降钙素基因相关肽对大鼠骨髓源性巨噬细胞破骨分化和骨吸收功能影响的实验研究

发布时间：2018-07-13 18:56

【摘要】：在临床上我们发现中枢神经系统损伤(如截瘫或者颅脑损伤)的患者若同时伴有四肢长骨骨折时,常常在骨折区出现大量骨痂的过度生长,严重的甚至出现异位骨化,其骨折愈合的速度也快于不伴中枢神经系统受损的四肢长骨骨折患者;相反的若是骨折患者同时伴有明显周围神经损伤时,骨折愈合的过程将出现明显的延长,延迟愈合甚至是骨不愈合的可能增高。大量文献表明中枢神经受到损伤后,神经肽类物质在外周血液中的浓度升高,这可能是导致骨痂过度生长的重要原因;这些神经肽类物质其中就包括降钙素基因相关肽(CGRP)和P物质等。降钙素基因相关肽(CGRP)是一种体内广泛分布的神经肽,研究发现其具有促成骨的作用,但是CGRP对骨吸收中破骨前体细胞即骨髓源性巨噬细胞(BMMs)的是否具有调控作用尚不清楚。因此我们课题组设计了相应实验,来探究CGRP对SD大鼠骨髓源性巨噬细胞(BMMs)破骨分化和骨吸收功能的影响,阐明其影响骨代谢的分子机制,为骨修复和骨重建提供新的思路。实验一降钙素基因相关肽对大鼠骨髓源性巨噬细胞破骨分化的影响目的:研究降钙素基因相关肽对大鼠骨髓源性巨噬细胞破骨分化的影响。方法:(1)采用差速贴壁的方法分离出SD大鼠骨髓源性巨噬细胞,原代培养并传代,而后加入适当浓度的s RANKL和M-CSF,进行大鼠破骨前体细胞的破骨分化诱导。(2)实验分组:A:空白对照组(不含CGRP);B:高剂量CGRP处理组(CGRP浓度为10-7mol/L);C:中剂量CGRP处理组(CGRP浓度为10-8mol/L);D:低剂量CGRP处理组(CGRP浓度为10-9mol/L)。(3)加入不同浓度CGRP后破骨分化诱导培养7天,通过抗酒石酸酸性磷酸酶染色法(TRAP染色)观察破骨细胞形态并对成熟的破骨细胞进行计数分析。(4)加入不同浓度CGRP后破骨分化诱导7天,通过RT-PCR方法检测破骨分化特异性特基因(RANK、TRAP、NFATc1)m RNA的表达。(5)对加入不同浓度CGRP破骨分化诱导7天后的破骨细胞进行处理,通过Western-blot检测破骨特征性TRAP和RANK蛋白的表达。结果:(1)TRAP染色显示,相对于空白对照组不同浓度CGPR处理组的成熟破骨细胞数目显著减少,有统计学差异(P0.05)并且随CGRP浓度的增高成熟破骨细胞数减少(P0.05),组间亦有明显差异(P0.05)。(2)RT-PCR检测破骨分化特异性特基因RANK、TRAP、NFATc1 m RNA和Western-blot测破骨特征性TRAP、RANK蛋白的表达,结果提示不同浓度CGRP组均明显低于空白对照组,具有统计学差异(P0.05)。结论:本实验通过体外骨髓源性巨噬细胞诱导培养破骨细胞的方法,观察到CGRP具有抑制破骨前体细胞破骨分化的作用,提示CGRP在骨修复及骨重建中可能发挥重要的作用。实验二降钙素基因相关肽对大鼠骨髓源性巨噬细胞破骨诱导后骨吸收功能的影响目的:研究降钙素基因相关肽对大鼠骨髓源性巨噬细胞破骨诱导后骨吸收功能的影响。方法:(1)采用差速贴壁的方法分离出SD大鼠骨髓源性巨噬细胞,原代培养并传代,而后加入适当浓度的s RANKL和M-CSF,进行大鼠破骨前体细胞的破骨分化诱导。(2)实验分组:A:空白对照组(不含CGRP);B:高剂量CGRP处理组(CGRP浓度为10-7mol/L);C:中剂量CGRP处理组(CGRP浓度为10-8mol/L);D:低剂量CGRP处理组(CGRP浓度为10-9mol/L)。(3)用不含CGRP的破骨诱导液培养骨髓源性巨噬细胞7天后,采用抗酒石酸酸性磷酸酶染色法(TRAP染色)观察破骨细胞形态,并对破骨细胞进行鉴别。(4)在不同时间点,通过WST-1法检测不同浓度CGRP对大鼠破骨前体细胞BMMs增殖率的影响。(5)加入不同浓度CGRP后将破骨前体细胞BMMs破骨分化诱导7天,通过RT-PCR检测破骨细胞骨吸收功能性基因MMP-9、Cathepsin K m RNA的表达。(6)将BMMs接种于骨磨片上,用含有不同浓度CGRP的破骨诱导液诱导7天后,对骨磨片行甲苯胺蓝染色检测CGRP对破骨细胞的骨吸收功能的影响。结果:(1)与空白对照组相比,各CGRP处理组对破骨前体细胞BMMs的增殖具有抑制作用,且随着CGRP浓度的升高,抑制作用更加明显。(2)RT-PCR结果显示,与空白对照组相比,CGRP处理组明显抑制破骨相关酶MMP-9和Cathepsin K m RNA的表达。(3)甲苯胺蓝骨磨片染色显示,与空白对照组相比,CGRP处理组的骨陷窝数目明显减少,具有统计学差异(P0.05),且CGRP处理组浓度与陷窝数量呈负相关性。结论:本实验通过体外骨髓源性巨噬细胞诱导培养破骨细胞的方法,观察到CGRP具有抑制破骨前体细胞增殖以及抑制骨吸收功能的作用,为CGRP临床治疗骨折提供了重要的理论依据。
[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.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R68

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