本文關(guān)鍵詞： 骨肉瘤 NRSN2 PI3K/AKT/mTOR信號(hào)通路 Wnt/β-catenin信號(hào)通路　出處：《新疆醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：目的:(1)通過(guò)檢測(cè)NRSN2在骨肉瘤組織及骨肉瘤細(xì)胞中的表達(dá)差異,研究NRSN2與骨肉瘤發(fā)生之間的相關(guān)性。(2)通過(guò)沉默/過(guò)表達(dá)NRSN2骨肉瘤細(xì)胞系的體內(nèi)體外實(shí)驗(yàn),探討NRSN2對(duì)骨肉瘤細(xì)胞增殖、分化的作用。(3)探討NRSN2在骨肉瘤細(xì)胞增殖、分化中的分子機(jī)制研究,確定NRSN2調(diào)控作用是否通過(guò)P13K/AKT/m TOR及Wnt/β-catenin信號(hào)通路。方法:(1)收集18例骨肉瘤和旁骨肉瘤組織標(biāo)本,采用實(shí)時(shí)定量PCR及免疫組織化學(xué)法檢測(cè)標(biāo)本中NRSN2的mRNA及蛋白表達(dá)水平,分析NRSN2在骨肉瘤和旁骨肉瘤組織標(biāo)本中的表達(dá)差異。(2)采用實(shí)時(shí)定量PCR及Western blot檢測(cè)體外培養(yǎng)的U20S、Saos2、MG63骨肉瘤細(xì)胞系中NRSN2的m RNA和蛋白表達(dá)水平,分析NRSN2在骨肉瘤細(xì)胞系中的表達(dá)差異。(3)選擇U20S、Saos2骨肉瘤細(xì)胞系(NRSN2相對(duì)高表達(dá)),構(gòu)建沉默NRSN2的U20S、Saos2骨肉瘤細(xì)胞系,Western blot驗(yàn)證沉默效果;體外培養(yǎng)沉默NRSN2的骨肉瘤細(xì)胞系,采用CCK8法檢測(cè)骨肉瘤細(xì)胞增殖、分化能力,通過(guò)軟瓊脂細(xì)胞克隆形成實(shí)驗(yàn),測(cè)量克隆細(xì)胞數(shù),沉默NRSN2的U2OS骨肉瘤細(xì)胞系接種裸鼠皮下,進(jìn)行異種皮下腫瘤形成實(shí)驗(yàn),測(cè)量瘤體重量,進(jìn)一步分析沉默NRSN2在體內(nèi)體外對(duì)骨肉瘤細(xì)胞增殖、分化的作用。(4)選擇MG63骨肉瘤細(xì)胞系(NRSN2相對(duì)低表達(dá)),構(gòu)建過(guò)表達(dá)NRSN2的MG63骨肉瘤細(xì)胞系,Western blot驗(yàn)證NRSN2過(guò)表達(dá)效果;體外培養(yǎng)過(guò)表達(dá)NRSN2的骨肉瘤細(xì)胞系,利用CCK8法檢測(cè)骨肉瘤細(xì)胞增殖、分化能力,通過(guò)軟瓊脂細(xì)胞克隆形成實(shí)驗(yàn),測(cè)量克隆細(xì)胞數(shù),過(guò)表達(dá)NRSN2的骨肉瘤細(xì)胞系接種裸鼠皮下,進(jìn)行異種皮下腫瘤形成實(shí)驗(yàn),測(cè)量瘤體重量,分析過(guò)表達(dá)NRSN2在體內(nèi)體外對(duì)骨肉瘤細(xì)胞增殖、分化的作用。(5)沉默NRSN2的U20S、Saos2骨肉瘤細(xì)胞系中,采用Wesrtern blot檢測(cè)PI3K/AKT/m TOR及Wnt/β-catenin信號(hào)通路相關(guān)蛋白Akt、p-Akt、mTOR、p-mTOR、GSK3β、p-GSK3β、nu-β-catenin的蛋白表達(dá)水平,進(jìn)一步采用實(shí)時(shí)定量PCR分別檢測(cè)Wnt/β-catenin信號(hào)通路目標(biāo)蛋白CCND1和c-myc的mRNA表達(dá)水平,分析沉默NRSN2對(duì)PI3K/AKT/m TOR及Wnt/β-catenin信號(hào)通路的調(diào)控。(6)過(guò)表達(dá)NRSN2的MG63骨肉瘤細(xì)胞系中,采用Wesrtern blot檢測(cè)Akt、p-Akt、mTOR、p-m TOR、GSK3β、p-GSK3β、β-catenin的蛋白表達(dá)水平,采用實(shí)時(shí)定量PCR檢測(cè)Wnt/β-catenin信號(hào)通路目標(biāo)蛋白CCND1和c-myc的m RNA表達(dá)水平,加入β-catenin抑制劑IWR-1-endo后采用CCK8法檢測(cè)骨肉瘤細(xì)胞增殖、分化能力,分析過(guò)表達(dá)NRSN2對(duì)PI3K/AKT/mTOR及Wnt/β-catenin信號(hào)通路的調(diào)控。結(jié)果:(1)18例骨肉瘤組織標(biāo)本中NRSN2的mRNA及蛋白表達(dá)水平均高于旁骨肉瘤組織標(biāo)本,差異有統(tǒng)計(jì)學(xué)意義(P0.01),提示:NRSN2與骨肉瘤相關(guān)。(2)U20S、Saos2、MG63骨肉瘤細(xì)胞系中NRSN2的mRNA和蛋白表達(dá)水平均高于hFOB1.19組,NRSN2與骨肉瘤細(xì)胞增殖、分化相關(guān),差異有統(tǒng)計(jì)學(xué)意義(均P0.01)。U20S和Saos2骨肉瘤細(xì)胞系相對(duì)高于MG63骨肉瘤細(xì)胞系,差異有統(tǒng)計(jì)學(xué)意義(P0.05),U20S和Saos2骨肉瘤細(xì)胞系間差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。(3)沉默NRSN2明顯抑制骨肉瘤細(xì)胞系U2OS、Saos2的增殖、分化能力(P0.01),重新上調(diào)NRSN2生物學(xué)作用后重新提高U2OS、Saos2骨肉瘤細(xì)胞系增殖、分化能力(P0.01),沉默NRSN2組克隆細(xì)胞數(shù)少于對(duì)照組(均P0.01),沉默NRSN2組裸鼠皮下形成的瘤體重量小于對(duì)照組(P0.01),未轉(zhuǎn)染和重新上調(diào)NRSN2組作為對(duì)照組。(4)過(guò)表達(dá)NRSN2使MG63骨肉瘤細(xì)胞系增殖、分化能力增強(qiáng)(P0.01),重新下調(diào)NRSN2生物學(xué)作用后MG63骨肉瘤細(xì)胞系增殖、分化能力下降(P0.01),過(guò)表達(dá)NRSN2組克隆細(xì)胞數(shù)多于對(duì)照組(均P0.01),過(guò)表達(dá)NRSN2組裸鼠皮下形成的瘤體重量大于對(duì)照組(P0.01),空質(zhì)粒和重新下調(diào)NRSN2組作為對(duì)照組。(5)沉默NRSN2的U2OS骨肉瘤細(xì)胞系中p-Akt、p-m TOR、p-GSK3β、nu-β-catenin的蛋白表達(dá)水平,CCND1及c-myc的mRNA表達(dá)水平均低于對(duì)照組(P0.01),未轉(zhuǎn)染組和重新上調(diào)NRSN2組作為對(duì)照組。(6)過(guò)表達(dá)NRSN2的MG63骨肉瘤細(xì)胞系中p-Akt、p-mTOR、p-GSK3β、nu-β-catenin的蛋白表達(dá)水平,CCND1及c-myc的mRNA表達(dá)水平均高于對(duì)照組(P0.01),加入β-catenin信號(hào)通路抑制劑IWR-1-endo后,MG63骨肉瘤細(xì)胞系增殖、分化能力比對(duì)照組明顯受到抑制(P0.01),空質(zhì)粒組作為對(duì)照組。結(jié)論:(1)通過(guò)檢測(cè)骨肉瘤組織及U20S、Saos2、MG63骨肉瘤細(xì)胞系中NRSN2的mRNA和蛋白表達(dá)水平,確定NRSN2與骨肉瘤之間的相關(guān)性。(2)通過(guò)沉默/過(guò)表達(dá)NRSN2骨肉瘤細(xì)胞系的體內(nèi)體外實(shí)驗(yàn)研究,進(jìn)一步證實(shí)NRSN2對(duì)骨肉瘤細(xì)胞增殖、分化有促進(jìn)作用。(3)通過(guò)NRSN2的分子機(jī)制研究,闡明NRSN2調(diào)控PI3k/AKT/mTOR及Wnt/β-catenin信號(hào)通路促進(jìn)骨肉瘤細(xì)胞增值、分化。
