【作者】 李焰；

【导师】 谭颖徽；

【作者基本信息】 第三军医大学 ， 外科学， 2011， 博士

【摘要】 周围神经所分泌的神经肽类物质对骨折修复重建和骨代谢具有重要生物学调控作用,部分神经肽类物质的促成骨作用在体内外实验中分别得以证实。降钙素基因相关肽（Calcitonin gene related peptide , CGRP）是其中研究较为详尽的一类,自从在骨痂中发现CGRP阳性神经纤维表达开始,体内实验通过转基因小鼠和基因敲除小鼠证明了CGRP的促成骨效应,体外实验也发现了成骨细胞表面有CGRP受体存在,证实了CGRP促进成骨细胞（Osteoblast,OB）增殖的生物学效应,并发现了其下游的环磷酸腺苷（Cyclic Adenosine monophosphatec, cAMP）,细胞内游离钙离子(Intracellular Ca²⁺, [Ca²⁺]_i)和丝裂原激活激酶（Mitogen-activated protein kinase MAPK）等信号通路。一氧化氮（Nitric oxide, NO）作为一种信号传导分子,是人体内常见的第二信使,研究证明NO能促进骨折愈合初期的血管生成,调节成骨细胞及破骨细胞活性,但目前尚无研究证明NO是否参与到CGRP对成骨细胞的调控信号通路中。体内试验发现一氧化氮合成酶（Nitric oxide synthase, NOS）的内皮型NOS（endothelial NOS, eNOS）,神经元型nNOS（neuronal NOS, nNOS）和诱导型NOS（inducible NOS, iNOS）三类亚型在骨折愈合过程中的不同时期均有表达,各自发挥不同的生理作用。人体对NO生成的调控主要通过以下两种方式进行,一是通过调节细胞内结构型NOS（constitutive NOS, cNOS）的酶活力,包括了eNOS和nNOS,二是通过调节iNOS基因表达,调节细胞内NO浓度。不同浓度的NO对成骨细胞有截然相反的生物学作用,cNOS所合成的持续低浓度的NO可以促进成骨细胞的增殖和骨基质分泌功能,同时促进前体破骨细胞向破骨细胞转换和提高破骨细胞的细胞活性。由细胞炎症因子所诱导iNOS产生的较高浓度NO对细胞有明显的细胞毒性作用,高浓度的NO则会抑制成骨细胞DNA合成、Ⅰ型胶原蛋白（CollegenⅠ）、骨钙素（Osteocalcin, OC）产生和碱性磷酸酶（Alkaline phosphatase,ALP）活性等。本实验旨在通过体外培养成骨样细胞株MG-63,验证CGRP对MG-63细胞的增殖分化的生物学作用;在明确CGRP对MG-63细胞的生物学作用的基础上,探讨CGRP对NOS酶活力和蛋白表达的调控,以及该调控对细胞NO生成的作用;在明确CGRP与NO生成的相关性后,探讨CGRP对NO生成的调控机制;最后探讨MG-63细胞中CGRP-NO调控的生物学意义。方法:1.细胞培养:人骨肉瘤细胞MG-63细胞株购买于ATCC公司,以5×10⁶/瓶密度接种于100ml培养瓶中培养传代,加入含10% FCS的高糖DMEM培养基,置于5% CO₂孵箱中37°C孵育72或96h传代,6-8代细胞用于实验。2. CGRP对MG-63细胞增殖和分化的作用:2.1增殖:MTT法检测CGRP对MG-63细胞增殖的量效作用(10^–10 -10^–7mol/L)和时效作用（24-96 h）;流式细胞仪检测110^–78 mol/L浓度的CGRP对MG-63细胞周期的时效作用（0-24 h）;RT-PCR检测10^–8 mol/L浓度的CGRP对MG-63的c-fos/c-jun mRNA表达时效作用（0-80 m）;2.2分化:改良Kaplow氏法检测CGRP对MG-63细胞ALP染色的量效作用(10–10 -10^–7mol/L);茜素红染色检测CGRP对MG-63钙化结节的量效作用(10–10 -10^–7mol/L);RT-PCR检测CGRP对MG-63细胞CollagenⅠmRNA表达的量效作用(10^–10 -10–7mol/L);ELISA检测CGRP对MG-63细胞OC表达的量效作用(10^–10 -10^–7mol/L)。3. CGRP对MG-63细胞NO的调控作用:Griess法检测10^–8 mol/L的CGRP对MG-63细胞培养液中NO浓度时效作用（0-48 h）;激光共聚焦DAF-FM DA荧光检测10^–8 mol/L的CGRP对MG-63细胞中NO浓度时效作用（0-4 h）;NOS酶活力试剂盒检测10^–8 mol/L的CGRP在作用1 h后对MG-63细胞eNOS、nNOS和iNOS亚型酶活力的调控作用;免疫荧光检测10^–8mol/L的CGRP对MG-63细胞中eNOS、nNOS和iNOS亚型表达的时效作用（0-48 h）。4. CGRP对MG-63细胞NO的调控机制:4.1 CGRP通过[Ca²⁺]_i对MG-63细胞eNOS酶活力的调控:激光共聚焦Fluo-3/AM荧光检测10^–8mol/L的CGRP对MG-63细胞中[Ca²⁺]_i的时效作用（0-15 m）;激光共聚焦DAF-FM DA荧光检测L型Ca²⁺通道抑制剂verpamil对CGRP诱导的细胞内NO浓度增高的抑制作用;Griess法检测verpamil对CGRP诱导的细胞培养液中NO浓度增高的抑制作用。4.2 CGRP对MG-63细胞iNOSmRNA表达的调控作用: Real time PCR检测CGRP对MG-63 iNOS mRNA表达的时效作用（0-48 h）;Realtime PCR检测,在炎症因子TNF-α和INF-γ诱导下CGRP对MG-63 iNOS mRNA表达的作用。5. MG-63中CGRP-NO调控作用的生物学意义:5.1增殖:MTT法检测eNOS抑制剂,50umol/L的L-NAME对MG-63细胞增殖的时效作用（24-96 h）;MTT法检测L-NAME对CGRP诱导的MG-63促增殖效应的抑制作用;流式细胞仪检测L-NAME对CGRP诱导MG-63细胞周期改变抑制作用;Real time PCR检测L-NAME对CGRP诱导MG-63细胞c-fos/c-jun mRNA表达改变的抑制作用。5.2分化: Real time PCR检测L-NAME对CGRP诱导MG-63细胞CollagenⅠmRNA表达表达改变的抑制作用;ELISA检测L-NAME对CGRP诱导MG-63细胞OC表达改变的抑制作用结果。