proliferation effect is stable. After RT-PCR detection, it is confirmed that MSCs expresses CGRP receptor mRNA. Western-blot detection confirms that MSCs expresses CGRP receptor protein.
The effect of second part CGRP on the proliferation of human bone marrow mesenchymal stem cells
Objective: To observe the changes in the proliferation of MSCs under the addition of exogenous CGRP, and to confirm the association between this change and the addition of CGRP.
Methods: the MSCs acquisition is still collected from healthy volunteers using bone marrow gradient centrifugation and adherent culture screening, MSCs cells. The group control group, CGRP group (according to the final concentration of CGRP added into 10-7mol/ L, 10-8mol/ L and 10-9mol/ L three group), by MTT method to detect cell proliferation curve in vitro; after amplification, the same phase point, cell morphology observed under light microscope; the cells were in the logarithmic growth period of 72 hours after third passages, the cell cycle detection, cells were observed in DNA synthesis and cell division of the ratio.
Results: after control culture, cell proliferation and cell density, the control group was lower than that in the experimental group, the cell morphology were typical, rules; cell cycle detection, each experimental group in cell division and cell proportion of pre DNA synthesis phase was significantly higher than the control group, the experimental group between the ratio of 10-8mol/, L10-9mol/ L 10-7mol/ L group, but there was no significant difference among the experimental groups; MTT group, during the logarithmic growth phase, the growth rate of the experimental group than the control group, the experimental group between the cell proliferation rate of 10-8mol/ L10-9mol/ / 10-7mol L group was statistically significant L.10-8mol/ L rate and also difference between two groups, the remaining two groups significant difference
Conclusion: using MTT method, it is confirmed that adding exogenous CGRP can promote the proliferation rate of MSCs cells in logarithmic proliferative phase. The loss cell assay confirmed that exogenous CGRP can increase the proportion of MSCs cells in DNA synthesis and pre mitosis.
The third part: the study of the effect of CGRP on the change of signal transduction in human bone marrow mesenchymal stem cells
Objective: To observe the changes in the intercellular communication connection of MSCs under the addition of exogenous CGRP, and to verify the correlation between the changes and the CGRP.
Methods: the MSCs acquisition is still collected from healthy volunteers using bone marrow gradient centrifugation and adherent culture screening, MSCs cells. The group control group, CGRP group and antagonist group, change radioimmunnity analysis was used to detect the intracellular signal molecule cAMP content; using CFDA dye, using laser scanning confocal microscope and gap junction the intercellular signal transduction ability changes of intercellular gap junction; detection molecule Cx43mRNA expression by real-time PCR.
Results: the result of radioimmunoassay showed that each group, in the CGRP group was among the highest content of cAMP, and there were significant differences between the rest two groups; the content of the antagonist binding agent group CGRP was higher than the control group, no significant difference between the two. After using confocal laser technology, combined with the biological activity of CFDA in group CGRP fluorescent dye, signal recovery rate compared with the control group and antagonist group, there was significant difference (P0.05); antagonist group restored significantly than the control group, but the difference between the two groups was not significant (P0.05). The expression of Cx43mRNA cells in three groups, CGRP group of high expression in inhibitor group and control group, there was significant difference of (P0.05); the expression of inhibitor group than the control group, there was no significant difference between the two.
Conclusion: CGRP can promote the intercellular gap junction of MSCs and promote the gene expression of gap connexin.
The fourth part: the effect of CGRP on the proliferation related factors of human bone marrow mesenchymal stem cells
Objective: To study the expression of IGF-1 and BMP-2 receptor mRNA related to proliferation and differentiation of MSCs in the presence of exogenous CGRP, and to investigate whether exogenous CGRP can induce MSCs to express mRNA in osteoblast inducing factor BMP-2.
Methods: MSCs access method for the isolation and culture of the same experimental group as control group, CGRP group and antagonist group, using fluorescence quantitative PCR technology to detect the proliferation related factor IGF-1 and its receptor mRNA expression; and osteogenic differentiation factor BMP-2 and its receptor mRNA expression.
Results: the relative quantitative expression of MSCs IGF-1 technology, CGRP group, IGF-1 receptor and BMP-2 receptor mRNA was higher than that of antagonist group and control group, and the difference was statistically significant. The three groups had no significant difference in the expression of BMP-2 mRNA, and the Ct value is more than 35.
Conclusion: exogenous CGRP can increase MSCs expression of IGF-1, IGF-1 receptor and BMP-2 receptor mRNA, but can not induce MSCs expression. BMP-2mRNA. mRNA BMP-2 average mRNA value of three groups is about 35, which can be considered negative expression.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R68;R329

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