[Abstract]:Objective: (1) through the detection of NRSN2 in osteosarcoma tissue and osteosarcoma cells in the differential expression, to study the correlation between NRSN2 and osteosarcoma. (2) in vivo and in vitro silencing / NRSN2 osteosarcoma cell line expression of NRSN2, on the proliferation of osteosarcoma cells, differentiation (3. Study) NRSN2 on the proliferation of osteosarcoma cells, molecular mechanism of differentiation, determine whether the regulation of NRSN2 by P13K/AKT/m TOR and Wnt/ -catenin signaling pathway. Methods: (1) collected 18 cases of bone sarcoma and adjacent osteosarcoma tissues, the expression level of mRNA protein and NRSN2 by real-time quantitative PCR and immunohistochemistry in the sample, analysis of differential expression of NRSN2 in osteosarcoma and adjacent bone sarcoma tissues. (2) using real-time quantitative PCR and Western blot cultured in vitro detection of U20S, Saos2, m RNA and NRSN2 MG63 protein expression in osteosarcoma cell lines of water Flat, differential expression of NRSN2 in osteosarcoma cell lines. (3) U20S, Saos2 osteosarcoma cell lines (NRSN2 relatively high expression), construction of NRSN2 silencing U20S, Saos2 osteosarcoma cell line, Western blot NRSN2 to verify the silence effect; silence of osteosarcoma cell lines cultured in vitro and the ability of using CCK8 the detection method of osteosarcoma cell proliferation, differentiation, cell clone formation assay in soft agar cloning, measuring cell number, U2OS osteosarcoma cell line was inoculated subcutaneously in nude mice. NRSN2, subcutaneous tumor xenograft formation experiment, measuring tumor weight, further analysis of the silencing of NRSN2 in vitro and in vivo on osteosarcoma cell proliferation and differentiation. (4) MG63 osteosarcoma cell lines (NRSN2 low expression), construct MG63 osteosarcoma cell lines expressing NRSN2, Western blot to verify the expression of NRSN2 in vitro effect; overexpression of human osteosarcoma cell line NRSN2, using the CCK8 method The ability to detect proliferation and differentiation of osteosarcoma cells, forming experiment by soft agar cloning, cloning of measuring cell number, over expression of osteosarcoma cell line NRSN2 were inoculated subcutaneously in nude mice subcutaneous tumor xenograft formation, experiment, measurement of tumor weight, analyzed the expression of NRSN2 in vivo in vitro proliferation of osteosarcoma cells, differentiation. (5) NRSN2 silencing U20S, Saos2 in osteosarcoma cell lines, using Wesrtern blot and Wnt/ TOR detection of PI3K/AKT/m beta -catenin signaling pathway related protein Akt, p-Akt, mTOR, p-mTOR, GSK3 beta, p-GSK3 beta, beta -catenin expression level of nu- protein were determined by real-time PCR detection of Wnt/ beta -catenin signaling pathway target CCND1 c-myc and protein expression levels of mRNA, NRSN2 analysis of the silence on the regulation of PI3K/AKT/m TOR and Wnt/ -catenin signaling pathway. (6) the expression of NRSN2 MG63 in osteosarcoma cell lines, using Wesrtern blot to detect A KT, p-Akt, mTOR, P-M, TOR, GSK3 beta, p-GSK3 beta, beta -catenin protein expression level, using M RNA real-time quantitative PCR detection of Wnt/ beta -catenin signaling pathway protein CCND1 and