结果:1. CGRP对MG-63细胞增殖和分化的作用:1.1增殖:MTT法检测发现10–8mol/L浓度CGRP作用组细胞在24-96 h的整个检测时段中均明显高于对照组（P<0.05或P<0.01）;CGRP实验组细胞增殖指数和S phase细胞比例在8 h高于对照组（P<0.01）,24 h无统计学差异;CGRP实验组细胞c-fos mRNA表达在20 m显著高于对照组（P<0.01）,c-jun mRNA表达在60 m显著低于对照组（P<0.01）。1.1分化: 10^–10-10^–7mol/L CGRP实验组细胞ALP染色光密度值高于对照组（P<0.05或P<0.01）;10^–10-10^–7mol/L CGRP实验组细胞茜素红染色计数高于对照组（P<0.01）;10^–9 -10^–7mol/L CGRP实验组细胞CollagenⅠmRNA表达高于对照组（P<0.05或P<0.01）;10^–10 -10^–7mol/L CGRP实验组细胞OC表达高于对照组（P<0.05或P<0.01）。2. CGRP对MG-63细胞NO的调控作用:CGRP实验组细胞培养液中的NO浓度在12和24 h显著高于对照组（P<0.01）;CGRP实验组细胞中的NO浓度在1、2和3 h显著高于对照组（P<0.05或P<0.01）;eNOS抑制组细胞的NOS酶活力显著低于CGRP作用组（P<0.01）,而nNOS和iNOS抑制组细胞酶活力与CGRP作用组无统计学差异。CGRP作用MG-63细胞0-48 h后,除eNOS染色在36 h高于对照组细胞（P<0.05）以外,eNOS、nNOS和iNOS三种蛋白表达均无统计学差异。3. CGRP对MG-63细胞NO的调控机制:3.1 CGRP通过[Ca²⁺]_i对MG-63细胞eNOS酶活力的调控:CGRP作用的实验组细胞较对照组在250-600 s时段中出现了一个特异性[Ca²⁺]_i波峰;[Ca²⁺]_i抑制组细胞内NO浓度在作用1 h时显著低于CGRP作用组（P<0.01）;[Ca²⁺]_i抑制组细胞培养液中NO浓度在作用12、24 h时显著低于CGRP作用组（P<0.01）。3.2 CGRP对MG-63细胞iNOSmRNA表达的调控作用:CGRP作用MG-63细胞的0-48 h内细胞iNOSmRNA表达无统计学差异;TNF-α与INF-γ联合组在24 h时细胞iNOS mRNA表达显著高于对照组（P<0.01）,CGRP实验组与联合诱导组之间无统计学差异。4. MG-63细胞中CGRP-NO调控作用的生物学意义:4.1增殖: 50umol/L L-NAME实验组细胞增殖率在48、72和96h显著低于对照组（P<0.01）,24 h时无统计学差异;L-NAME抑制组细胞增殖率在24 h时显著低于CGRP作用组（P<0.01）;在作用24 h后,L-NAME抑制组细胞G2 phase细胞比例显著低于CGRP作用组（P<0.01）,IP与CGRP作用组相比无统计学差异;在作用20min后,L-NAME抑制组细胞c-fos mRNA表达显著低于CGRP作用组（P<0.01）,而c-jun mRNA表达无统计学差异。4.2分化:在作用24 h后,L-NAME抑制组细胞CollagenⅠmRNA与OC的表达,与CGRP作用组之间无统计学差异。结论1. CGRP对MG-63细胞具有显著的促增殖效应,以10^–8mol/L浓度作用最为明显;CGRP对MG-63的促增殖效应体现在激活AP-1的c-fos mRNA表达,加速细胞进入有丝分裂期,从而提高细胞增殖指数;CGRP促进MG-63细胞分化,细胞ALP染色、矿化结节形成、CollagenⅠmRNA表达及OC分泌均有显著提高。2. CGRP能够激活MG-63细胞eNOS酶活力,升高细胞内NO浓度,从而使培养液中NO产物浓度增加;CGRP对MG-63细胞的NOS各亚型的蛋白表达无明确的调控作用。3. CGRP对细胞NO的上调作用主要是通过增加细胞内的[Ca²⁺]_i,激活细胞eNOS酶活力所完成的,而与iNOS mRNA表达调控无关;CGRP对在炎症因子诱导下MG-63细胞iNOS mRNA表达无调控作用。4.通过抑制细胞内eNOS酶,有效抑制了CGRP对MG-63细胞c-fos mRNA表达上调作用,抑制CGRP对MG-63细胞的促增殖效应;抑制细胞内eNOS酶对CGRP诱导的细胞分化标志物CollagenⅠ及OC的上调作用无意义。更多还原

【Abstract】 Neuropeptide which is secreted by peripheral nerve plays an important role in fracture healing and bone metabolism. Calcitonin gene-related peptide （CGRP）, a 37-amino acid peptide generated by tissue-specific alternative splicing of the calcitonin gene, has been shown to be expressed in nerve fibers during bone development and regeneration, and CGRP-knockout mice as well as functional studies have demonstrated that CGRP is an important factor during bone formation and repair. CGRP receptors have been identified both in vitro osteoblast-like cell models as well as downstream signal transduction pathways, such as the cyclic adenosine monophosphate （cAMP）, intracellular Ca²⁺ and mitogen-activated protein kinase （MAPK） pathways.Nitric oxide （NO） has been reported to have biphasic effects on osteoblastic bone regeneration. These biological characteristics of NO are determined by the mechanisms of NO action. NO secreted from constitutive NOS （cNOS, including eNOS and nNOS）, which is constitutively expressed at low levels in its tissues of origin