【作者】 刘伟；

【导师】 刘宗平；

【作者基本信息】 扬州大学 ， 临床兽医学， 2016， 博士

【摘要】 骨保护素(osteoprotegerin, OPG)发现于上世纪九十年代,它作为肿瘤坏死因子受体(TNF)超家族成员之一,能通过阻断核因子κB受体活化因子配体(RANKL)和RANK结合来完成诱骗受体功能,从而抑制破骨细胞(OCs)的分化和活性。本课题组最新研究发现OPG可诱导体外成熟破骨细胞发生凋亡,这种凋亡可能是OPG抑制成熟破骨细胞活性的主要途径,然而OPG在OCs存活以及凋亡中的确切作用仍不清楚。随着我国养殖业集约化和规模化发展,动物骨营养不良性疾病的发生越来越多,目前仍缺乏高效的防控药物。本文以原代分离小鼠骨髓诱导分化为OCs及破骨细胞前体细胞(OPCs)为研究对象,OCs分化成熟后添加不同浓度OPG,通过TACS-XL Blue法(TUNEL凋亡检测衍生法)、实时荧光定量PCR (QRT-PCR)、蛋白免疫印迹(Western blot)免疫共沉淀(Co-immunoprecipitation, Co-IP)等技术手段,揭示OPG对OCs和OPCs凋亡中形态、功能、相关基因及关键信号蛋白的影响,以期阐明OPG诱导OCs和OPCs凋亡的分子机制,为相关药物的研发提供理论依据和新思路。研究内容如下：1.OPG诱导破骨细胞及其前体细胞凋亡为了研究OPG对体外培养小鼠OCs和OPCs凋亡的影响,取8-12周龄Balb/cJ小鼠的骨髓细胞,体外培养过程中采用巨噬细胞集落刺激因子(M-CSF)+RANKL联合诱导分化为OCs和OPCs,作为OCs和OPCs细胞模型；同时运用仅添加M-CSF的培养基诱导为单一OPCs细胞模型,在体外培养5d,随后双细胞因子处理组去除RANKL,在两种模型中均分别加入不同浓度OPG((0、20、40、80ng/mL)进行处理。通过Annexin V-FITC荧光染色法,AnnexinV-PI流式法及TACS-XL Blue法,检钡OPCs的早期凋亡、晚期凋亡以及OCs的凋亡情况。结果显示,与对照组相比,OPG处理组OPCs的凋亡率显著增加(P<0.05),其中早期凋亡率及晚期凋亡率在40ng/mLOPG处理组达到最高值。与对照组相比,OPG处理组OCs的凋亡率显著增加,呈剂量—效应关系(P<0.05)。2.OPG对破骨细胞凋亡相关基因的影响为了研究OPG诱导OCs凋亡中凋亡相关基因的变化,采用M-CSF+RANKL诱导原代小鼠骨髓细胞分化形成OCs,在体外培养5d,去除RANKL,各组分别添加不同浓度的OPG (0、20、40、80ng/mL)。培养结束后收集OCs,提取总RNA和总蛋白,QRT-PCR和Western blot法检测凋亡相关基因转录及蛋白表达水平的变化。结果发现,OPG处理使得OCs中促凋亡基因Bax的转录水平显著升高(P<0.05),抑凋亡基因Bcl-2基因的转录水平极显著降低(P<0.01),呈剂量—效应关系。同时,Western blot结果表明,与对照组相比,随着OPG浓度的增加,Bax的表达量上升、Bcl-2的表达量下降,Bax/Bcl-2极显著增加(P<0.01)。表明OPG能通过促进促凋亡基因表达和抑制抑凋亡基因表达来诱导破骨细胞凋亡。3.OPG诱导OCs和OPCs凋亡的Fas/FasL死亡受体途径为了研究OPG诱导OCs和OPCs凋亡对Fas/FasL死亡受体途径的影响,在上述双因子处理诱导而来的OCs和OPCs中加入相应浓度的OPG和/或Fas/FasL死亡受体通路拮抗剂进行处理。通过QRT-PCR技术及Western blot等技术,研究OPG对OCs和OPCs的Fas/FasL死亡受体途径相关蛋白的转录和表达水平的影响。此外,酶联免疫吸附测定(ELISA)及Co-IP法检测可溶性FasL (sFasL)在细胞上清中浓度的变化,以及其与OPG的结合情况。结果显示,与对照组相比,20和40 ng/mL OPG能显著增加Fas的转录及表达水平和caspase-8的活化程度,但80 ng/mL OPG处理会显著地降低Fas的转录及表达水平(P<0.05)。同时,与对照组相比,OPG处理显著增加FasL的转录及表达水平,但上清中的sFasL含量显著减少(P<0.05)。运用Fas/FasL死亡受体途径的拮抗剂与OPG共处理能极显著减弱OPG处理引起的caspase-8和caspase-3活化增强(P<0.01)。Co-IP法检测发现OPG能与sFasL结合。这一结合会阻滞sFasL对OCs和OPCs凋亡的抑制作用。4.OPG诱导破骨细胞凋亡的线粒体途径为了研究OPG诱导OCs和OPCs凋亡的线粒体途径,在上述双因子处理诱导而来的OCs和OPCs中分别添加相应浓度的OPG。培养结束后收集细胞,通过Western blot法检测线粒体凋亡途径相关蛋白细胞色素C (cyt. c)的分布、caspase-9和caspase-3的活化水平,通过免疫荧光技术检测凋亡相关因子(AIF)和线粒体核酸内切酶G (Endo G)的核转位。结果发现,OPG处理能诱导OCs和OPCs中cyt. c从线粒体释放到胞浆,激活caspase-9和caspase-3,AIF和Endo G发生核转位。表明OPG能通过线粒体凋亡途径诱导OCs和OPCs发生凋亡。更多还原

【Abstract】 Osteoprotegerin (OPG), as a member of the tumor necrosis factor (TNF) receptor superfamily, has been found early in 1990s. OPG is known to inhibit differentiation and activation of osteoclasts (OCs) by functioning as a decoy receptor blocking interactions between Receptor Activator for Nuclear Factor-KB (RANK) and RANKL. Our team found that OPG could induce the apoptosis of mature OCs cultivated in vitro. It might be the main route that OPG suppressed the activity of OCs. However, the exact role of OPG in the survival/apoptosis of OCs remains unclear. Malnutrition in bone in large animal and mass breeding