c-myc expression levels, detection of osteosarcoma cell proliferation by CCK8 method, adding beta -catenin inhibitors after IWR-1-endo differentiation ability analysis the expression of NRSN2 on the regulation of PI3K/AKT/mTOR and Wnt/ beta -catenin signaling pathway. Results: (1) 18 cases of mRNA and NRSN2 protein in osteosarcoma tissues the expression level was higher than that of adjacent osteosarcoma tissues, the difference was statistically significant (P0.01), suggested that NRSN2 associated with osteosarcoma. (2) U20S, Saos2, expression the level of mRNA and NRSN2 MG63 protein in osteosarcoma cell lines were higher than that of group hFOB1.19, NRSN2 and osteosarcoma cell proliferation, differentiation, the difference was statistically significant (P0.01).U20S and Saos2 osteosarcoma cell line of MG63 is higher than that of osteosarcoma cell lines, difference There was statistical significance (P0.05), there were no significant differences between U20S and Saos2 in osteosarcoma cell line (P0.05). (3) silencing NRSN2 inhibited osteosarcoma cell line U2OS, Saos2 proliferation, differentiation ability (P0.01), to improve the biological function of NRSN2 up-regulated U2OS, Saos2 proliferation of osteosarcoma cells, differentiation capacity (P0.01), silent group NRSN2 cell clone number is less than the control group (P0.01), the tumor weight of NRSN2 silencing groups nude mice formed less than that of the control group (P0.01), transfected and re raised NRSN2 group as the control group. (4) overexpression of NRSN2 to proliferation of human osteosarcoma MG63 cells, enhance the ability of differentiation (P0.01), to the down-regulation of NRSN2 biological effects of proliferation of line MG63 osteosarcoma cells, differentiation ability (P0.01), NRSN2 overexpression group cloned cells more than the control group (P0.01), expression of tumor weight in NRSN2 group nude mice formed greater than that of the control group (P0.01), empty The new plasmid and down-regulation of NRSN2 group as the control group. (5) p-Akt, U2OS osteosarcoma cell line NRSN2 in P-M silencing TOR, p-GSK3 beta, beta -catenin expression level of nu- protein, the expression level of CCND1 and c-myc mRNA were lower than the control group (P0.01), non transfection group and up-regulated NRSN2 group as a control group. (6) the over expression of p-Akt and MG63 in osteosarcoma cell line NRSN2 in p-mTOR, p-GSK3 beta, beta -catenin expression level of nu- protein, the expression level of CCND1 and c-myc mRNA were higher than the control group (P0.01), the addition of the beta -catenin signaling pathway inhibitor IWR-1-endo, MG63 proliferation of osteosarcoma cells, differentiation ability than the control group was significantly inhibited (P0.01), empty plasmid group as the control group. Conclusion: (1) through the detection of osteosarcoma tissue and U20S, Saos2, mRNA and protein expression level of NRSN2 MG63 in osteosarcoma cell lines, to determine the correlation between NRSN2 and osteosarcoma. (2) through the silence / expression In vivo and in vitro studies of NRSN2 osteosarcoma cell line further confirm that NRSN2 can promote the proliferation and differentiation of osteosarcoma cells. (3) through the molecular mechanism of NRSN2, it is clarified that NRSN2 regulates PI3k/AKT/mTOR and Wnt/ beta -catenin signaling pathway to promote the proliferation and differentiation of osteosarcoma cells.

