本文選題：骨轉(zhuǎn)移　切入點：骨破壞　出處：《華中科技大學》2016年博士論文

【摘要】：研究背景及目的骨是惡性腫瘤最常累及的轉(zhuǎn)移部位之一,骨轉(zhuǎn)移的出現(xiàn)嚴重影響患者的生活質(zhì)量,并常與不良預后相關。隨著對骨轉(zhuǎn)移發(fā)生機制及對骨微環(huán)境中“惡性循環(huán)”的深入了解,骨轉(zhuǎn)移的治療開啟了新篇章。腫瘤細胞誘導的破骨細胞活性增強被認為是導致骨轉(zhuǎn)移骨破壞的主要機制。腫瘤細胞于骨微環(huán)境內(nèi)釋放多種活性因子,包括PTHrP、 TNF-α、IL-1β、IL-6和RANKL等,它們可以直接作用于破骨前體細胞,或間接通過促進成骨細胞分泌RANKL激活破骨前體細胞內(nèi)的NF-κB等通路,誘導破骨細胞活化,增強溶骨作用。艾拉莫德是一種抗風濕新藥,臨床前研究表明其具有抗炎、抗細胞因子、抑制NF-κB活性的作用。由于活化的破骨細胞是骨轉(zhuǎn)移骨破壞的主要執(zhí)行者,而炎癥因子是骨微環(huán)境內(nèi)增強破骨細胞活性的重要組分之一。我們推測艾拉莫德可能通過抗炎癥因子及抑制破骨細胞激活的作用發(fā)揮對骨轉(zhuǎn)移骨破壞的治療效果。因此,我們擬分別在體內(nèi)體外實驗中研究艾拉莫德對骨轉(zhuǎn)移骨破壞的影響及其可能的作用機制。實驗方法1、建立大鼠脛骨腫瘤種植模型,研究艾拉莫德對乳腺癌骨轉(zhuǎn)移骨破壞的影響。于大鼠左側(cè)脛骨內(nèi)接種大鼠乳腺癌Walker 256細胞建立腫瘤種植模型。分組給予安慰劑或艾拉莫德治療。運用X線攝片、HE染色的方法對大鼠脛骨骨破壞情況進行評價,運用TRAP染色對大鼠脛骨內(nèi)破骨細胞活化水平進行檢測,同時在模型建立前后檢測大鼠左后肢機械痛閾的變化。2、體外培養(yǎng)破骨前體細胞,研究艾拉莫德對破骨細胞分化激活的影響及其機制。分離并培養(yǎng)小鼠骨髓單核巨噬細胞,加入RANKL誘導其向破骨細胞分化,同時予以艾拉莫德處理,用TRAP染色的方法檢測艾拉莫德對破骨細胞分化的影響。Western blot免疫印跡法檢測RANKL刺激后細胞內(nèi)RANK受體下游NF-κB和MAPK通路的激活狀態(tài),以及轉(zhuǎn)錄因子c-Fos和NFATc1的表達水平。最后用qRT-PCR法檢測細胞內(nèi)NFATc1及其靶基因mRNA的表達水平。3、研究艾拉莫德對乳腺癌細胞炎癥因子表達、細胞增殖、侵襲等生物學行為的影響。qRT-PCR法檢測艾拉莫德對人乳腺癌MDA-MB-231細胞炎癥因子表達的影響,并運用Western blot免疫印跡法檢測細胞內(nèi)NF-κB p65的激活情況。隨后在兩種人乳腺癌細胞MDA-MB-231和MCF-7中檢測艾拉莫德對其增殖、遷移、侵襲的影響。用CCK-8法檢測艾拉莫德對細胞增殖的作用,流式細胞術檢測艾拉莫德對兩種細胞周期分布的影響,劃痕實驗及Transwell侵襲實驗檢測艾拉莫德對細胞遷移、侵襲的影響。實驗結(jié)果1、完成給藥后,大鼠左側(cè)脛骨X線檢查和HE染色的結(jié)果顯示接受艾拉莫德治療的大鼠脛骨骨破壞程度較安慰劑組減輕,同時治療后大鼠的左后肢機械痛覺過敏情況較安慰劑組改善。對大鼠左側(cè)脛骨的TRAP染色發(fā)現(xiàn)艾拉莫德治療組大鼠的脛骨內(nèi)活化的破骨細胞數(shù)目少于安慰劑組。2、體外實驗表明艾拉莫德顯著抑制了破骨細胞的分化。并且細胞活力檢測提示此抑制效應并非由艾拉莫德對細胞生長的抑制引起。信號通路的相關檢測結(jié)果顯示艾拉莫德顯著抑制了RANKL誘導的破骨前體細胞內(nèi)NF-κB和MAPK通路的激活,并下調(diào)了轉(zhuǎn)錄因子c-Fos和NFATc1的水平,減少了破骨細胞功能相關基因Cathepsin K、MMP-9和TRAP的表達。3、qRT-PCR和Western blot結(jié)果顯示艾拉莫德使MDA-MB-231細胞表達TNF-α、IL-1β、IL-6的水平下降,但對RANKL的表達沒有顯著影響,同時藥物處理后細胞內(nèi)NF-κBp65的磷酸化水平也下降。此外,艾拉莫德只在高濃度(30μg/ml)時輕度抑制了MDA-MB-231以及MCF-7細胞的增殖,而與對照組相比,艾拉莫德對兩種乳腺癌細胞的細胞周期分布、遷移和侵襲能力均沒有顯著影響。實驗結(jié)論1、艾拉莫德使乳腺癌骨轉(zhuǎn)移大鼠的骨破壞程度減輕,并使其機械性痛覺過敏癥狀得到一定改善。2、艾拉莫德緩解骨破壞的機制之一可能是其抑制了RANKL誘導的破骨前體細胞內(nèi)NF-κB通路和MAPK通路的激活,從而下調(diào)了破骨細胞特異性基因的表達,影響了破骨細胞的活化,削弱了其溶骨作用。3、艾拉莫德緩解骨破壞的另一可能機制是通過NF-κB相關機制下調(diào)了乳腺癌細胞表達炎癥因子(TNF-α、IL-1β、IL-6)的水平,減少了骨微環(huán)境中激活破骨細胞的刺激因素,而不直接顯著影響乳腺癌細胞RANKL的分泌、細胞的增殖、遷移和侵襲能力。
[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.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R737.9

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