animals have become more and more serious with the further development of animal husbandry industry in China. It becoming more and more important to using the recombinant OPG and OPG gene therapy technology to prevent bone disease in animal. But the inhibition effect of OPG on the survival of OCs have not been well elucidated. All these make it difficult to develop and use OPG related drug to cure the nutrition metabolic bone disease of livestock and poultry. In this study, isolated murine bone marrow borrow were induced to OCs and osteoclast precursor cells (OPCs), different concentrations of OPG were added when the OCs were mature. TACS-XL Blue assay (derivative method of TUNEL apoptosis detection), QRT-PCR, Western blot, Co-IP etc. were all performed to clarify the effect of OPG on the morphological changes, functions, related genes expression, and key signaling proteins of apoptosis in OCs and OPCs. The aim of this study was to determine the precise mechanism of apoptosis induced by OPG in OCs and OPCs. This would provide theoretical basis and new treatment strategy for the development and application of related drugs. All test carried out were showed below.1. OPG induces OCs and OPCs apoptosisIn order to figure out the influence of OPG on the apoptosis of murine OCs and OPCs cultured in vitro, bone marrow cells were harvested from 8-12-week-old male Balb/cJ mice and incubated with M-CSF+RANKL for osteoclastogenesis. Meanwhile, bone marrow cells incubated with only M-CSF were used to induce the OPCs model. After 5 days cultivation, RANKL was removed, different concentration of OPG (0,20,40 and 80 ng/ml) were added. Annexin V-FITC immunofluorescent assay, flow cytometry using Annexin V-FITC and propidium iodide (PI) double staining kit, and TACS-XL Blue assay were performed to determine the early apoptosis and late apoptosis rate of OPCs, apoptotic morphological changes of OCs, respectively. The results showed that, compared to control group, OPG treatment would significantly increase the apoptotic rate of OPCs (P<0.05). The early apoptotic rate and late apoptotic rate reached the peak in 40 ng/mL OPG treatment group. OPG treatment significantly increased the apoptotic rate of OCs in a dose-dependent manner, compared to control group (P<0.05).2. The effect of OPG on apoptotic genes in OCsThe aim of this study was to detect the alteration of apoptotic genes induced by OPG in OCs. Murine bone marrow were incubated with M-CSF+RANKL for osteoclastogenesis. After 5 days cultivation,0,20,40 and 80 ng/ml of OPG were added to different groups of cells, after removing RANKL. OCs were collected, RNA and total protein were isolated, and then levels of gene transcription and protein expression of apoptotic genes were monitored via QRT-PCR and Western blot. The results showed that OPG treatment would significantly promote the