本文選題：喹硫平　切入點：破骨細(xì)胞　出處：《第三軍醫(yī)大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景:進(jìn)展期乳腺癌、肺癌及前列腺癌等惡性腫瘤具有發(fā)生骨轉(zhuǎn)移的傾向。腫瘤細(xì)胞轉(zhuǎn)移至骨組織后可直接或間接激活破骨細(xì)胞并導(dǎo)致骨組織破壞,進(jìn)一步引發(fā)頑固性疼痛、神經(jīng)壓迫癥狀、病理性骨折及高鈣血癥等并發(fā)癥的發(fā)生,嚴(yán)重影響患者的生存及生活質(zhì)量。與其他組織不同,骨組織主要由較為堅硬的礦物質(zhì)構(gòu)成,骨組織對于腫瘤的侵襲應(yīng)具有更強的抗性。目前認(rèn)為:腫瘤骨轉(zhuǎn)移導(dǎo)致的骨損傷并非由腫瘤細(xì)胞直接引起而是由破骨細(xì)胞介導(dǎo)的骨吸收導(dǎo)致的。為了破壞骨組織,腫瘤細(xì)胞必須具有激活破骨細(xì)胞的相關(guān)特性,進(jìn)而導(dǎo)致骨組織的損傷。大量研究表明:腫瘤細(xì)胞可以通過釋放PTHr P、IL-1、IL-6和IL-11等因子作用于成骨細(xì)胞產(chǎn)生核因子κB受體活化因子配體(receptor activator of NF-κB ligand,RANKL)。RANKL目前被認(rèn)為是刺激破骨細(xì)胞活化最主要、最有效的刺激因子。破骨細(xì)胞來源于骨髓造血干細(xì)胞,是骨吸收的效應(yīng)細(xì)胞,腫瘤細(xì)胞轉(zhuǎn)移至骨組織引發(fā)的溶骨性損傷與其對破骨細(xì)胞的激活能力密切相關(guān)。因此,抑制破骨細(xì)胞分化的藥物能夠有效防止腫瘤骨轉(zhuǎn)移引發(fā)的溶骨性骨損傷及相關(guān)并發(fā)癥的發(fā)生。目前,臨床上用于抗骨損傷的藥物主要包括雙磷酸鹽類藥物、激素類藥物及RANKL單克隆抗體等。盡管上述藥物對于溶骨性骨損傷的治療均具有一定的療效,但這些藥物的應(yīng)用都存在一定的局限性及副作用的發(fā)生,如骨壞死、骨肉瘤發(fā)生、栓塞及食管刺激等。因此,尋求能夠更為安全、有效地用于治療腫瘤骨轉(zhuǎn)移等疾病引發(fā)骨損傷的藥物成為近些年關(guān)注及研究的熱點。喹硫平(Quetiapine,QUE)是經(jīng)FDA認(rèn)證的非典型抗精神病藥物。課題組前期研究發(fā)現(xiàn):QUE能夠通過作用于MAPK通路促進(jìn)神經(jīng)前體細(xì)胞向少突膠質(zhì)細(xì)胞的分化,同時有研究表明:QUE對NF-κB通路也具有一定的調(diào)控作用,而MAPK和NF-κB通路均是破骨細(xì)胞分化過程中的關(guān)鍵通路。于是我們嘗試驗證QUE能否通過抑制MAPK和NF-κB通路抑制RANKL誘導(dǎo)的破骨細(xì)胞前體細(xì)胞向破骨細(xì)胞分化,以及保護(hù)腫瘤細(xì)胞骨轉(zhuǎn)移引發(fā)的溶骨性骨損傷。期望通過本研究為腫瘤骨轉(zhuǎn)移等溶骨性疾病的治療提供新的藥物選擇和治療策略。研究目的:1.利用RANKL誘導(dǎo)的RAW 264.7細(xì)胞和小鼠骨髓來源巨噬細(xì)胞(Bone marrow-derived macrophages,BMMs)向破骨細(xì)胞分化模型,體外觀察非典型抗精神病藥物QUE對破骨細(xì)胞分化是否具有抑制作用。2.利用腫瘤細(xì)胞骨轉(zhuǎn)移動物模型,在體進(jìn)一步驗證QUE能否抑制腫瘤細(xì)胞介導(dǎo)的破骨細(xì)胞活化并起到骨保護(hù)作用。3.利用RANKL誘導(dǎo)的RAW 264.7細(xì)胞向破骨細(xì)胞分化模型,對QUE抑制破骨細(xì)胞分化的分子機制進(jìn)行初步探討。