降钙素基因相关肽对氧化损伤的小鼠成骨细胞的保护作用研究
发布时间：2018-09-12 10:40
【摘要】：机体在正常代谢中的不同阶段均会产生或多或少的活性氧(Reactive oxygen species,ROS)[1];当机体遭受创伤时,自身抗氧化能力下降、ROS产生增多,同时机体自身防御机制启动,产生大量的IL-6、IL-1β、TNF-α等炎症因子,当ROS和各种炎症因子同时作用于成骨细胞时,成骨细胞的增殖、分化水平降低,骨创伤愈合减缓,甚至停滞[2]。骨创伤愈合是一个复杂的连续的病理生理过程,包括多种分子信号、细胞因子的参与。[3]有实验证实,神经系统参与骨重塑。在机体骨折或者其他外伤时,外周肽能神经可以通过一些神经肽影响破骨细胞的形成,进而影响骨折或者其他外伤的愈合。降钙素基因相关肽(Calcitonin gene-related peptide,CGRP)是一种由特定神经元的可变剪接生成的37个氨基酸组成,是骨修复和发育过程中神经纤维所表达的一种重要的神经肽。[4]本课题组前期研究[4]发现,CGRP在骨创伤愈合的过程中,部分通过促进成骨细胞增殖分化来发挥其促进修复作用,本实验将进一步证实是否有部分是通过保护成骨细胞氧化损伤来实现其促进修复作用。[5]本实验拟构建小鼠成骨细胞的氧化损伤模型,通过对ROS的含量、超氧化物歧化酶(Superoxide dismutase,SOD)活性以及炎症因子白细胞介素(Interleukin,IL)-6、IL-1β、肿瘤坏死因子(Tumor necrosis factor,TNF)-α水平的检测,来探索CGRP对小鼠成骨细胞的抗氧化损伤保护作用及其相关机制。研究方法:1.体外分离、培养、鉴定原代Balb/c小鼠成骨细胞。2.在体外用过氧化氢(H_2O_2)构建小鼠成骨细胞氧化损伤模型。3.使用不同浓度的CGRP(10-6、10-7、10-8、10-9、10-10mol/L)预处理在体外培养的小鼠成骨细胞1h后,CCK(Cell counting kit)-8法检测各组细胞活性并筛选优势浓度。4.使用SOD试剂盒对H_2O_2和CGRP处理后的小鼠成骨细胞进行SOD活性检测。5.使用ROS试剂盒对H_2O_2和CGRP处理后的小鼠成骨细胞进行ROS含量检测。6.使用小鼠IL-6、IL-1β、TNF-α定量分析酶联免疫吸附测定(Enzyme-linked immuno sorbent assay,ELISA)试剂盒检测H_2O_2和CGRP处理后的小鼠成骨细胞。结果:1.成功分离并纯化扩增Balb/c小鼠成骨细胞。2.在体外小鼠成骨细胞培养基中加入H_2O_2,成功构建小鼠成骨细胞氧化损伤模型。使用CCK-8法检测细胞活性,小鼠成骨细胞在含不同浓度(10-1、10-2、10-3、10-4、10-5mol/L)H_2O_2的培养液的条件下培养12、24、36、48h后,当培养液使用的是含10-4mol/L的H_2O_2培养液培养时,细胞的增殖活性开始受到抑制(P0.01)。3.CCK-8法结果显示,小鼠成骨细胞在含不同浓度(10-6、10-7、10-8、10-9、10-10mol/L)CGRP的培养液的条件下预处理1h后,当使用的培养液是含10-8mol/L的CGRP培养液培养时,细胞的增殖活性最高(P0.01)。4.SOD试剂盒检测结果提示,CGRP组SOD活性与空白对照组相比,明显升高(P0.05);H_2O_2组SOD活性较空白对照组相比受到明显抑制(P0.05);CGRP+H_2O_2组SOD活性相对于H_2O_2组是显著升高的(P0.01)。5.ROS试剂盒检测结果提示,H_2O_2处理后,ROS含量相对于对照组显著增高(P0.01);先使用10-8 mol/L CGRP预处理后再用H_2O_2处理,其ROS含量与单独使用H_2O_2处理相比显著降低(P0.01)。6.小鼠IL-6、IL-1β、TNF-αELISA试剂盒结果提示,使用10-4mol/L H_2O_2的培养液处理后,小鼠成骨细胞IL-6、IL-1β、TNF-α分泌与空白对照组相比,明显增高(P0.01);而预处理使用含10-8 mol/L CGRP的培养液后接着再使用H_2O_2处理,IL-6、IL-1β、TNF-α分泌水平相对于H_2O_2组明显降低(P0.05)。结论:1.使用贴壁筛选手段可以成功的从Balb/c小鼠颅骨获取所需要的成骨细胞2.H_2O_2能够成功构建小鼠成骨细胞氧化损伤模型,H_2O_2对小鼠成骨细胞会造成氧化损伤3.CGRP对小鼠成骨细胞具有促进增殖,保护氧化损伤作用,其优势浓度为10-8mol/L
[Abstract]:The body produces more or less reactive oxygen species (ROS) at different stages of normal metabolism; when the body suffers from trauma, its own antioxidant capacity decreases, ROS production increases, and the body's self-defense mechanism starts, producing a large number of inflammatory factors, such as IL-6, IL-1beta, TNF-a, when ROS and various inflammatory factors work together. Bone wound healing is a complex and continuous pathophysiological process involving multiple molecular signals and cytokines. Peptidyl nerves can affect osteoclast formation and fracture or other traumatic healing through some neuropeptides. Calcitonin gene-related peptide (CGRP) is a 37-amino-acid compound produced by the variable splicing of specific neurons and is expressed in nerve fibers during bone repair and development. [4] Preliminary study of our group [4] found that CGRP partially promotes osteoblast proliferation and differentiation in the process of bone wound healing, and this experiment will further confirm whether CGRP partially promotes osteoblast repair by protecting the oxidative damage of osteoblasts. In order to explore the anti-oxidative damage of CGRP on mouse osteoblasts, the oxidative damage model of mouse osteoblasts was established by detecting the content of ROS, the activity of superoxide dismutase (SOD), the levels of inflammatory factors interleukin (IL) - 6, IL-1beta and tumor necrosis factor (TNF) - alpha. METHODS: 1. Isolation, culture and identification of primary Balb / c mouse osteoblasts in vitro. 2. Establishment of mouse osteoblasts oxidative damage model by hydrogen peroxide (H_2O_2) in vitro. 