喹硫平抑制破骨细胞分化的作用观察及机制研究

发布时间：2018-03-13 19:19

  本文选题：喹硫平　切入点：破骨细胞　出处：《第三军医大学》2015年博士论文　论文类型：学位论文

【摘要】：研究背景:进展期乳腺癌、肺癌及前列腺癌等恶性肿瘤具有发生骨转移的倾向。肿瘤细胞转移至骨组织后可直接或间接激活破骨细胞并导致骨组织破坏,进一步引发顽固性疼痛、神经压迫症状、病理性骨折及高钙血症等并发症的发生,严重影响患者的生存及生活质量。与其他组织不同,骨组织主要由较为坚硬的矿物质构成,骨组织对于肿瘤的侵袭应具有更强的抗性。目前认为:肿瘤骨转移导致的骨损伤并非由肿瘤细胞直接引起而是由破骨细胞介导的骨吸收导致的。为了破坏骨组织,肿瘤细胞必须具有激活破骨细胞的相关特性,进而导致骨组织的损伤。大量研究表明:肿瘤细胞可以通过释放PTHr P、IL-1、IL-6和IL-11等因子作用于成骨细胞产生核因子κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)。RANKL目前被认为是刺激破骨细胞活化最主要、最有效的刺激因子。破骨细胞来源于骨髓造血干细胞,是骨吸收的效应细胞,肿瘤细胞转移至骨组织引发的溶骨性损伤与其对破骨细胞的激活能力密切相关。因此,抑制破骨细胞分化的药物能够有效防止肿瘤骨转移引发的溶骨性骨损伤及相关并发症的发生。目前,临床上用于抗骨损伤的药物主要包括双磷酸盐类药物、激素类药物及RANKL单克隆抗体等。尽管上述药物对于溶骨性骨损伤的治疗均具有一定的疗效,但这些药物的应用都存在一定的局限性及副作用的发生,如骨坏死、骨肉瘤发生、栓塞及食管刺激等。因此,寻求能够更为安全、有效地用于治疗肿瘤骨转移等疾病引发骨损伤的药物成为近些年关注及研究的热点。喹硫平(Quetiapine,QUE)是经FDA认证的非典型抗精神病药物。课题组前期研究发现:QUE能够通过作用于MAPK通路促进神经前体细胞向少突胶质细胞的分化,同时有研究表明:QUE对NF-κB通路也具有一定的调控作用,而MAPK和NF-κB通路均是破骨细胞分化过程中的关键通路。于是我们尝试验证QUE能否通过抑制MAPK和NF-κB通路抑制RANKL诱导的破骨细胞前体细胞向破骨细胞分化,以及保护肿瘤细胞骨转移引发的溶骨性骨损伤。期望通过本研究为肿瘤骨转移等溶骨性疾病的治疗提供新的药物选择和治疗策略。研究目的:1.利用RANKL诱导的RAW 264.7细胞和小鼠骨髓来源巨噬细胞(Bone marrow-derived macrophages,BMMs)向破骨细胞分化模型,体外观察非典型抗精神病药物QUE对破骨细胞分化是否具有抑制作用。2.利用肿瘤细胞骨转移动物模型,在体进一步验证QUE能否抑制肿瘤细胞介导的破骨细胞活化并起到骨保护作用。3.利用RANKL诱导的RAW 264.7细胞向破骨细胞分化模型,对QUE抑制破骨细胞分化的分子机制进行初步探讨。研究方法:1.选用目前使用较多的DMEM和α-MEM培养基培养RAW 264.7细胞,观察上述培养基对RAW 264.7细胞增殖、存活的影响,从而选择适合培养基用于后续实验研究。用10、30、50、100、200ng/ml浓度RANKL重组蛋白作用于RAW 264.7细胞,遴选用于促进破骨细胞分化的最佳RANKL使用浓度。2.分别利用差速贴壁法、梯度离心法等方法获取BMMs,并使用不同浓度RANKL和M-CSF重组蛋白作用于BMMs,选择最优的细胞获取方法和适宜的细胞因子使用浓度。3.使用不同浓度QUE(1μM、10μM、25μM、50μM、100μM)作用于RAW 264.7细胞并培养48小时,CCK-8检测细胞增殖情况,选择适合的QUE药物使用浓度。利用前期建立的RANKL诱导的破骨细胞分化模型,观察QUE对破骨细胞分化是否具有抑制作用。使用不同浓度QUE(1μM、10μM、25μM、50μM)作用于RANKL诱导的破骨细胞分化模型,利用实时荧光定量PCR检测QUE对破骨细胞分化过程中相关m RNA表达的影响。4.RANKL诱导的破骨细胞分化过程中,分别于培养的第1天、第2天、第3天和第4天加入QUE,观察QUE抑制破骨细胞分化的有效作用时期。5.购买商品化免疫缺陷小鼠,胫骨髓腔内肿瘤细胞注射法构建人类乳腺癌MDA-MB-231肿瘤细胞骨转移动物模型。给予QUE(10mg/kg/d)腹腔注射处理6周,X线检测QUE对肿瘤骨转移引发骨损伤的保护作用,HE和TRAP染色观察肿瘤细胞侵袭情况及QUE对肿瘤骨转移引发破骨细胞活化的抑制作用。6.利用RANKL诱导的破骨细胞分化模型,采用western blot和免疫细胞化学染色等方法观察QUE对RANKL诱导的破骨细胞分化过程中MAPK和NF-κB等通路的作用情况。结果:1.相比于α-MEM培养基,DMEM培养基更有利于RAW 264.7细胞的增殖、存活及形态的维持。使用不同浓度的RANKL重组蛋白作用于RAW 264.7细胞,当浓度低于30ng/ml时,RANKL诱导破骨细胞分化作用比较有限。但当浓度高于50ng/ml时,RANKL诱导破骨细胞分化的作用明显增强,且50ng/ml到200ng/ml之间差异不明显。RAW 264.7细胞经RANKL(50ng/ml)诱导分化5天后,经TRAP染色可见大量TRAP阳性多核破骨样细胞形成。2.相比于梯度离心法和磁珠分选法,差速贴壁法简单、易行,通过该方法分离得到的BMMs细胞经RANKL和M-CSF共同诱导7天后可最终分化为TRAP阳性的多核破骨样细胞。当RANKL浓度为50ng/ml、M-CSF浓度为25ng/ml时,其对BMMs向破骨细胞分化具有较好的促进作用。3.经CCK-8检测,1μM到50μM浓度QUE对RAW 264.7细胞没有明显毒性作用。用50μM浓度的QUE分别作用于RAW 264.7细胞3天、5天,作用于BMMs细胞3天、5天和7天,50μM浓度QUE对上述细胞的增殖均没有显著影响。4.相比于阳性对照组,1μM和10μM浓度QUE对RANKL诱导的破骨细胞分化无显著影响,但当浓度为25μM和50μM时,QUE能够显著抑制RANKL诱导的RAW264.7细胞向破骨细胞的分化。用50μM浓度QUE作用于RANKL和M-CSF共同诱导的BMMs向破骨细胞分化模型,7天后对上述细胞进行TRAP染色。结果发现:50μM浓度QUE同样能够有效抑制BMMs向破骨细胞的分化。经实时荧光定量PCR检测发现:25μM和50μM浓度QUE可以有效抑制RANKL诱导的破骨细胞分化过程中破骨细胞分化相关基因的表达。5.RANKL诱导的破骨细胞分化模型中,当第1天和第2天加入50μM浓度QUE时,QUE能够有效抑制破骨细胞的分化,而在分化后期(第3天、第4天)加入QUE则抑制破骨细胞分化作用不明显。6.QUE处理乳腺癌骨转移动物模型6周后,X线检测结果提示:QUE能够明显减轻肿瘤骨转移引发的骨损伤,起到骨保护作用;HE及TRAP染色结果显示:实验组及对照组均可见大量肿瘤细胞侵袭。与对照组相比,QUE处理组肿瘤侵袭区域与正常骨组织交界处破骨细胞的激活数量明显减少。7.Western blot检测QUE对破骨细胞分化过程中MAPK通路的作用。结果表明:QUE对p38、ERK和JNK的磷酸化都有明显的抑制作用。8.RANKL诱导的破骨细胞分化模型中,在RANKL作用下的NF-κB通路中IκBα的降解明显,而QUE可明显抑制IκBα降解的发生,并同时抑制p65的磷酸化水平。免疫细胞化学染色显示:在15到60分钟时间段内RANKL可以促进p65的浆核转移,而加入QUE处理后可明显抑制浆核转移的发生。结论:1.成功建立RANKL诱导的RAW 264.7细胞和BMMs向破骨细胞分化模型,为后续观察QUE对破骨细胞分化的作用及相关机制研究打下了良好的实验基础。2.在RANKL诱导的RAW 264.7细胞向破骨细胞分化模型以及RANKL和M-CSF共同诱导的BMMs向破骨细胞分化模型中,QUE均能够明显抑制破骨细胞前体细胞向破骨细胞的分化,并能够抑制破骨细胞分化过程中相关m RNA的表达。3.QUE对破骨细胞分化的抑制作用主要作用于破骨细胞分化的早期阶段。4.人类乳腺癌MDA-MB-231细胞介导的肿瘤骨转移动物模型中,QUE能够明显抑制肿瘤骨转移引发的破骨细胞活化,进而减轻肿瘤骨转移引发的溶骨性骨损伤。5.QUE抑制破骨细胞分化的作用与其对RANKL诱导的破骨细胞分化过程中MAPK和NF-κB信号通路的抑制作用相关。
