艾拉莫德在乳腺癌骨转移骨破坏中的抑制作用及其机制研究

发布时间：2018-03-31 05:29

本文选题：骨转移　切入点：骨破坏　出处：《华中科技大学》2016年博士论文

【摘要】：研究背景及目的骨是恶性肿瘤最常累及的转移部位之一,骨转移的出现严重影响患者的生活质量,并常与不良预后相关。随着对骨转移发生机制及对骨微环境中“恶性循环”的深入了解,骨转移的治疗开启了新篇章。肿瘤细胞诱导的破骨细胞活性增强被认为是导致骨转移骨破坏的主要机制。肿瘤细胞于骨微环境内释放多种活性因子,包括PTHrP、 TNF-α、IL-1β、IL-6和RANKL等,它们可以直接作用于破骨前体细胞,或间接通过促进成骨细胞分泌RANKL激活破骨前体细胞内的NF-κB等通路,诱导破骨细胞活化,增强溶骨作用。艾拉莫德是一种抗风湿新药,临床前研究表明其具有抗炎、抗细胞因子、抑制NF-κB活性的作用。由于活化的破骨细胞是骨转移骨破坏的主要执行者,而炎症因子是骨微环境内增强破骨细胞活性的重要组分之一。我们推测艾拉莫德可能通过抗炎症因子及抑制破骨细胞激活的作用发挥对骨转移骨破坏的治疗效果。因此,我们拟分别在体内体外实验中研究艾拉莫德对骨转移骨破坏的影响及其可能的作用机制。实验方法1、建立大鼠胫骨肿瘤种植模型,研究艾拉莫德对乳腺癌骨转移骨破坏的影响。于大鼠左侧胫骨内接种大鼠乳腺癌Walker 256细胞建立肿瘤种植模型。分组给予安慰剂或艾拉莫德治疗。运用X线摄片、HE染色的方法对大鼠胫骨骨破坏情况进行评价,运用TRAP染色对大鼠胫骨内破骨细胞活化水平进行检测,同时在模型建立前后检测大鼠左后肢机械痛阈的变化。2、体外培养破骨前体细胞,研究艾拉莫德对破骨细胞分化激活的影响及其机制。分离并培养小鼠骨髓单核巨噬细胞,加入RANKL诱导其向破骨细胞分化,同时予以艾拉莫德处理,用TRAP染色的方法检测艾拉莫德对破骨细胞分化的影响。Western blot免疫印迹法检测RANKL刺激后细胞内RANK受体下游NF-κB和MAPK通路的激活状态,以及转录因子c-Fos和NFATc1的表达水平。最后用qRT-PCR法检测细胞内NFATc1及其靶基因mRNA的表达水平。3、研究艾拉莫德对乳腺癌细胞炎症因子表达、细胞增殖、侵袭等生物学行为的影响。qRT-PCR法检测艾拉莫德对人乳腺癌MDA-MB-231细胞炎症因子表达的影响,并运用Western blot免疫印迹法检测细胞内NF-κB p65的激活情况。随后在两种人乳腺癌细胞MDA-MB-231和MCF-7中检测艾拉莫德对其增殖、迁移、侵袭的影响。用CCK-8法检测艾拉莫德对细胞增殖的作用,流式细胞术检测艾拉莫德对两种细胞周期分布的影响,划痕实验及Transwell侵袭实验检测艾拉莫德对细胞迁移、侵袭的影响。实验结果1、完成给药后,大鼠左侧胫骨X线检查和HE染色的结果显示接受艾拉莫德治疗的大鼠胫骨骨破坏程度较安慰剂组减轻,同时治疗后大鼠的左后肢机械痛觉过敏情况较安慰剂组改善。对大鼠左侧胫骨的TRAP染色发现艾拉莫德治疗组大鼠的胫骨内活化的破骨细胞数目少于安慰剂组。2、体外实验表明艾拉莫德显著抑制了破骨细胞的分化。并且细胞活力检测提示此抑制效应并非由艾拉莫德对细胞生长的抑制引起。信号通路的相关检测结果显示艾拉莫德显著抑制了RANKL诱导的破骨前体细胞内NF-κB和MAPK通路的激活,并下调了转录因子c-Fos和NFATc1的水平,减少了破骨细胞功能相关基因Cathepsin K、MMP-9和TRAP的表达。3、qRT-PCR和Western blot结果显示艾拉莫德使MDA-MB-231细胞表达TNF-α、IL-1β、IL-6的水平下降,但对RANKL的表达没有显著影响,同时药物处理后细胞内NF-κBp65的磷酸化水平也下降。此外,艾拉莫德只在高浓度(30μg/ml)时轻度抑制了MDA-MB-231以及MCF-7细胞的增殖,而与对照组相比,艾拉莫德对两种乳腺癌细胞的细胞周期分布、迁移和侵袭能力均没有显著影响。实验结论1、艾拉莫德使乳腺癌骨转移大鼠的骨破坏程度减轻,并使其机械性痛觉过敏症状得到一定改善。2、艾拉莫德缓解骨破坏的机制之一可能是其抑制了RANKL诱导的破骨前体细胞内NF-κB通路和MAPK通路的激活,从而下调了破骨细胞特异性基因的表达,影响了破骨细胞的活化,削弱了其溶骨作用。3、艾拉莫德缓解骨破坏的另一可能机制是通过NF-κB相关机制下调了乳腺癌细胞表达炎症因子(TNF-α、IL-1β、IL-6)的水平,减少了骨微环境中激活破骨细胞的刺激因素,而不直接显著影响乳腺癌细胞RANKL的分泌、细胞的增殖、迁移和侵袭能力。
[Abstract]:Background and purpose: bone metastasis of malignant tumor is one of the most frequently involved, bone metastases appear serious influence patient's quality of life, and often associated with poor prognosis. The bone metastasis mechanism and the bone microenvironment in a "vicious spiral" in-depth understanding of the treatment of bone metastases of bone broken opens a new chapter. Cell activity of tumor cell induced enhancement is thought to be the main mechanism of bone destruction of bone metastasis. Tumor cells in the bone microenvironment in the release of a variety of active factors, including PTHrP, TNF- alpha, IL-1 beta, IL-6 and RANKL, they can be directly used for osteoclast precursor cells, or indirectly by promoting osteoblast secretion RANKL activation of osteoclast precursor cells within the NF- kappa B pathway, induce osteoclast activation, enhanced osteolysis. Ira Maude is a kind of anti rheumatic drugs, preclinical studies have demonstrated the anti-inflammatory, anti cytokine, inhibiting NF- NF kappa B activity. The activity of osteoclasts is mainly performed bone destruction of bone metastases, and inflammatory cytokines in the bone microenvironment to enhance osteoclast activity in the group one. We speculated that Ira Maude might play bone destruction on bone metastasis therapy through anti-inflammatory factor and inhibition of osteoclast activation. Therefore, we proposed respectively in vivo and in vitro studies on the effect of Ira Maude bone metastatic bone destruction and its possible mechanism. Methods 1, the establishment of rat model of tibial cancer implant, bone destruction of Ira Maude effect in bone metastasis of breast cancer in rats. The left tibia inoculation of rat breast carcinoma Walker 256 cells to establish the tumor model group. Placebo or Ira Maude treatment. X-ray examination using HE staining method to evaluate the damage of tibia in rats, using TRAP staining The rat tibial osteoclast activation levels were detected at the same time, changes in the.2 model rats were detected before and after the establishment