rhIGF1对成骨细胞和破骨细胞相互作用的调节作用

rhIGF1对成骨细胞和破骨细胞相互作用的调节作用天津医科大学硕士学位论文中文摘要目的在成骨细胞(ostelblast，OB)及破骨细胞(Osteoclast,OC)的表面均表达有胰岛素样生长因子1(insulin like growth factor 1,IGF—I)的受体，IGF．I对这两种细胞也都具有激活作用。

IGF．1在调节骨代谢平衡中具有重要作用，被认为是存在于RANKL／OPG系统之上，调节成骨细胞与破骨细胞相互作用的核心分子。

为此，本文拟探讨不同浓度的rhlGF．I对成骨细胞和破骨细胞的直接作用，及对成骨细胞和破骨细胞相互作用的调节。

方法1．利用50ng／ml的RANKL诱导小鼠破骨前体细胞系分化为成熟破骨细胞。

TRAP(抗酒石酸酸性磷酸酶染色)鉴定。

2．以50ng／ml rhlGF．1分别干预成细胞以及分化成熟的破骨细胞，干预时间分别为：6min、20min、60mira Western．blotting检测rhlGF．1 R活化情况。

3．分别加入0、2、4、6ug／ml的兔抗。

rhlGF-I IgG中和培养基中的rhlGF-I，利用Western-blotting进行抗体中和浓度的筛选。

4．重新接种成骨细胞和破骨细胞，用O、10、50、lOOnedlIll的rhlGF。

1分别干预成骨细胞、破骨细胞，12小时后终止培养并收集培基，之后实验分为三组：A组：rhIGF．1直接干预组。

B组：条件培养基交互培养组：破骨细胞经rhIGF．1 干预后的条件培养基(OC conditioned medium after rhlGF．1 treatment，OCCM)；成骨细胞经rhlGF．1干预后的条件培养基(OB conditioned medium after rMGF．1 treatment，OBCM)。

滤过OCCM、OBCM后两细胞培基互换交互培养12h。

C组：rhlGF-l中和后交互培养组：OCCM、OBCM中加入4ug／ml的抗rhlGF．I IgG，中和后再用于交互培养12h。

(1)ELISA检测各组OB上清液中RANKL、OPG及BGP含量。

(2)实时荧光定量PCR检测各组OB中Bgp，Opg，RanM及OC中Ck，Mmp一9、Rank mRNA的表达水平。

结果1．50ng／ml的RANKL诱导培养8天后，RAW264．7出现TRAP阳性多核细胞。

2．50ng／ml rMGF．1分别干预成骨细胞、破骨细胞6、20、60rain后，Western．blotting检测随着rhlGF—l干预时间的延长磷酸化rhlGF．1R的量逐渐增II天津医科大学硕士学位论文加，而未磷酸化d：lIGF-1R逐渐减少。

3．未加rhlGF．1干预空白对照组无磷酸化rhlGF-1R；rhlGF．1干预后可以检测到大量磷酸化rhlGF-1R；rhlGF—l干预后培基中加入2ug／ml抗体中和后可检测到少量磷酸化rhlGF一1R，而加入4、6ug／ml的抗体中和后未检测到磷酸化rhlGF．1R。

同时随着中和抗体浓度的增加，非磷酸化IGF．IR也增加。

4．(1)当rhlGF一1单独干预成骨细胞时，测定上清液中RANKL、OPG及BGP 含量，结果显示不同浓度的IGF．1对成骨细胞合成RANKL、OPG及BGP无影响：而当用OCCM及抗体中和后的OCCM培养成骨细胞时，发现三种细胞因子在上清液中的含量均在rhlGF．1初始干预浓度为10、50ng／mL时明显高于其他组，并以低剂量组——即rhIGF．1初始浓度为]0ng／mL时最高。