[Abstract]:Background: advanced breast cancer, lung cancer and prostate cancer and other malignant tumors with prone bone metastasis. Tumor cell metastasis to bone tissue after direct or indirect activation of osteoclasts and lead to bone destruction, further lead to intractable pain, nerve compression symptoms, pathological fracture and other complications of severe hypercalcemia. Effect of patient survival and quality of life. Unlike other tissues, bone tissue is mainly composed of relatively hard mineral composition, bone tissue should have a stronger resistance to tumor invasion. The tumor bone transfer bone damage is not caused by tumor cells directly caused by absorption but by osteoclast mediated bone lead in order to destroy the bone tissue, tumor cells must have the relevant characteristics of the activation of osteoclasts, resulting in bone tissue injury. Many studies indicate that tumor cells by release PTHr P, IL-1, IL-6 and IL-11 and other factors on function of osteoblast derived receptor activator of nuclear factor kappa B ligand (receptor activator of B ligand RANKL NF- K,.RANKL) is currently considered to stimulate osteoclast activation is most main, the most effective stimulating factor. Osteoclasts derived from bone marrow hematopoietic stem cells that is the effector cell of bone resorption, tumor cell metastasis to bone tissue caused by osteolytic injury and on osteoclast activation is closely related. Therefore, drugs to inhibit the differentiation of osteoclast can effectively prevent the osteolytic bone injury and complications of metastatic bone tumor. Currently, clinical drugs for anti bone injury mainly includes bisphosphonates, hormone drugs and RANKL monoclonal antibodies. Although these drugs have certain curative effect for the treatment of bone injury solution, but the application of these drugs are There are certain limitations and side effects occur, such as bone necrosis, osteosarcoma, embolism and esophageal stimulation. Therefore, to seek more secure, effective for the treatment of metastatic bone tumor and other diseases caused by drugs of bone injury has become the focus of attention and research in recent years. - Liu Ping (Quetiapine, QUE) is atypical FDA certified antipsychotic medications. Previous studies showed that QUE can act on MAPK pathway to promote neural progenitor cells to oligodendrocyte differentiation, while studies have shown that: QUE has some effects on the regulation of NF- B pathway, while MAPK and NF- B pathway is the key pathway in the process of osteoclast differentiation. So we try to verify whether QUE can inhibit RANKL induced osteoclast through inhibition of MAPK and NF- B pathway of precursor cells into osteoclast differentiation and bone metastasis of tumor cells, protect the induced osteolysis Bone injury. Expect to provide drug selection and treatment strategies for new treatment through the study of osteolytic bone metastasis of the disease. Objective: macrophage RAW 264.7 cells and 1. from the bone marrow of mice induced by RANKL (Bone marrow-derived macrophages, BMMs) to osteoclast