【摘要】：在臨床上我們發(fā)現(xiàn)中樞神經(jīng)系統(tǒng)損傷(如截癱或者顱腦損傷)的患者若同時(shí)伴有四肢長(zhǎng)骨骨折時(shí),常常在骨折區(qū)出現(xiàn)大量骨痂的過(guò)度生長(zhǎng),嚴(yán)重的甚至出現(xiàn)異位骨化,其骨折愈合的速度也快于不伴中樞神經(jīng)系統(tǒng)受損的四肢長(zhǎng)骨骨折患者;相反的若是骨折患者同時(shí)伴有明顯周圍神經(jīng)損傷時(shí),骨折愈合的過(guò)程將出現(xiàn)明顯的延長(zhǎng),延遲愈合甚至是骨不愈合的可能增高。大量文獻(xiàn)表明中樞神經(jīng)受到損傷后,神經(jīng)肽類物質(zhì)在外周血液中的濃度升高,這可能是導(dǎo)致骨痂過(guò)度生長(zhǎng)的重要原因;這些神經(jīng)肽類物質(zhì)其中就包括降鈣素基因相關(guān)肽(CGRP)和P物質(zhì)等。降鈣素基因相關(guān)肽(CGRP)是一種體內(nèi)廣泛分布的神經(jīng)肽,研究發(fā)現(xiàn)其具有促成骨的作用,但是CGRP對(duì)骨吸收中破骨前體細(xì)胞即骨髓源性巨噬細(xì)胞(BMMs)的是否具有調(diào)控作用尚不清楚。因此我們課題組設(shè)計(jì)了相應(yīng)實(shí)驗(yàn),來(lái)探究CGRP對(duì)SD大鼠骨髓源性巨噬細(xì)胞(BMMs)破骨分化和骨吸收功能的影響,闡明其影響骨代謝的分子機(jī)制,為骨修復(fù)和骨重建提供新的思路。實(shí)驗(yàn)一降鈣素基因相關(guān)肽對(duì)大鼠骨髓源性巨噬細(xì)胞破骨分化的影響目的:研究降鈣素基因相關(guān)肽對(duì)大鼠骨髓源性巨噬細(xì)胞破骨分化的影響。方法:(1)采用差速貼壁的方法分離出SD大鼠骨髓源性巨噬細(xì)胞,原代培養(yǎng)并傳代,而后加入適當(dāng)濃度的s RANKL和M-CSF,進(jìn)行大鼠破骨前體細(xì)胞的破骨分化誘導(dǎo)。(2)實(shí)驗(yàn)分組:A:空白對(duì)照組(不含CGRP);B:高劑量CGRP處理組(CGRP濃度為10-7mol/L);C:中劑量CGRP處理組(CGRP濃度為10-8mol/L);D:低劑量CGRP處理組(CGRP濃度為10-9mol/L)。(3)加入不同濃度CGRP后破骨分化誘導(dǎo)培養(yǎng)7天,通過(guò)抗酒石酸酸性磷酸酶染色法(TRAP染色)觀察破骨細(xì)胞形態(tài)并對(duì)成熟的破骨細(xì)胞進(jìn)行計(jì)數(shù)分析。(4)加入不同濃度CGRP后破骨分化誘導(dǎo)7天,通過(guò)RT-PCR方法檢測(cè)破骨分化特異性特基因(RANK、TRAP、NFATc1)m RNA的表達(dá)。(5)對(duì)加入不同濃度CGRP破骨分化誘導(dǎo)7天后的破骨細(xì)胞進(jìn)行處理,通過(guò)Western-blot檢測(cè)破骨特征性TRAP和RANK蛋白的表達(dá)。結(jié)果:(1)TRAP染色顯示,相對(duì)于空白對(duì)照組不同濃度CGPR處理組的成熟破骨細(xì)胞數(shù)目顯著減少,有統(tǒng)計(jì)學(xué)差異(P0.05)并且隨CGRP濃度的增高成熟破骨細(xì)胞數(shù)減少(P0.05),組間亦有明顯差異(P0.05)。(2)RT-PCR檢測(cè)破骨分化特異性特基因RANK、TRAP、NFATc1 m RNA和Western-blot測(cè)破骨特征性TRAP、RANK蛋白的表達(dá),結(jié)果提示不同濃度CGRP組均明顯低于空白對(duì)照組,具有統(tǒng)計(jì)學(xué)差異(P0.05)。結(jié)論:本實(shí)驗(yàn)通過(guò)體外骨髓源性巨噬細(xì)胞誘導(dǎo)培養(yǎng)破骨細(xì)胞的方法,觀察到CGRP具有抑制破骨前體細(xì)胞破骨分化的作用,提示CGRP在骨修復(fù)及骨重建中可能發(fā)揮重要的作用。實(shí)驗(yàn)二降鈣素基因相關(guān)肽對(duì)大鼠骨髓源性巨噬細(xì)胞破骨誘導(dǎo)后骨吸收功能的影響目的:研究降鈣素基因相關(guān)肽對(duì)大鼠骨髓源性巨噬細(xì)胞破骨誘導(dǎo)后骨吸收功能的影響。方法:(1)采用差速貼壁的方法分離出SD大鼠骨髓源性巨噬細(xì)胞,原代培養(yǎng)并傳代,而后加入適當(dāng)濃度的s RANKL和M-CSF,進(jìn)行大鼠破骨前體細(xì)胞的破骨分化誘導(dǎo)。(2)實(shí)驗(yàn)分組:A:空白對(duì)照組(不含CGRP);B:高劑量CGRP處理組(CGRP濃度為10-7mol/L);C:中劑量CGRP處理組(CGRP濃度為10-8mol/L);D:低劑量CGRP處理組(CGRP濃度為10-9mol/L)。(3)用不含CGRP的破骨誘導(dǎo)液培養(yǎng)骨髓源性巨噬細(xì)胞7天后,采用抗酒石酸酸性磷酸酶染色法(TRAP染色)觀察破骨細(xì)胞形態(tài),并對(duì)破骨細(xì)胞進(jìn)行鑒別。(4)在不同時(shí)間點(diǎn),通過(guò)WST-1法檢測(cè)不同濃度CGRP對(duì)大鼠破骨前體細(xì)胞BMMs增殖率的影響。(5)加入不同濃度CGRP后將破骨前體細(xì)胞BMMs破骨分化誘導(dǎo)7天,通過(guò)RT-PCR檢測(cè)破骨細(xì)胞骨吸收功能性基因MMP-9、Cathepsin K m RNA的表達(dá)。(6)將BMMs接種于骨磨片上,用含有不同濃度CGRP的破骨誘導(dǎo)液誘導(dǎo)7天后,對(duì)骨磨片行甲苯胺藍(lán)染色檢測(cè)CGRP對(duì)破骨細(xì)胞的骨吸收功能的影響。結(jié)果:(1)與空白對(duì)照組相比,各CGRP處理組對(duì)破骨前體細(xì)胞BMMs的增殖具有抑制作用,且隨著CGRP濃度的升高,抑制作用更加明顯。(2)RT-PCR結(jié)果顯示,與空白對(duì)照組相比,CGRP處理組明顯抑制破骨相關(guān)酶MMP-9和Cathepsin K m RNA的表達(dá)。(3)甲苯胺藍(lán)骨磨片染色顯示,與空白對(duì)照組相比,CGRP處理組的骨陷窩數(shù)目明顯減少,具有統(tǒng)計(jì)學(xué)差異(P0.05),且CGRP處理組濃度與陷窩數(shù)量呈負(fù)相關(guān)性。結(jié)論:本實(shí)驗(yàn)通過(guò)體外骨髓源性巨噬細(xì)胞誘導(dǎo)培養(yǎng)破骨細(xì)胞的方法,觀察到CGRP具有抑制破骨前體細(xì)胞增殖以及抑制骨吸收功能的作用,為CGRP臨床治療骨折提供了重要的理論依據(jù)。
