本文選題：骨肉瘤 + RIPK4��； 參考：《蘭州大學(xué)》2017年碩士論文

【摘要】：目的:探討受體相互作用蛋白激酶4(receptor interacting protein kinase 4,RIPK4)在骨肉瘤組織和細(xì)胞中的表達(dá)及其臨床意義;進(jìn)一步研究RIPK4對(duì)骨肉瘤MG-63、U-2OS細(xì)胞上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)的影響及分子機(jī)制。方法:(1)應(yīng)用免疫組織化學(xué)法檢測(cè)36例骨肉瘤和15例骨軟骨瘤組織中RIPK4和β-catenin蛋白的表達(dá)情況,并分析二者與骨肉瘤患者的臨床病理特征及預(yù)后的相關(guān)性;(2)分別提取4例骨肉瘤和正常骨組織、骨肉瘤MG-63和U-2OS細(xì)胞、人正常成骨細(xì)胞hFOB1.19中蛋白,采用蛋白質(zhì)免疫印跡法進(jìn)一步驗(yàn)證RIPK4骨肉瘤組織和細(xì)胞中表達(dá)水平。(3)采用脂質(zhì)體法將特異性針對(duì)RIPK4基因的小分子干擾RNA(small interfering RNA,siRNA)分別轉(zhuǎn)入骨肉瘤MG-63、U-2OS細(xì)胞,成功構(gòu)建RIPK4基因沉默表達(dá)的骨肉瘤MG-63、U-2OS細(xì)胞系,采用蛋白質(zhì)印跡法檢測(cè)沉默RIPK4基因表達(dá)后骨肉瘤MG-63、U-2OS細(xì)胞系中RIPK4表達(dá)的變化;采用CCK-8法檢測(cè)沉默RIPK4基因后對(duì)骨肉瘤MG-63、U-2OS細(xì)胞增殖活力的影響;Transwell小室法檢測(cè)骨肉瘤MG-63、U-2OS細(xì)胞侵襲和遷移能力的變化;倒置光學(xué)顯微鏡下觀察骨肉瘤MG-63、U-2OS細(xì)胞形態(tài)的變化。最后,懫用蛋白質(zhì)印跡法和免疫熒光染色法檢測(cè)骨肉瘤MG-63、U-2OS細(xì)胞中EMT相關(guān)分子E-鈣黏蛋白(E-cadherin)、N-鈣黏蛋白(N-cadherin)、波形蛋白(vimentin)以及β-catenin總蛋白和核蛋白的表達(dá)變化。結(jié)果:(1)骨肉瘤組織中RIPK4、β-catenin陽(yáng)性表達(dá)率分別為63.9%(23/36)、75.0%(27/36),顯著高于其在骨軟骨瘤組織中的陽(yáng)性表達(dá)率6.7%(1/15)、20.0%(3/15)(P值均0.05)。RIPK4和β-catenin在骨肉瘤組織的表達(dá)均與Enneking分期密切相關(guān)(P值均0.05);RIPK4的表達(dá)還與腫瘤大小相關(guān)(P0.05)。骨肉瘤組織中RIPK4的表達(dá)與β-catenin表達(dá)之間呈正相關(guān)(r=0.634,P0.01)。Kaplan-Meier生存分析發(fā)現(xiàn),RIPK4、β-catenin陽(yáng)性表達(dá)組中患者的總生存時(shí)間均短于陰性表達(dá)組(P值均0.05)。單因素COX回歸分析發(fā)現(xiàn),年齡、Enneking分期、轉(zhuǎn)移、RIPK4蛋白的陽(yáng)性表達(dá)與患者總生存時(shí)間相關(guān)(P值均0.05)。(2)蛋白質(zhì)印跡法結(jié)果顯示,RIPK4無(wú)論是在骨肉組織還是骨肉細(xì)胞中的表達(dá)水平均高于正常骨組織和正常成骨細(xì)胞。(3)RIPK4-siRNA轉(zhuǎn)染骨肉瘤MG-63、U-2OS細(xì)胞后,RIPK4的表達(dá)水平顯著下調(diào)(P0.05);RIPK4-siRNA轉(zhuǎn)染組MG-63、U-2OS細(xì)胞的增殖、遷移以及侵襲能力明顯降低(P值均0.05);倒置光學(xué)顯微鏡下觀察發(fā)現(xiàn)RIPK4-siRNA轉(zhuǎn)染后,骨肉瘤MG-63、U-2OS細(xì)胞由間質(zhì)形態(tài)轉(zhuǎn)變成上皮細(xì)胞形態(tài)。此外,蛋白質(zhì)印跡法結(jié)果顯示:RIPK4-siRNA轉(zhuǎn)染組MG-63、U-2OS細(xì)胞中E-cadherin的表達(dá)水平顯著上調(diào)(P0.05),而N-cadherin、vimentin以及β-catenin總蛋白和核蛋白的表達(dá)水平明顯降低(P0.05)。同時(shí),免疫熒光染色也得到了相同結(jié)論。結(jié)論:(1)骨肉瘤組織中RIPK4、β-catenin表達(dá)明顯上調(diào),且與骨肉瘤患者的病理特征及預(yù)后密切相關(guān);同時(shí),二者表達(dá)呈正相關(guān),提示RIPK4和β-catenin可能共同協(xié)調(diào)骨肉瘤發(fā)生發(fā)展。(2)沉默RIPK4可能通過(guò)抑制Wnt信號(hào)通路逆轉(zhuǎn)骨肉瘤MG-63、U-2OS細(xì)胞EMT的發(fā)生。
[Abstract]:Objective: To investigate the effects of receptor interacting protein kinase 4 (receptor interacting protein kinase 4, RIPK4) expression in osteosarcoma tissues and cells and its clinical significance; further study of RIPK4 on osteosarcoma MG-63 and U-2OS cells of epithelial mesenchymal transition (epithelial-mesenchymal transition, EMT) and the effects of molecular mechanisms. Methods: (1) expression RIPK4 and -catenin protein was detected by immunohistochemistry in 36 cases of osteosarcoma and 15 cases of osteochondroma tissues, and to analyze the correlation between clinicopathologic features and prognosis of two patients with osteosarcoma; (2) were extracted from 4 cases of osteosarcoma and normal bone tissues, osteosarcoma MG-63 and U-2OS cells, human normal bone cells hFOB1.19 protein, using Western blotting further confirmed the expression level of RIPK4 in osteosarcoma tissues and cells. (3) using Lipofectamine specific RIPK4 gene targeted small molecule RNA (siRNA small interfering interference RNA) were transferred