同时，当用抗体中和后的OCCM培养成骨细胞时，三种细胞因子在上清液中的表达在低剂量组明显高于OCCM培养的成骨细胞组。

(2)实时荧光定量PCR检测结果显示rhlGF．1直接干预OB、OC时，OB中Bgp，Opg、．Rankl及OC中Ck、Mmp一9，．Rank mRNA的表达水平在不同剂量组与未加rhlGF．1干预的空白组之间无明显差别；而OCCM与抗体中和后 OCCM培养的OB组及OBCM与抗体中和后OBCM培养的OC在rhlGF．1干预浓度为10ng／rrd时Bgp，Opg、Rankl及Ck、Mmp一9，Rank mRNA的表达水平显著高于空白组与其他剂量组，统计学分析表明前者与其它各组表达水平存在显著差异(p<0．05)。

结论综合上述各项指标的检测结果显示，rhlGF．1直接干预成、破骨细胞时作用不明显；当有另一细胞参与后，各项检测指标的变化均非常显著，以低剂量组最为明显，但不是剂量依赖性。

鉴于rhlGF．1已经被证实有显著的体内促成骨活性，因此本研究推测rhlGF．1在对成骨细胞的刺激上可能首先作用于破骨细胞，引起破骨细胞及其活动微环境的改变，进而影响到成骨细胞的生长和功能；同理对破骨细胞也是一样，要先作用于成骨细胞引起成骨细胞及其活动微环境的改变，进而影响到破骨细胞的生长和功能。

