本文關鍵詞： 降鈣素基因相關肽 間充質(zhì)干細胞 增殖 骨形態(tài)發(fā)生蛋白-2 胰島素樣生長因子-1　出處：《第三軍醫(yī)大學》2008年博士論文　論文類型：學位論文

【摘要】： 降鈣素基因相關肽(CGRP)是一種在骨組織中廣泛分布的神經(jīng)肽,已發(fā)現(xiàn),CGRP與骨組織的增殖、塑形密切相關。通過組織學研究,在骨折愈合試驗中觀察到,骨組織中,含CGRP的神經(jīng)纖維主要分布在骨生成和塑形活躍的區(qū)域,例如在長骨的干骺端分布就比骨干部位高約10倍,且神經(jīng)分布及CGRP含量的變化與骨折痊愈的過程緊密相關。研究證實,CGRP能顯著促進成骨細胞增殖,增強成骨活性。 骨髓間充質(zhì)干細胞(MSCs)是成骨細胞重要的來源干細胞,也是骨組織工程的主要種子細胞來源。MSCs的增值能力,直接影響骨的生長、塑形和修復,也對組織工程骨的構建效能和成骨能力起著決定性的作用。因此,對影響MSCs增殖的因素的研究,一直是骨科界和組織工程學界研究的重點。 目前的研究已經(jīng)發(fā)現(xiàn),MSCs在骨組織的分布,主要集中在干骺端的紅骨髓中,這與CGRP能的神經(jīng)分布相符合,相關研究也發(fā)現(xiàn),CGRP能促進骨髓有核細胞集落的形成。但是關于CGRP對MSCs的增殖的影響及其機制,仍有大量研究需要深入。 第一部分人骨髓間充質(zhì)干細胞表面CGRP受體存在證據(jù) 目的:尋找人骨髓MSCs表面存在CGRP受體的確切證據(jù)(從基因及蛋白質(zhì)表達水平) 方法:原材料采自健康志愿者骨髓,應用梯度離心法及貼壁培養(yǎng)篩選,獲得MSCs細胞。體外培養(yǎng)擴增后,取對數(shù)生長期細胞,采用RT-PCR技術進行細胞CGRP受體mRNA表達檢測;采用雜交瘤技術,獲得兔抗人CGRP受體蛋白抗體,利用該抗體,使用Westernblot技術進行MSCs表達CGRP受體蛋白檢測。 結果:采用梯度離心及貼壁培養(yǎng)篩選,細胞純度較高,表面標記穩(wěn)定,且與文獻報道相符;經(jīng)RT-PCR檢測結果,證實人骨髓MSCs表達CGRP受體mRNA;采用雜交瘤技術獲得的兔抗人CGRP受體抗體,結構穩(wěn)定,滴度較高,能較好的滿足Westernblot試驗需要;采用Westernblot的DAB顯色法,檢測出人MSCs表達CGRP受體蛋白,且表達量較大。 結論:采用梯度離心及貼壁培養(yǎng)法獲得MSCs純度較高,增殖效果穩(wěn)定;經(jīng)過RT-PCR檢測,證實MSCs表達CGRP受體mRNA;Western-blot檢測證實,MSCs表達CGRP受體蛋白。 第二部分CGRP對人骨髓間充質(zhì)干細胞增殖的作用 目的:觀察在添加外源性CGRP的情況下,MSCs增殖的改變,并證實這種改變與添加CGRP間的關聯(lián)性 方法:MSCs獲取仍采自健康志愿者骨髓,應用梯度離心法及貼壁培養(yǎng)篩選,獲得MSCs細胞。分組采用對照組、CGRP組(根據(jù)CGRP添加的終濃度分為10-7mol/ L、10-8mol/ L和10-9mol/ L三組),以MTT法檢測細胞增殖曲線變化;體外培養(yǎng)擴增后,取相同時相點,光鏡下進行細胞形態(tài)觀察;將各組細胞在第三代傳代后72小時的對數(shù)生長期,進行細胞周期檢測,觀察各組細胞處在DNA合成期及細胞分裂前期的比例。 