【摘要】：機(jī)體在正常代謝中的不同階段均會(huì)產(chǎn)生或多或少的活性氧(Reactive oxygen species,ROS)[1];當(dāng)機(jī)體遭受創(chuàng)傷時(shí),自身抗氧化能力下降、ROS產(chǎn)生增多,同時(shí)機(jī)體自身防御機(jī)制啟動(dòng),產(chǎn)生大量的IL-6、IL-1β、TNF-α等炎癥因子,當(dāng)ROS和各種炎癥因子同時(shí)作用于成骨細(xì)胞時(shí),成骨細(xì)胞的增殖、分化水平降低,骨創(chuàng)傷愈合減緩,甚至停滯[2]。骨創(chuàng)傷愈合是一個(gè)復(fù)雜的連續(xù)的病理生理過程,包括多種分子信號(hào)、細(xì)胞因子的參與。[3]有實(shí)驗(yàn)證實(shí),神經(jīng)系統(tǒng)參與骨重塑。在機(jī)體骨折或者其他外傷時(shí),外周肽能神經(jīng)可以通過一些神經(jīng)肽影響破骨細(xì)胞的形成,進(jìn)而影響骨折或者其他外傷的愈合。降鈣素基因相關(guān)肽(Calcitonin gene-related peptide,CGRP)是一種由特定神經(jīng)元的可變剪接生成的37個(gè)氨基酸組成,是骨修復(fù)和發(fā)育過程中神經(jīng)纖維所表達(dá)的一種重要的神經(jīng)肽。[4]本課題組前期研究[4]發(fā)現(xiàn),CGRP在骨創(chuàng)傷愈合的過程中,部分通過促進(jìn)成骨細(xì)胞增殖分化來發(fā)揮其促進(jìn)修復(fù)作用,本實(shí)驗(yàn)將進(jìn)一步證實(shí)是否有部分是通過保護(hù)成骨細(xì)胞氧化損傷來實(shí)現(xiàn)其促進(jìn)修復(fù)作用。[5]本實(shí)驗(yàn)擬構(gòu)建小鼠成骨細(xì)胞的氧化損傷模型,通過對(duì)ROS的含量、超氧化物歧化酶(Superoxide dismutase,SOD)活性以及炎癥因子白細(xì)胞介素(Interleukin,IL)-6、IL-1β、腫瘤壞死因子(Tumor necrosis factor,TNF)-α水平的檢測(cè),來探索CGRP對(duì)小鼠成骨細(xì)胞的抗氧化損傷保護(hù)作用及其相關(guān)機(jī)制。研究方法:1.體外分離、培養(yǎng)、鑒定原代Balb/c小鼠成骨細(xì)胞。2.在體外用過氧化氫(H_2O_2)構(gòu)建小鼠成骨細(xì)胞氧化損傷模型。3.使用不同濃度的CGRP(10-6、10-7、10-8、10-9、10-10mol/L)預(yù)處理在體外培養(yǎng)的小鼠成骨細(xì)胞1h后,CCK(Cell counting kit)-8法檢測(cè)各組細(xì)胞活性并篩選優(yōu)勢(shì)濃度。4.使用SOD試劑盒對(duì)H_2O_2和CGRP處理后的小鼠成骨細(xì)胞進(jìn)行SOD活性檢測(cè)。5.使用ROS試劑盒對(duì)H_2O_2和CGRP處理后的小鼠成骨細(xì)胞進(jìn)行ROS含量檢測(cè)。6.使用小鼠IL-6、IL-1β、TNF-α定量分析酶聯(lián)免疫吸附測(cè)定(Enzyme-linked immuno sorbent assay,ELISA)試劑盒檢測(cè)H_2O_2和CGRP處理后的小鼠成骨細(xì)胞。結(jié)果:1.成功分離并純化擴(kuò)增Balb/c小鼠成骨細(xì)胞。2.在體外小鼠成骨細(xì)胞培養(yǎng)基中加入H_2O_2,成功構(gòu)建小鼠成骨細(xì)胞氧化損傷模型。使用CCK-8法檢測(cè)細(xì)胞活性,小鼠成骨細(xì)胞在含不同濃度(10-1、10-2、10-3、10-4、10-5mol/L)H_2O_2的培養(yǎng)液的條件下培養(yǎng)12、24、36、48h后,當(dāng)培養(yǎng)液使用的是含10-4mol/L的H_2O_2培養(yǎng)液培養(yǎng)時(shí),細(xì)胞的增殖活性開始受到抑制(P0.01)。3.CCK-8法結(jié)果顯示,小鼠成骨細(xì)胞在含不同濃度(10-6、10-7、10-8、10-9、10-10mol/L)CGRP的培養(yǎng)液的條件下預(yù)處理1h后,當(dāng)使用的培養(yǎng)液是含10-8mol/L的CGRP培養(yǎng)液培養(yǎng)時(shí),細(xì)胞的增殖活性最高(P0.01)。4.SOD試劑盒檢測(cè)結(jié)果提示,CGRP組SOD活性與空白對(duì)照組相比,明顯升高(P0.05);H_2O_2組SOD活性較空白對(duì)照組相比受到明顯抑制(P0.05);CGRP+H_2O_2組SOD活性相對(duì)于H_2O_2組是顯著升高的(P0.01)。5.ROS試劑盒檢測(cè)結(jié)果提示,H_2O_2處理后,ROS含量相對(duì)于對(duì)照組顯著增高(P0.01);先使用10-8 mol/L CGRP預(yù)處理后再用H_2O_2處理,其ROS含量與單獨(dú)使用H_2O_2處理相比顯著降低(P0.01)。6.小鼠IL-6、IL-1β、TNF-αELISA試劑盒結(jié)果提示,使用10-4mol/L H_2O_2的培養(yǎng)液處理后,小鼠成骨細(xì)胞IL-6、IL-1β、TNF-α分泌與空白對(duì)照組相比,明顯增高(P0.01);而預(yù)處理使用含10-8 mol/L CGRP的培養(yǎng)液后接著再使用H_2O_2處理,IL-6、IL-1β、TNF-α分泌水平相對(duì)于H_2O_2組明顯降低(P0.05)。結(jié)論:1.使用貼壁篩選手段可以成功的從Balb/c小鼠顱骨獲取所需要的成骨細(xì)胞2.H_2O_2能夠成功構(gòu)建小鼠成骨細(xì)胞氧化損傷模型,H_2O_2對(duì)小鼠成骨細(xì)胞會(huì)造成氧化損傷3.CGRP對(duì)小鼠成骨細(xì)胞具有促進(jìn)增殖,保護(hù)氧化損傷作用,其優(yōu)勢(shì)濃度為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
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R782

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