and is regulated at the level of enzymatic activity by changes in free intracellular Ca²⁺ concentration ([Ca²⁺]_i). In osteoblasts, eNOS is widely and constitutively expressed; iNOS is only induced by stimulation with pro-inflammatory cytokines, and nNOS is hardly expressed at all. Low NO concentrations, constitutively produced by osteoblasts, have been shown to act as an autocrine stimulator of osteoblast growth and cytokine production, while high NO concentrations, which are stimulated by pro-inflammatory cytokines, have been shown to have inhibitory effects on osteoblast growth and differentiation. In contrast, NO is secreted from iNOS in larger quantities and for prolonged time periods, resulting in cytotoxicity due to oxygen-derived free radicals and lipid peroxidation.This study was designed to verify the effect of CGRP on the osteoblasts’proliferation and differentiation in vitro; to clear the effect of CGRP on cell NO prodcution, NOS activity and expression; to explore the the regulation mechanism of CGRP on NO production; at last, to find the biological meaning of CGRP-NO signaling pathway in osteoblasts.Methods1. Cell culture: MG-63 cell （ATCC） were cultured in Dulbecco’s modified Eagle’s Medium supplemented with 10% fetal calf serum. Cells were subcultured every 72 h in a humidified 5% CO2 incubator. Cells from passages 6–8 were used in the experiments.2. Effect of CGRP on proliferation and differentiation of MG-63:2.1 Proliferation: Time-effect （24-96 h） and dose-effect (10^–10 -10^–7mol/L) of CGRP on cell viability of MG-63 were determinated by MTT method; time-effect （0-24 h） of CGRP on cell cycle was assayed using flow cytometry; time-effect （0-80 m） of CGRP on expression of c-fos/c-jun mRNA in cell was detected by RT-PCR.2.2 Differentiation: Dose-effect (10^–10 -10^–7mol/L) of CGRP on cell ALP was stained by reforming Kaplow method; dose-effect (10^–10 -10^–7mol/L) of CGRP on cell calcifacaiton was stained by alizarin bordeaux; dose-effect (10^–10 -10^–7mol/L) of CGRP on expression of CollagenⅠmRNA in cell was detected by RT-PCR; dose-effect (10^–10 -10–7mol/L) of CGRP on expression of OC in cell culture supernatant was detected by ELISA.3. Effect of CGRP on NO production in MG-63: Time-effect （0-48 h） of 10^–8 mol/L CGRP on NO production in cell culture supernatants was detected by Griess reaction; time-effect （0-4 h） of 10^–8 mol/L CGRP on NO concentration in cell was detected by DAF-FM DA fluorescence detection; effects of 10^–8 mol/L CGRP on eNOS, nNOS and iNOS activity in cell were detected by NOS enzyme kit; time-effects （0-48 h） of 10^–8 mol/L CGRP on expression of eNOS, nNOS and iNOS were stained and detected by immunoflorescence.4. Regulation of CGRP on NO production in MG-63:4.1 Regulation of CGRP on activity of eNOS via [Ca²⁺]_i : Time-effect （0-15 m） of 10^–8 mol/L