[Abstract]:Clinically, we find that patients with central nervous system injury, such as paraplegia or craniocerebral injury, are often accompanied by long bone fractures in the extremities, often in a large number of excessively growing callus in the fracture area, serious or even ectopic ossification, and the rate of fracture healing is faster than that of the extremities fracture of the extremities without the impairment of the central nervous system. Conversely, if a fracture patient is accompanied by an obvious peripheral nerve injury, the process of fracture healing will appear significantly prolonged, delayed union or even a possible increase in bone nonunion. A large number of documents suggest that the concentration of neuropeptides in the peripheral blood increases after the injury of the central nervous system, which may lead to excessive growth of the callus. The important reasons are that these neuropeptides include calcitonin gene related peptide (CGRP) and substance P. The calcitonin gene related peptide (CGRP) is a widely distributed neuropeptide in the body, and it has been found to have the role of inducing bone, but CGRP has a effect on the bone resorption of osteoclast, namely, bone marrow derived macrophages (BMMs). There is no clear regulation. Therefore, we have designed a corresponding experiment to explore the effect of CGRP on bone destruction and bone resorption of bone marrow derived macrophages (BMMs) in SD rats, elucidate the molecular mechanism of its effect on bone metabolism, and provide a new way for bone repair and bone reconstruction. The effect of the osteoclast differentiation of sex macrophage Objective: To study the effect of calcitonin gene related peptide on the osteoclast differentiation of rat bone marrow derived macrophages. Methods: (1) the bone marrow derived macrophages of SD rats were separated by differential adherence, and the primary culture and generation were carried out, then the appropriate concentration of s RANKL and M-CSF were added to the rat bone marrow precursor. Osteoclast differentiation induction. (2) experimental groups: A: blank control group (without CGRP); B: high dose CGRP treatment group (CGRP concentration 10-7mol/L); C: medium dose CGRP treatment group (CGRP concentration 10-8mol/L); D: low dose CGRP treatment group (3) after adding different concentrations of osteoclast differentiation induction culture for 7 days, through the acidity of tartaric acid The morphology of osteoclasts was observed by phosphatase staining (TRAP staining) and the mature osteoclasts were counted and analyzed. (4) after adding different concentrations of CGRP, the osteoclast differentiation was induced for 7 days, and the expression of the specific specific gene of osteoclast (RANK, TRAP, NFATc1) m RNA was detected by the RT-PCR method. (5) the rupture of the osteoclast with different concentrations of osteoclast was induced for 7 days. Bone cells were treated and the expression of osteoclast characteristic TRAP and RANK protein were detected by Western-blot. Results: (1) TRAP staining showed that the number of mature osteoclasts in the CGPR treatment group with different concentrations in the blank control group decreased significantly (P0.05) and the number of mature osteoclasts decreased with the increase of CGRP concentration (P0.05), and the number of mature osteoclasts decreased with the concentration of CGRP (P0.05). There were also significant differences (P0.05). (2) RT-PCR detected osteoclast specific specific gene RANK, TRAP, NFATc1 m RNA and Western-blot to detect bone characteristic TRAP and RANK protein expression. The results showed that the different concentrations