【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R738

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2 尚永軍;TIM-3在骨肉瘤侵襲轉(zhuǎn)移中的作用及機(jī)制研究[D];延邊大學(xué);2015年

3 禚文昆;MiR-20a調(diào)節(jié)EGR2對(duì)骨肉瘤的影響[D];第四軍醫(yī)大學(xué);2015年

4 高琰;CD44在卵巢癌/骨肉瘤進(jìn)展中的作用及上轉(zhuǎn)換納米逆轉(zhuǎn)腫瘤耐藥的研究[D];鄭州大學(xué);2016年

5 趙加力;B7-H3在骨肉瘤微環(huán)境中的表達(dá)及其作用機(jī)制[D];蘇州大學(xué);2016年

6 林博川;MicroRNA-184在骨肉瘤阿霉素耐藥中的功能與機(jī)制研究[D];南方醫(yī)科大學(xué);2016年

7 包擁政;miR-664調(diào)控靶基因SOX7促進(jìn)MG-63骨肉瘤細(xì)胞體外侵襲和遷移機(jī)理研究[D];南方醫(yī)科大學(xué);2016年

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9 閆廣寧;骨肉瘤干細(xì)胞自我更新的調(diào)控機(jī)制及意義[D];第三軍醫(yī)大學(xué);2016年

10 程思;S100A9在骨肉瘤中的表達(dá)和對(duì)骨肉瘤功能作用的影響及相關(guān)機(jī)制的研究[D];重慶醫(yī)科大學(xué);2016年

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1 靳曉建;新輔助化療結(jié)合保肢手術(shù)治療骨肉瘤的臨床療效分析[D];山西醫(yī)科大學(xué);2015年

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3 陳記軍;骨肉瘤患者血清中特異性microRNA表達(dá)譜的篩選及其臨床意義的研究[D];南京大學(xué);2013年

4 康學(xué)華;骨肉瘤中WIF-1基因啟動(dòng)子區(qū)異常甲基化及mRNA的表達(dá)研究[D];青島大學(xué);2015年

5 李騰飛;HIF-1α和PTEN在骨肉瘤中的表達(dá)及其臨床意義[D];南華大學(xué);2015年

6 關(guān)國(guó)鋒;CXCR4靶點(diǎn)近紅外光學(xué)分子成像檢測(cè)骨肉瘤原位及肺轉(zhuǎn)移腫瘤病灶的研究[D];第四軍醫(yī)大學(xué);2015年

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8 榮小麗;LIMK1基因穩(wěn)定轉(zhuǎn)染骨肉瘤耐藥細(xì)胞系的建立及其增殖侵襲能力的研究[D];吉林大學(xué);2016年

9 關(guān)紅亞;miR-192靶向TCF7基因?qū)侨饬黾?xì)胞增殖和侵襲的抑制作用[D];鄭州大學(xué);2016年

10 閆紅衛(wèi);骨肉瘤臨床診治的影像學(xué)檢查優(yōu)化探討[D];廣西醫(yī)科大學(xué);2016年

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