关键词：重组人胰岛素样生长因子1 成骨细胞破骨细胞ELISA实时荧光定量PCRIII天津医科大学硕士学位论文AbstractIGF 一1 was concemed 懿the key factor in regulating homeostasis of the bone metabolism and the interaction between osteoblast and osteoclast ，beyond RANKL ／OPG pathway ．There ale rhlGF 一1R being expressed on the cell membrane of either osteoblast and osteoclast ，and both of them could be activated by rhlGF-1．The aim of this article is try to discuss the interact regulating action between osteoblastand osteoclast under the treatment of rhIGF ．1．Method1．Osteoclast precursor cell line RAW264．7 was induced differentiation into osteoclasts by RANKL ，certificated by TRAP staining ．2．rhIGF 一1 50ng ／ml were added to osteoblasts and RAW264．7-derived osteoclast respectively ，intervention time were ：6rain,20min ，60min ．Western-blotting Was applied to confirm the activation of rhIGF 一1 receptor ．3．Rabbit anti —rhlGF-I IgG were added to the medium wi 廿l the fmal concentrations of O ，2，4，6 ug ／ml ，Western-blotting to select the optimized neutralizing concentration of rhlGF ．I ．4．Re-inoculated osteoblasts and osteoclasts ，added 0，1 0，50，1 00 ng ／ml of rhIGF·1 respectively to interfere osteoblasts and osteoelasts ，osteoblast·conditioned medium(OBCM)and osteoclast-eonditioned medium(OCCM)with 打lIGF-1 were collected after 12 hours ，then the experiment WaS divided into three groups ：A ： rhIGF-l directly treated groups ；B ：Conditioned medium c ross·cultured groups ： Conditioned medium 、wrc collected and filtered ．then exchanged the medium andcominuc to culture 0B and 0C for 12h ：C ：Conditioiled medium with rhIGF 一1 neutralization cross-cultured groups ：conditioned medium were collected and added 4ug ／ml anti-rhI(、F-I IgG ，exchanged the medium and continue to culture OB and OC for 12h ．(1)RANKI ，，OPG and BGP in the medium were examined by ELISA ．(2)Bgp ，Opg 、Rankl mRNA in OB and Ck 、Mmp 一9、Rank mRNA in OC were quantitatively determined by Real-time polymerase chain reaction ．Result ．．IV天津医科大学硕士学位论文1．Under the induction of RANKL，谢tll 50ng／ml for 8 days，RAW264．7 cells were induced into multiple-nucleus osteoclasts．2．Western—blotting results confirmed that rhlGF—I could result in rhlGF-1 Rphosphorylation in both OB and OC．And the amount of phosphorylated rhIGF·1R were increased as the intcrfere time extended．3．Western—blotting results confirmed that the amount of phosphorylated rhIGF-1R were does dependtly decreaSed in the presence of anti-rhlGF．I．And when anti-rhIGF-I reached 4,6ug／ml，activated rhlGF—lreceptor ocould not be detected4．(1)After OB were treated晰m different dose of rhlGF一1，to examined the content of RANKL and BGP in OBCM by ELISA，and the results showed that there were no significant difference among different groups．HoweveL when OB cultured wim OCCM and OCCM after antibody neutralizing．we found that three cell factors expression were significantly higer than other groups in the presence of rhlGF-1 at1 0，50ng／ml，it WaS highest rhlGF-1 at lOng／mL；In low dose group，the three cytokines in C group were significantly higher than that in B group．(2)Real-time PCR results showed that in A group，the mRNA expression levels ofBgp，Opg，Rankl in OB and Ck、Mmp-9、Rank in OC were no significant difference among differem groups；However,there were significantly higher than other dose ofgroups when rhIGF-1 reached 1 0ng／m1．Statistical analysis showed that the differences is significant(p<0．05)．ConclusionIn the conclusion，results above showed that when different dose of rMGF．1were added to OB and OC，the effect were not significant；However，when cultured OB、^，ith OCCM and rhlGF-l neutralized OCCM or cultured 0C with 0BCM and rhIGF-1 neutralized OBCM，the changes were very significant．It Was the most obvious in the low dose group．Therefore，our study suggested that rhlGF一1 may primarily caused the change of OC，then alterted OC activities and its micro—environmental changemem subsequently affected the growth and functions of OB．Which might be happened same to the OC．Keywords：rhlGF．1 osteoblast osteoclast ELISA Real-time PCRV天津医科大学硕士学位论文缩略语缩写英文全称中文全称Ab antibody 抗体Acr aerylamide 丙烯酰胺AP ammoni啪persulfate 过硫酸铵BCA bicinchonimic acid 二辛可酸BGP bone Gla protein 骨钙素BME 13 I]-mercaptoethanol 15一巯基乙醇bp basepair 碱基对BSA bovine serulTl albumin 牛血清白蛋白Ck CathepsinK 组织蛋白酶K ddH20 distilled twice water 双蒸水DNA Deoxyribonucleic acid 脱氧核糖核酸ds DNA double strand DNA 双链DNAdNTP 2'-deoxynucleoside-5’-triphosphate 脱氧核苷三磷酸EB ethidium bromide 溴化乙锭EDTA ethylene diaminetetraacetic acid 乙二胺四乙酸His histidine 组氨酸HRP horseradish peoxidase 辣根过氧化物酶IGF-1 Insulin—like growth factor l 胰岛素样生长因子．1 IGF-1R Insulin—like growth factor 1 receptor 胰岛素样生长因子．1受体Mmp一9 matrix metallopeptidase 9 基质金属蛋白酶-9OB Osteoblast 成骨细胞OC Osteoclast 破骨细胞OPG Osteoprotegeri 骨保护素PBS phosphate buffer solution 磷酸盐缓冲液PCR polymerase chain reaction 聚合酶链反应核因子IcB受体活化因子Rank Receptor activator ofnuclear factorkappa-BRANKL Receptor activator of nuclear factor kappa-B ligand核因子邸受体活化因子配基VIII天津医科大学硕士学位论文RNA ribonucleic acid 核糖核酸SDS sodium dodecyl sulfate 十二烷基硫酸钠SDS-PAGE SDS-polyacrylamide gel electrophoresisSDS聚丙烯酰胺凝胶电泳TEMED N，N，N’N'-tetra methyl ethy．1ene diamine N，N，N’N’四甲基L--胺"I ris tris(hydroxymethyl)aminomethane 三(羟甲基)IX天津医科大学硕士学位论文刖舌研究现状、成果随着社会老龄化的到来，骨质疏松症的发病率逐年增高，其所造成的病理性骨折等并发症已成为严重影响公众健康的社会问题。