3. Pretreatment of mouse osteoblasts with different concentrations of CGRP (10-6, 10-7, 10-8, 10-9, 10-10-10mol/L) for 1 hour after in vitro culture, CCK (Cell Cell SOD activity of mouse osteoblasts treated with H_2O_2 and CGRP was detected with SOD kit. ROS content of mouse osteoblasts treated with H_2O_2 and CGRP was detected with ROS kit. 6. Enzyme-linked immunosorbent assay (ELISA) was performed with mouse IL-6, IL-1beta and TNF-alpha. Enzyme-linked immuno sorbent assay (ELISA) kit was used to detect mouse osteoblasts treated with H_2O_2 and CGRP. Results: 1. Balb/c mouse osteoblasts were successfully isolated and amplified. 2. The oxidative damage model of mouse osteoblasts was successfully constructed by adding H_2O_2 into the culture medium of mouse osteoblasts in vitro. CCK-8 method was used to detect the fineness of osteoblasts. Cell viability. After 12,24,36,48 hours of incubation with different concentrations (10-1,10-2,10-3,10-4,10-5 mol/L) of H_2O_2, the proliferation activity of mouse osteoblasts was inhibited (P 0.01). 3. CCK-8 assay showed that mouse osteoblasts were cultured with different concentrations of H_2O_2 (10-1,10-2,10-3,10-4,10-5 mol/L). (10-6,10-7,10-8,10-9,10-10 mol/L) CGRP culture medium pretreated for 1 hour, when the culture medium was CGRP culture medium containing 10-8 mol/L, the cell proliferation activity was the highest (P 0.01). 4. SOD kit test results showed that the activity of SOD in CGRP group was significantly higher than that in blank control group (P 0.05); the activity of SOD in H_2O_2 group was higher than that in blank control group (P 0.05). The SOD activity of CGRP+H_2O_2 group was significantly higher than that of H_2O_2 group (P 0.01). The results of ROS kit test showed that the ROS content of CGRP+H_2O_2 group was significantly higher than that of control group (P 0.01); the ROS content of CGRP+H_2O_2 group was higher than that of H_2O_2 group after 10-8 mol/L CGRP pretreatment and then H_2O_2 pretreatment. The results of mouse IL-6, IL-1beta and TNF-alpha ELISA kit suggested that the secretion of IL-6, IL-1beta and TNF-alpha in mouse osteoblasts treated with 10-4mol/L H_2O_2 was significantly higher than that in the control group (P 0.01). After pretreatment, the culture medium containing 10-8 mol/L CGRP was used and then treated with H_2O_2, IL-6, TNF-1beta, IL-1beta, and TNF-alpha. The level of alpha secretion was significantly lower than that of H_2O_2 group (P 0.05). CONCLUSION: 1. Osteoblasts 2.H_2O_2 can be successfully obtained from Balb/c mice skull by adherent screening method, and the oxidative damage model of mouse osteoblasts can be successfully constructed. H_2O_2 can cause oxidative damage to mouse osteoblasts. 3. CGRP can promote the oxidative damage of mouse osteoblasts. Proliferation and protection of oxidative damage, its dominant concentration is 10-8mol/L
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R782
本文编号：2238747
[Abstract]:The body produces more or less reactive oxygen species (ROS) at different stages of normal metabolism; when the body suffers from trauma, its own antioxidant capacity decreases, ROS production increases, and the body's self-defense mechanism starts, producing a large number of inflammatory factors, such as IL-6, IL-1beta, TNF-a, when ROS and various inflammatory factors work together. Bone wound healing is a complex and continuous pathophysiological process involving multiple molecular signals and cytokines. Peptidyl nerves can affect osteoclast formation and fracture or other traumatic healing through some neuropeptides. Calcitonin gene-related peptide (CGRP) is a 37-amino-acid compound produced by the variable splicing of specific neurons and is expressed in nerve fibers during bone repair and development. [4] Preliminary study of our group [4] found that CGRP partially promotes