transcriptional level of Bax gene (P<0.05), highly significant suppress the transcriptional level of Bcl-2 gene (P<0.01), in dose-dependent manner. Simultaneously, the results of Western blot showed that, compared to control group, the expression of Bax ascended, the expression of Bcl-2 descended, the ratio of Bax/Bcl-2 increased highly significant (P<0.01), according to the increasing of concentration of OPG. These data indicated that OPG induced OCs apoptosis by accelerating the expression of pro-apoptotic genes and inhibiting the anti-apoptotic genes.3. OPG induced OCs and OPCs apoptosis via Fas/FasL pathwayTo determine the molecular mechanism of Fas/FasL signalling pathway involved in apoptosis induced by OPG in OCs and OPCs, murine bone marrow were incubated with M-CSF+RANKL for OC and OPC. After cultivation, OPG of relative concentration and/or the antagonist of Fas/FasL pathway were added to different groups of cells. QRT-PCR and Western blot were used to detect the effect of OPG on the levels of transcription and expression of key signaling proteins involved in Fas/FasL pathway in OCs and OPCs. In addition, the change of soluble FasL (sFasL) concentration in the cell supernatant and the binding of sFasL were detected by ELISA and Co-IP, respectively. The results demonstrated that, compared to control group, the transcription and expression levels of Fas and activated caspase-8 was increased by both 20 ng/mL and 40 ng/mL of OPG, but these were markedly decreased at 80 ng/mL (P<0.05). In the meantime, transcriptional and expression levels of FasL increased with increasing doses of OPG, sFasL in the supernatant significantly decreased (P<0.05). The co-treatment of antagonist of Fas/FasL pathway with OPG would highly significant attenuate the activation of caspase-8 and caspase-3 (P<0.01). Co-IP assay found that sFasL would bind OPG. The binding would block the inhibition of the apoptosis of OCs and OPCs.4. OPG induced OCs and OPCs apoptosis through mitochondrial apoptotic pathwayTo investigate the role of mitochondrial pathway in apoptosis induced by OPG in OCs and OPCs, OCs and OPCs induced by method showed above were treated with OPG with corresponding concentrations. Cells were collected at the end of cultivation, Western blot was used to detect the distribution of cytochrome C (cyt. c), related proteins of mitochondrial apoptotic pathway, activation of caspase-9 and caspase-3. Immunofluorescence staining technique was used to detect nuclear translocation of Apoptosis Inducing Factor (AIF) and Endoribonuclease G (Endo G). The results showed that OPG treatment could induce the release of cyt. c from mitochondrion to cytoplasm, the activation of caspase-8 and caspase-3, and the nuclear translocation of AIF and Endo-G. All these results clarified that OPG would induced OCs and OPCs through mitochondrial apoptotic pathway.更多还原