differentiation model in vitro to observe atypical antipsychotic drugs QUE on osteoclast differentiation is inhibited by.2. bone metastasis animal model of tumor cell, osteoclast differentiation model in vivo to further verify whether QUE can inhibit osteoclast mediated tumor cell activation and play a protective effect on bone.3. induced by RANKL RAW 264.7 cells and preliminary study on the molecular mechanism of inhibition of QUE osteoclast differentiation. Methods: 1. selection of currently used DMEM and alpha -MEM cultured RAW 264.7 cells, observed the culture medium of RAW 264.7 fine The effect of cell proliferation, survival, and choose the suitable medium for subsequent experiments. With the effect of RANKL recombinant protein 10,30,50100200ng/ml concentrations in RAW 264.7 cells, the selection of the best RANKL for promoting osteoclast differentiation using.2. concentration respectively by differential adhesion method, gradient centrifugation method to obtain BMMs, and use the effect of different concentrations of protein RANKL and M-CSF recombinant in BMMs, select the optimal cell access method and the appropriate cytokine concentration of.3. with different concentrations of QUE (1 M, 10 M, 25 M, 50 M, 100 M) on RAW 264.7 cells were cultured for 48 hours, CCK-8 cell proliferation was detected, choose to use the concentration of QUE for drugs. Osteoclast differentiation model using pre established by RANKL, to observe whether QUE can inhibit the differentiation of osteoclasts. Using different concentrations of QUE (1 M, 10 M, 25 M, 50 M) on RANKL induced The differentiation of osteoclast mediated model, using real-time fluorescence quantitative PCR to detect the expression of QUE of m in osteoclast differentiation effect of RNA on.4.RANKL induced osteoclast differentiation process, respectively, on the first day, training second days, third days and fourth days to join QUE, observe QUE inhibits osteoclast differentiation the effective period of the purchase of commercial.5. immunodeficiency mice, animal model of human breast cancer cell line MDA-MB-231 bone metastasis of tibial intramedullary tumor cell injection. QUE (10mg/kg/d) by intraperitoneal injection for 6 weeks, the protective effect of bone injury caused by X-ray detection QUE on bone metastasis, HE and TRAP staining and invasion of tumor cells QUE of bone metastasis caused by.6. inhibition of osteoclast differentiation model of RANKL induced osteoclast activation, by Western blot and immunocytochemical staining were used to observe the QUE of R Effect of osteoclast differentiation induced by ANKL in MAPK and NF- kappa B pathway. Results: 1. compared to alpha -MEM medium, DMEM medium is more conducive to the proliferation of RAW 264.7 cells, maintaining the survival and morphology. Using different concentrations of recombinant RANKL protein in RAW 264.7 cells, when the concentration is lower than 30ng/ml when RANKL induced osteoclast differentiation effect is limited. But when the concentration is higher than 50ng/ml, the effects of RANKL induced osteoclast differentiation was enhanced, and 50ng/ml to RANKL 200ng/ml no significant difference between the.RAW 264.7 cell (50ng/ml) differentiation after 5 days by TRAP staining showed TRAP positive multinucleated osteoclast like cells compared to the formation of.2. gradient