CGRP on [Ca²⁺]_i in MG-63 was dectected by Fluo-3/AM fluorescence detection; depressive effect of verpamil on CGRP induced NO concentration in MG-63 was detected by DAF-FM DA fluorescence detection; depressive effect of verpamil on CGRP induced NO production in cell culture supernatants was detected by Griess reaction.4.2 Regulation of CGRP on expression of iNOS mRNA : Time-effect （24-96 h） of 10^–8 mol/L CGRP on expression of iNOS mRNA in cell was detected by Real time PCR; after inducted by TNF-αand INF-γ, effect of CGRP on expression of iNOS mRNA in MG-63 was detected by Real time PCR.5. Biological meaning of CGRP-ON regulation in MG-63:5.1 Proliferation: Time-effect （24-96 h） of L-NAME, inhibitor of eNOS on CGRP induced cell proliferation was detected by MTT method; depresive effect of L-NAME on CGRP induced cell cycle acceleration was assayed using flow cytometry; depresive effect of L-NAME on CGRP induced up-expression of c-fos/c-jun mRNA in cell was detected by Real time PCR.5.2 Differentiation: Depresive effect of L-NAME on CGRP induced up-expression of CollagenⅠmRNA in cell was detected by Real time PCR; depresive effect of L-NAME on CGRP induced up-expression of OC in cell culture supernatant was detected by ELISA. Results:1. Effect of CGRP on proliferation and differentiation of MG-63:1.1 Proliferation: In MTT assay, growth rate of cell in 10^–10 -10^–7mol/L CGRP experimental groups were significantly higher than control group （P<0.05 or P<0.01）; IP and the proportion of S phase cells in CGRP experimental groups were high at 8 h, but no statistical difference at 24 h; expression of c-fos mRNA in experimental groups’cell were significant higher than control group at 20 m （P<0.01）,expression of c-jun mRNA were lower than control group at 60m （P<0.01）.1.2 Differentiation: OD value of ALP stain in 10^–10 -10^–7mol/L CGRP experimental groups were high than control group （P<0.05 or P<0.01）,number of alizarin bordeaux stain in 10^–10 -10^–7mol/L CGRP experimental groups were higher than control group （P<0.01）,expression of CollagenⅠmRNA in 10–9 -10^–7mol/L CGRP experimental groups were higher than control group （P<0.05 or P<0.01）,expression of OC in cell culture supernatant in 10^–10 -10^–7mol/L CGRP experimental groups were high than control group （P<0.05 or P<0.01）.2. Effect of CGRP on NO production in MG-63: After induced by CGRP, production in cell culture supernatant in CGRP experimental groups were higher than control group （P<0.01） at 12 and 24 h,NO concentration in cell in CGRP experimental groups were higher than control group （P<0.05 or P<0.01） at 1, 2 and 3 h,activity of NOS in inhibited eNOS group was significant lower than CGRP reduced group （P<0.01）, but no statistical difference in inhibited nNOS and iNOS groups. After CGRP indeced 0-48 h, except for expression of eNOS