into osteosarcoma MG-63, U-2OS cells, the successful construction of osteosarcoma MG-63 expression of RIPK4 gene silencing, U-2OS cell line was detected by using Western blotting, silencing of RIPK4 gene expression in human osteosarcoma MG-63, expression of RIPK4 in U-2OS cell line; MG-63 silencing RIPK4 gene on osteosarcoma detection using CCK-8 method, U-2OS cell proliferation activity; osteosarcoma MG-63 were determined by Transwell. The changes of U-2OS cell invasion and migration of osteosarcoma MG-63; observed under an inverted microscope, U-2OS changes in cell morphology. Finally, Zhi by Western blotting and immunofluorescence staining of osteosarcoma U-2OS cells in MG-63 assay. EMT related molecules of E- cadherin (E-cadherin), N- cadherin (N-cadherin), vimentin (vimentin) and the expression of beta -catenin total protein and nuclear protein changes. Results: (1) osteosarcoma RIPK4 in the tumor tissues, beta -catenin positive expression rate was 63.9% (23/36), 75% (27/36), significantly higher than the positive expression in osteochondroma tissues was 6.7% (1/15), 20% (3/15) (P 0.05) expression of.RIPK4 and beta -catenin in osteosarcoma tissues were associated with Enneking stage closely related (P 0.05); the expression of RIPK4 was correlated with tumor size (P0.05). The expression of RIPK4 in osteosarcoma and beta -catenin expression was positively correlated (r=0.634, P0.01).Kaplan-Meier survival analysis showed that RIPK4, the total survival time of patients with positive expression in group B -catenin were shorter than the negative group (P 0.05). The single factor COX regression analysis showed that age, Enneking staging, metastasis, RIPK4 protein expression was correlated with survival time (P < 0.05). (2) the results of Western blotting showed that RIPK4 in osteosarcoma tissue or blood cells in the expression level Were higher than that of normal bone tissue and normal osteoblast cells. (3) RIPK4-siRNA was transfected into osteosarcoma MG-63 and U-2OS cells, the expression level of RIPK4 was significantly reduced (P0.05); RIPK4-siRNA MG-63 transfection group, U-2OS cell proliferation, migration and invasion ability decreased significantly (P < 0.05); RIPK4-siRNA transfection was observed by inverted optical microscope after MG-63 of osteosarcoma, U-2OS cells by mesenchymal morphology into epithelial cells. In addition, the results of Western blotting showed that RIPK4-siRNA MG-63 transfection group, the expression level of E-cadherin in U-2OS cells was significantly up-regulated (P0.05), N-cadherin, vimentin and the expression level of beta -catenin total protein and nuclear protein decreased significantly (P0.05). At the same time, immunofluorescence staining also got the same conclusion. Conclusion: (1) RIPK4 in osteosarcoma, beta -catenin expression, which is closely related with the pathological characteristics and prognosis of patients with osteosarcoma; At the same time, the two was a positive correlation, suggesting that RIPK4 and -catenin may coordinate beta osteosarcoma development. (2) silencing RIPK4 could inhibit the Wnt signaling pathway and reverses osteosarcoma U-2OS cells MG-63, EMT.

【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R738

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