結果:經(jīng)過對照培養(yǎng),細胞增殖同時相點,對照組細胞細胞密度低于各實驗組,細胞形態(tài)各組均較為典型、規(guī)則;細胞周期檢測,各實驗組處于細胞分裂前期及DNA合成期細胞比例明顯高于對照組,各實驗組間該比例為,10-8mol/ L10-9mol/ L組10-7mol/ L,但各實驗組間差異無統(tǒng)計學意義;MTT組檢測,在對數(shù)生長期,各實驗組增殖速率高于對照組,實驗組間細胞增殖速率為10-8mol/ L10-9mol/ L組10-7mol/ L。10-8mol/ L速率與另外兩實驗組間差異有統(tǒng)計學意義,其余兩組間無顯著性差異 結論:采用MTT法,證實添加外源性CGRP能促進對數(shù)增殖期MSCs細胞增殖速度;采用流失細胞法檢測,證實,外源性CGRP能提高MSCs細胞處于DNA合成和有絲分裂前期的比例。 第三部分: CGRP對人骨髓間充質(zhì)干細胞細胞信號傳導的變化的影響的研究 目的:觀察在添加外源性CGRP的情況下,MSCs胞間通訊連接的改變,并驗證改變與CGRP的關聯(lián)性 方法:MSCs獲取仍采自健康志愿者骨髓,應用梯度離心法及貼壁培養(yǎng)篩選,獲得MSCs細胞。分組采用對照組、CGRP組和拮抗劑組,放射免疫法檢測各組胞間信號分子cAMP含量改變;使用CFDA染料,應用激光共聚焦技術觀察細胞間縫隙連接和胞間信號傳導能力變化;采用熒光定量PCR技術檢測縫隙連接分子Cx43mRNA表達變化。 結果:放免法檢測結果顯示,各組間,以CGRP組胞間cAMP含量最高,與其余兩組間存在統(tǒng)計學差異;CGRP結合拮抗劑組的含量高于對照組,后兩者間無統(tǒng)計學差異。采用激光共聚焦技術,結合CFDA生物活性染料,顯示CGRP組熒光信號恢復幅度較對照組及拮抗劑組大,差異有顯著性(P0.05);拮抗劑組恢復幅度較對照組大,但兩組間差異不具有顯著性(P0.05)。三組細胞Cx43mRNA表達,CGRP組表達量高于抑制劑組及對照組,差異有顯著性(P0.05);抑制劑組表達量高于對照組,兩者間差異無顯著性。 結論:CGRP能促進MSCs胞間縫隙連接,促進縫隙連接蛋白的基因表達。 第四部分:CGRP對人骨髓間充質(zhì)干細胞細胞增殖相關因子的作用 目的:研究在添加外源性CGRP的情況下,與MSCs增殖及分化相關的細胞因子IGF-1、BMP-2的受體mRNA表達變化;研究外源性CGRP是否造成MSCs對成骨誘導因子BMP-2的mRNA表達。 方法:MSCs獲取、分離及培養(yǎng)方法同前。實驗分組為對照組、CGRP組和拮抗劑組,采用熒光定量PCR技術分別檢測增殖相關因子IGF-1及其受體mRNA表達;以及成骨分化因子BMP-2及其受體mRNA表達。 結果:采用相對定量技術,CGRP組MSCs表達IGF-1、IGF-1受體以及BMP-2受體mRNA量高于拮抗劑組及對照組,且差異有統(tǒng)計學意義。而三組在表達BMP-2 mRNA無顯著差異,且Ct值≥35。 結論:外源性CGRP能夠提高MSCs表達IGF-1、IGF-1受體以及BMP-2受體mRNA,而不能誘導MSCs表達BMP-2mRNA。三組BMP-2的mRNA平均Ct值均在35左右,可以認為陰性表達。
[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.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R68;R329

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