研究方法:1.選用目前使用較多的DMEM和α-MEM培養(yǎng)基培養(yǎng)RAW 264.7細(xì)胞,觀察上述培養(yǎng)基對RAW 264.7細(xì)胞增殖、存活的影響,從而選擇適合培養(yǎng)基用于后續(xù)實驗研究。用10、30、50、100、200ng/ml濃度RANKL重組蛋白作用于RAW 264.7細(xì)胞,遴選用于促進(jìn)破骨細(xì)胞分化的最佳RANKL使用濃度。2.分別利用差速貼壁法、梯度離心法等方法獲取BMMs,并使用不同濃度RANKL和M-CSF重組蛋白作用于BMMs,選擇最優(yōu)的細(xì)胞獲取方法和適宜的細(xì)胞因子使用濃度。3.使用不同濃度QUE(1μM、10μM、25μM、50μM、100μM)作用于RAW 264.7細(xì)胞并培養(yǎng)48小時,CCK-8檢測細(xì)胞增殖情況,選擇適合的QUE藥物使用濃度。利用前期建立的RANKL誘導(dǎo)的破骨細(xì)胞分化模型,觀察QUE對破骨細(xì)胞分化是否具有抑制作用。使用不同濃度QUE(1μM、10μM、25μM、50μM)作用于RANKL誘導(dǎo)的破骨細(xì)胞分化模型,利用實時熒光定量PCR檢測QUE對破骨細(xì)胞分化過程中相關(guān)m RNA表達(dá)的影響。4.RANKL誘導(dǎo)的破骨細(xì)胞分化過程中,分別于培養(yǎng)的第1天、第2天、第3天和第4天加入QUE,觀察QUE抑制破骨細(xì)胞分化的有效作用時期。5.購買商品化免疫缺陷小鼠,脛骨髓腔內(nèi)腫瘤細(xì)胞注射法構(gòu)建人類乳腺癌MDA-MB-231腫瘤細(xì)胞骨轉(zhuǎn)移動物模型。給予QUE(10mg/kg/d)腹腔注射處理6周,X線檢測QUE對腫瘤骨轉(zhuǎn)移引發(fā)骨損傷的保護(hù)作用,HE和TRAP染色觀察腫瘤細(xì)胞侵襲情況及QUE對腫瘤骨轉(zhuǎn)移引發(fā)破骨細(xì)胞活化的抑制作用。6.利用RANKL誘導(dǎo)的破骨細(xì)胞分化模型,采用western blot和免疫細(xì)胞化學(xué)染色等方法觀察QUE對RANKL誘導(dǎo)的破骨細(xì)胞分化過程中MAPK和NF-κB等通路的作用情況。結(jié)果:1.相比于α-MEM培養(yǎng)基,DMEM培養(yǎng)基更有利于RAW 264.7細(xì)胞的增殖、存活及形態(tài)的維持。使用不同濃度的RANKL重組蛋白作用于RAW 264.7細(xì)胞,當(dāng)濃度低于30ng/ml時,RANKL誘導(dǎo)破骨細(xì)胞分化作用比較有限。但當(dāng)濃度高于50ng/ml時,RANKL誘導(dǎo)破骨細(xì)胞分化的作用明顯增強,且50ng/ml到200ng/ml之間差異不明顯。RAW 264.7細(xì)胞經(jīng)RANKL(50ng/ml)誘導(dǎo)分化5天后,經(jīng)TRAP染色可見大量TRAP陽性多核破骨樣細(xì)胞形成。2.相比于梯度離心法和磁珠分選法,差速貼壁法簡單、易行,通過該方法分離得到的BMMs細(xì)胞經(jīng)RANKL和M-CSF共同誘導(dǎo)7天后可最終分化為TRAP陽性的多核破骨樣細(xì)胞。當(dāng)RANKL濃度為50ng/ml、M-CSF濃度為25ng/ml時,其對BMMs向破骨細(xì)胞分化具有較好的促進(jìn)作用。3.經(jīng)CCK-8檢測,1μM到50μM濃度QUE對RAW 264.7細(xì)胞沒有明顯毒性作用。用50μM濃度的QUE分別作用于RAW 264.7細(xì)胞3天、5天,作用于BMMs細(xì)胞3天、5天和7天,50μM濃度QUE對上述細(xì)胞的增殖均沒有顯著影響。4.