centrifugation and magnetic cell sorting method, differential attachment method is simple, easy, the BMMs cells were treated with RANKL and M-CSF after 7 days of induction can be differentiated into TRAP positive nucleus separated by this method Osteoclast like cells. When the RANKL concentration is 50ng/ml, the concentration of M-CSF is 25ng/ml, the BMMs on osteoclast differentiation can promote.3. detected by CCK-8. The 1 M to 50 M concentration of QUE has no obvious toxic effect on RAW 264.7 cells. With 50 M concentration of QUE in RAW respectively. 264.7 cells for 3 days, 5 days, in BMMs cells for 3 days, 5 days and 7 days, 50 M concentration of QUE on the proliferation of these cells were not affected by.4. compared to the positive control group, 1 M and 10 M concentration of QUE had no significant effect on osteoclast differentiation induced by RANKL, but when the concentration is 25 M and 50 M, QUE can significantly inhibit RANKL induced differentiation of RAW264.7 cells into osteoclasts. With 50 M concentration of QUE in RANKL and M-CSF BMMs induced osteoclast differentiation model, 7 days after the TRAP staining of these cells. The results showed that: 50. The concentration of M QUE can also inhibit BMMs To the differentiation of osteoclasts. By real-time fluorescence quantitative PCR detection showed that osteoclast differentiation model expression of.5.RANKL 25 M and 50 M QUE concentration of osteoclast differentiation process effectively inhibited in RANKL induced osteoclast differentiation related genes induced in first days and second days, when the 50 M the concentration of QUE, QUE can effectively inhibit osteoclast differentiation, and differentiation in late (third days, fourth days) adding QUE can inhibit the differentiation of osteoclasts has no obvious effect on.6.QUE treatment of bone metastasis of breast cancer animal model after 6 weeks, the X-ray detection results showed that QUE can significantly reduce bone injury caused by mild bone tumor transfer to bone protective effect; HE and TRAP staining showed that the experimental group and the control group had a lot of tumor cell invasion. Compared with the control group, QUE treatment group tumor invasion area and normal bone tissue at the junction of the number of activation of osteoclasts was significantly reduced. Effect of 7.Western blot QUE on osteoclast differentiation process of MAPK pathway. The results showed that the QUE of p38, ERK and phosphorylation of JNK in osteoclast differentiation model has obvious inhibitory effect on.8.RANKL induced degradation of NF- in the B pathway in the presence of RANKL in I kappa B alpha, and QUE inhibited I kappa B alpha degradation occurred, and also inhibited the phosphorylation of p65. Immunocytochemical staining showed that in the 15 to 60 minute period of RANKL can promote the transfer of nuclear p65 slurry, and adding QUE treatment can inhibit cytoplasm nucleus metastasis. Conclusion: 1. constructed RANKL induced RAW 264.7 cells and BMMs osteoclast differentiation model for follow-up observation of QUE on osteoclast differentiation effect and mechanism of the experimental basis of.2. well in RANKL induced RAW 264.7 cells into osteoclast differentiation model and RANKL and M-CSF 鍚岃�卞�肩殑BMMs鍚戠牬楠ㄧ粏鑳炲垎鍖栨ā鍨嬩腑,QUE鍧囪兘澶熸槑鏄炬姂鍒剁牬楠ㄧ粏鑳炲墠浣撶粏鑳炲悜鐮撮�ㄧ粏鑳炵殑鍒嗗專�

本文编号：1607786