osteoblast proliferation and differentiation in the process of bone wound healing, and this experiment will further confirm whether CGRP partially promotes osteoblast repair by protecting the oxidative damage of osteoblasts. In order to explore the anti-oxidative damage of CGRP on mouse osteoblasts, the oxidative damage model of mouse osteoblasts was established by detecting the content of ROS, the activity of superoxide dismutase (SOD), the levels of inflammatory factors interleukin (IL) - 6, IL-1beta and tumor necrosis factor (TNF) - alpha. METHODS: 1. Isolation, culture and identification of primary Balb / c mouse osteoblasts in vitro. 2. Establishment of mouse osteoblasts oxidative damage model by hydrogen peroxide (H_2O_2) in vitro. 3. Pretreatment of mouse osteoblasts with different concentrations of CGRP (10-6, 10-7, 10-8, 10-9, 10-10-10mol/L) for 1 hour after in vitro culture, CCK (Cell Cell SOD activity of mouse osteoblasts treated with H_2O_2 and CGRP was detected with SOD kit. ROS content of mouse osteoblasts treated with H_2O_2 and CGRP was detected with ROS kit. 6. Enzyme-linked immunosorbent assay (ELISA) was performed with mouse IL-6, IL-1beta and TNF-alpha. Enzyme-linked immuno sorbent assay (ELISA) kit was used to detect mouse osteoblasts treated with H_2O_2 and CGRP. Results: 1. Balb/c mouse osteoblasts were successfully isolated and amplified. 2. The oxidative damage model of mouse osteoblasts was successfully constructed by adding H_2O_2 into the culture medium of mouse osteoblasts in vitro. CCK-8 method was used to detect the fineness of osteoblasts. Cell viability. After 12,24,36,48 hours of incubation with different concentrations (10-1,10-2,10-3,10-4,10-5 mol/L) of H_2O_2, the proliferation activity of mouse osteoblasts was inhibited (P 0.01). 3. CCK-8 assay showed that mouse osteoblasts were cultured with different concentrations of H_2O_2 (10-1,10-2,10-3,10-4,10-5 mol/L). (10-6,10-7,10-8,10-9,10-10 mol/L) CGRP culture medium pretreated for 1 hour, when the culture medium was CGRP culture medium containing 10-8 mol/L, the cell proliferation activity was the highest (P 0.01). 4. SOD kit test results showed that the activity of SOD in CGRP group was significantly higher than that in blank control group (P 0.05); the activity of SOD in H_2O_2 group was higher than that in blank control group (P 0.05). The SOD activity of CGRP+H_2O_2 group was significantly higher than that of H_2O_2 group (P 0.01). The results of ROS kit test showed that the ROS content of CGRP+H_2O_2 group was significantly higher than that of control group (P 0.01); the ROS content of CGRP+H_2O_2 group was higher than that of H_2O_2 group after 10-8 mol/L CGRP pretreatment and then H_2O_2 pretreatment. The results of mouse IL-6, IL-1beta and TNF-alpha ELISA kit suggested that the secretion of IL-6, IL-1beta and TNF-alpha in mouse osteoblasts treated with 10-4mol/L H_2O_2 was significantly higher than that in the control group (P 0.01). After pretreatment, the culture medium containing 10-8 mol/L CGRP was used and then treated with H_2O_2, IL-6, TNF-1beta, IL-1beta, and TNF-alpha. The level of alpha secretion was significantly lower than that of H_2O_2 group (P 0.05). CONCLUSION: 1. Osteoblasts 2.H_2O_2 can be successfully obtained from Balb/c mice skull by adherent screening method, and the oxidative damage model of mouse osteoblasts can be successfully constructed. H_2O_2 can cause oxidative damage to mouse osteoblasts. 3. CGRP can promote the oxidative damage of mouse osteoblasts. Proliferation and protection of oxidative damage, its dominant concentration is 10-8mol/L
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R782
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