of the left hindlimb mechanical threshold, osteoclast precursor cells were cultured in vitro and the effect of Ira Maude on the activation of osteoclast differentiation and its mechanism. The separation and culture of mouse bone marrow mononuclear macrophages induced by addition of RANKL to the osteoclast differentiation, also be Ira Maude, on the activation of RANK receptor intracellular downstream NF- kappa B and MAPK pathway to detect effect of RANKL stimulation.Western blot blot to osteoclast differentiation by TRAP staining method after the detection of Ira Maude, and the transcription factor c-Fos and NFATc1 expression level. Finally, cells were detected by qRT-PCR method NFATc1 and its target gene mRNA expression levels of.3, research on Ira Maude expression on cancer cells, inflammatory factors of breast cell proliferation, invasion and other biological behavior changes. Effect of detection of Ira Maude qRT-PCR method on the expression of MDA-MB-231 cell cytokines of breast cancer, and the use of activated Western cells were detected by blot blotting in NF- kappa B p65. Then the migration on the proliferation, in two the detection of MDA-MB-231 and MCF-7 in human breast cancer cells invasion. The influence of Ira Maude effect was detected by CCK-8 Ira Maude on cell proliferation, flow cytometry and Ira Maude of two kinds of cell cycle distribution, wound healing assay and Transwell invasion assay Ira Maude on cell migration, invasion and effect. The experimental results of 1, completed after administration, the rats of left tibia X-ray examination and HE staining results showed that the rats received the Ira Maude treatment of tibial bone the extent of the damage than in the placebo group reduced, and improved after treatment in rats with left hindlimb mechanical hyperalgesia compared with the placebo group. The rats left tibial TRAP The number of osteoclasts were found in Ira Maude treated rats than in the placebo group in the tibia of activated.2 in vitro experiments indicated that Ira Maude significantly inhibited osteoclast differentiation and cell viability detection. The inhibitory effect not by Ira Maude on cell growth inhibition caused by signal pathway. The results of correlation detection showed that Ira Maude was significantly inhibited RANKL induced activation of osteoclast precursor cells in NF- kappa B and MAPK pathway, and down-regulation of c-Fos transcription factors and NFATc1 levels decreased osteoclast function related gene expression of.3 MMP-9 and K Cathepsin, TRAP, qRT-PCR and Western blot showed that Ella Maud MDA-MB-231 cells expression of TNF- alpha, IL-1 beta, IL-6 levels decreased, but no significant effect on the expression of RANKL, at the same time after drug treatment for intracellular NF- kappa Bp65 phosphorylation was decreased. In addition, Alamo only In the high concentration (30 g/ml) slightly inhibited MDA-MB-231 and MCF-7 cell proliferation, compared with the control group, Ira Maude on the cell cycle of two breast cancer cell distribution, migration and invasion are not significant. Conclusions: 1, the degree of bone destruction Ira Maude breast cancer metastasis in rat bone loss the mechanical hyperalgesia symptoms were improved.2, Alamo de relieve mechanism of bone destruction may be the inhibition of the activation of RANKL induced osteoclast precursor cells within the NF- B pathway and MAPK pathway, thus reduced the expression of osteoclast specific genes, affect the activation of osteoclasts, weakening the bone resorption function of.3, Ira Maude ease another possible mechanism of bone destruction by NF- kappa B mechanism by breast cancer cells and expression of inflammatory cytokines (TNF- alpha, IL-1 beta, IL-6) levels, reduced bone microenvironment Activation of osteoclast stimulates and does not directly affect the secretion of RANKL, cell proliferation, migration and invasion of breast cancer cells.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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