of CGRP group were significantly lower than those in the blank control group, with statistical difference (P0.05). Conclusion: this experiment was induced by bone marrow derived macrophages in vitro. To guide the cultivation of osteoclast, the effect of CGRP on osteoclast differentiation of osteoclast cells was observed, suggesting that CGRP may play an important role in bone repair and bone reconstruction. Experiment two the effect of calcitonin gene related peptide on bone resorption after bone marrow induced bone marrow induced bone destruction in rats: the study of calcitonin gene phase The effect of peptide on bone resorption after osteoclast induced bone marrow macrophages in rats. Methods: (1) the bone marrow derived macrophages of SD rats were separated by differential adherence, and the primary culture and generation were used, and then the appropriate concentration of s RANKL and M-CSF were added to the osteoclast differentiation induction of the rat osteoclast cells. (2) the experimental group: A: blank The control group (without CGRP), B: high dose CGRP treatment group (CGRP concentration was 10-7mol/L), C: medium dose CGRP treatment group (CGRP concentration 10-8mol/L), D: low dose CGRP treatment group (CGRP concentration). (3) culture of bone marrow derived megagaric cells with no osteoclast induction solution for 7 days, using tartaric acid acid phosphatase staining (staining) view The morphology of bone cells was detected and the osteoclasts were identified. (4) at different time points, the effect of different concentrations of CGRP on the proliferation rate of BMMs in rat osteoclast cells was detected by WST-1. (5) after adding different concentrations of CGRP, the osteoclast differentiation of the osteoclast cells was induced for 7 days, and RT-PCR was used to detect the bone resorption functional gene MMP-9, Ca The expression of thepsin K m RNA. (6) inoculating BMMs on the bone grinding plate and using the osteoclast induction solution containing different concentrations of CGRP to induce the bone resorption of the osteoclast by the staining of toluidine blue on the bone mill. Results: (1) the proliferation of the BMMs in the osteoclast cells was inhibited by each CGRP treatment group compared with the blank control group. The inhibitory effect was more obvious with the increase of CGRP concentration. (2) RT-PCR results showed that compared with the blank control group, the CGRP treatment group obviously inhibited the expression of osteoclast related enzyme MMP-9 and Cathepsin K m RNA. (3) toluidine blue bone grinding plate staining showed that the number of bone lacunae in the CGRP treatment group decreased significantly compared with the blank control group. There was a negative correlation between the study difference (P0.05), and the concentration of CGRP treatment group was negatively correlated with the number of lacunae. Conclusion: this experiment shows that CGRP can inhibit the proliferation of osteoclast cells and inhibit bone resorption by inducing the culture of osteoclasts from bone marrow derived macrophages in vitro. It provides an important theoretical basis for the fracture of CGRP in the clinical treatment of fracture. According to it.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R68

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