was higher than control （P<0.05） at 36 h, there was no stastical difference in eNOS, nNOS and iNOS experission.3. Regulation of CGRP on NO production in MG-63:3.1 Regulation of CGRP on activity of eNOS via [Ca²⁺]_i : After induced by CGRP, [Ca²⁺]_i in experimental groups’cells increased from 250-600 s contrast with control; NO concentration in [Ca²⁺]_i inhibited group was lower than CGRP induced group （P<0.01）, NO production in culture supernatant in [Ca²⁺]_i inhibited group was lower than CGRP induced group （P<0.01）.3.2 Regulation of CGRP on expression of iNOS mRNA : After induced by CGRP for 48 h, there was no difference of iNOS mRNA expression between experimental group and control group; after inducted by TNF-αand INF-γfor 48 h, expression of iNOS mRNA in experimental group cells was significant higher than control （P<0.01）, but no difference between TNF-αand INF-γinduced group and CGRP+ TNF-α+INF-γgroup.4. Biological meaning of CGRP-ON regulation in MG-63:4.1 Proliferation: Growth rate of cell in L-NAME experimental groups were significantly lower than control group at 48, 72 and 96 h（P<0.01）, but no difference at 24 h; growth rate of cell in L-NAME inhibited group was lower than CGRP induced group at 24 h （P<0.01）; proportion of G2 phase cells in L-NAME inhibited group were lower than CGRP induced group （P<0.01）, but no statistical difference in IP; expression of c-fos mRNA in L-NAME inhibited group were lower than CGRP induced group at 20 m （P<0.01）,and no difference in c-jun mRNA. 4.2 Differentiation: After induced by CGRP for 24 h, there was no difference of CollagenⅠmRNA and OC expression between L-NAME inhibited group and CGRP induced group .Conclusion:1. CGRP up-regulates proliferation of MG-63 in vitro, and 10^–8mol/L is the most effective concentration; effect of CGRP on MG-63 proliferation reflected in the activation of AP-1, expression of c-fos mRNA, and accelerates cell into mitosis to raise the proliferation of cell; CGRP up-regulates activity of ALP, mineralized nodule formation, expression of CollagenⅠmRNA and OC to simulate differentiation of MG-63.2. CGRP stimulates activity of eNOS in MG-63 cell, increases concentration of intracellular NO, and increases production of NO in culture medium; CGRP dose not regulate expression of NOS isoforms in MG-63.3. CGRP increases intracellular NO concentration via up-regulating [Ca²⁺]i to stimulate activity of eNOS in MG-63, but not regulating expression of iNOS mRNA; even stimulated by inflammatory cytokines, expression of iNOS mRNA can not be effected by CGRP.4. CGRP induced c-fos mRNA up-regulation can be inhibited via inhibiting eNOS activity; this inhibiting effect also be found in CGRP induced proliferation and cell cycle change; but it not be found in expression of CollagenⅠmRNA and OC, which reflect differentiation of MG-63.更多还原