相比于陽性對照組,1μM和10μM濃度QUE對RANKL誘導(dǎo)的破骨細(xì)胞分化無顯著影響,但當(dāng)濃度為25μM和50μM時,QUE能夠顯著抑制RANKL誘導(dǎo)的RAW264.7細(xì)胞向破骨細(xì)胞的分化。用50μM濃度QUE作用于RANKL和M-CSF共同誘導(dǎo)的BMMs向破骨細(xì)胞分化模型,7天后對上述細(xì)胞進(jìn)行TRAP染色。結(jié)果發(fā)現(xiàn):50μM濃度QUE同樣能夠有效抑制BMMs向破骨細(xì)胞的分化。經(jīng)實時熒光定量PCR檢測發(fā)現(xiàn):25μM和50μM濃度QUE可以有效抑制RANKL誘導(dǎo)的破骨細(xì)胞分化過程中破骨細(xì)胞分化相關(guān)基因的表達(dá)。5.RANKL誘導(dǎo)的破骨細(xì)胞分化模型中,當(dāng)?shù)?天和第2天加入50μM濃度QUE時,QUE能夠有效抑制破骨細(xì)胞的分化,而在分化后期(第3天、第4天)加入QUE則抑制破骨細(xì)胞分化作用不明顯。6.QUE處理乳腺癌骨轉(zhuǎn)移動物模型6周后,X線檢測結(jié)果提示:QUE能夠明顯減輕腫瘤骨轉(zhuǎn)移引發(fā)的骨損傷,起到骨保護(hù)作用;HE及TRAP染色結(jié)果顯示:實驗組及對照組均可見大量腫瘤細(xì)胞侵襲。與對照組相比,QUE處理組腫瘤侵襲區(qū)域與正常骨組織交界處破骨細(xì)胞的激活數(shù)量明顯減少。7.Western blot檢測QUE對破骨細(xì)胞分化過程中MAPK通路的作用。結(jié)果表明:QUE對p38、ERK和JNK的磷酸化都有明顯的抑制作用。8.RANKL誘導(dǎo)的破骨細(xì)胞分化模型中,在RANKL作用下的NF-κB通路中IκBα的降解明顯,而QUE可明顯抑制IκBα降解的發(fā)生,并同時抑制p65的磷酸化水平。免疫細(xì)胞化學(xué)染色顯示:在15到60分鐘時間段內(nèi)RANKL可以促進(jìn)p65的漿核轉(zhuǎn)移,而加入QUE處理后可明顯抑制漿核轉(zhuǎn)移的發(fā)生。結(jié)論:1.成功建立RANKL誘導(dǎo)的RAW 264.7細(xì)胞和BMMs向破骨細(xì)胞分化模型,為后續(xù)觀察QUE對破骨細(xì)胞分化的作用及相關(guān)機制研究打下了良好的實驗基礎(chǔ)。2.在RANKL誘導(dǎo)的RAW 264.7細(xì)胞向破骨細(xì)胞分化模型以及RANKL和M-CSF共同誘導(dǎo)的BMMs向破骨細(xì)胞分化模型中,QUE均能夠明顯抑制破骨細(xì)胞前體細(xì)胞向破骨細(xì)胞的分化,并能夠抑制破骨細(xì)胞分化過程中相關(guān)m RNA的表達(dá)。3.QUE對破骨細(xì)胞分化的抑制作用主要作用于破骨細(xì)胞分化的早期階段。4.人類乳腺癌MDA-MB-231細(xì)胞介導(dǎo)的腫瘤骨轉(zhuǎn)移動物模型中,QUE能夠明顯抑制腫瘤骨轉(zhuǎn)移引發(fā)的破骨細(xì)胞活化,進(jìn)而減輕腫瘤骨轉(zhuǎn)移引發(fā)的溶骨性骨損傷。5.QUE抑制破骨細(xì)胞分化的作用與其對RANKL誘導(dǎo)的破骨細(xì)胞分化過程中MAPK和NF-κB信號通路的抑制作用相關(guān)。
[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瀵肩殑BMMs鍚戠牬楠ㄧ粏鑳?yōu)鍒嗗寲妯″瀷涓?QUE鍧囪兘澶熸槑鏄炬姂鍒剁牬楠ㄧ粏鑳?yōu)鍓嶄綋缁嗚優(yōu)鍚戠牬楠ňl嗚優(yōu)鐨勫垎鍖，

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