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

【摘要】：目的:(1)通過檢測NRSN2在骨肉瘤組織及骨肉瘤細胞中的表達差異,研究NRSN2與骨肉瘤發(fā)生之間的相關性。(2)通過沉默/過表達NRSN2骨肉瘤細胞系的體內體外實驗,探討NRSN2對骨肉瘤細胞增殖、分化的作用。(3)探討NRSN2在骨肉瘤細胞增殖、分化中的分子機制研究,確定NRSN2調控作用是否通過P13K/AKT/m TOR及Wnt/β-catenin信號通路。方法:(1)收集18例骨肉瘤和旁骨肉瘤組織標本,采用實時定量PCR及免疫組織化學法檢測標本中NRSN2的mRNA及蛋白表達水平,分析NRSN2在骨肉瘤和旁骨肉瘤組織標本中的表達差異。(2)采用實時定量PCR及Western blot檢測體外培養(yǎng)的U20S、Saos2、MG63骨肉瘤細胞系中NRSN2的m RNA和蛋白表達水平,分析NRSN2在骨肉瘤細胞系中的表達差異。(3)選擇U20S、Saos2骨肉瘤細胞系(NRSN2相對高表達),構建沉默NRSN2的U20S、Saos2骨肉瘤細胞系,Western blot驗證沉默效果;體外培養(yǎng)沉默NRSN2的骨肉瘤細胞系,采用CCK8法檢測骨肉瘤細胞增殖、分化能力,通過軟瓊脂細胞克隆形成實驗,測量克隆細胞數(shù),沉默NRSN2的U2OS骨肉瘤細胞系接種裸鼠皮下,進行異種皮下腫瘤形成實驗,測量瘤體重量,進一步分析沉默NRSN2在體內體外對骨肉瘤細胞增殖、分化的作用。(4)選擇MG63骨肉瘤細胞系(NRSN2相對低表達),構建過表達NRSN2的MG63骨肉瘤細胞系,Western blot驗證NRSN2過表達效果;體外培養(yǎng)過表達NRSN2的骨肉瘤細胞系,利用CCK8法檢測骨肉瘤細胞增殖、分化能力,通過軟瓊脂細胞克隆形成實驗,測量克隆細胞數(shù),過表達NRSN2的骨肉瘤細胞系接種裸鼠皮下,進行異種皮下腫瘤形成實驗,測量瘤體重量,分析過表達NRSN2在體內體外對骨肉瘤細胞增殖、分化的作用。(5)沉默NRSN2的U20S、Saos2骨肉瘤細胞系中,采用Wesrtern blot檢測PI3K/AKT/m TOR及Wnt/β-catenin信號通路相關蛋白Akt、p-Akt、mTOR、p-mTOR、GSK3β、p-GSK3β、nu-β-catenin的蛋白表達水平,進一步采用實時定量PCR分別檢測Wnt/β-catenin信號通路目標蛋白CCND1和c-myc的mRNA表達水平,分析沉默NRSN2對PI3K/AKT/m TOR及Wnt/β-catenin信號通路的調控。(6)過表達NRSN2的MG63骨肉瘤細胞系中,采用Wesrtern blot檢測Akt、p-Akt、mTOR、p-m TOR、GSK3β、p-GSK3β、β-catenin的蛋白表達水平,采用實時定量PCR檢測Wnt/β-catenin信號通路目標蛋白CCND1和c-myc的m RNA表達水平,加入β-catenin抑制劑IWR-1-endo后采用CCK8法檢測骨肉瘤細胞增殖、分化能力,分析過表達NRSN2對PI3K/AKT/mTOR及Wnt/β-catenin信號通路的調控。結果:(1)18例骨肉瘤組織標本中NRSN2的mRNA及蛋白表達水平均高于旁骨肉瘤組織標本,差異有統(tǒng)計學意義(P0.01),提示:NRSN2與骨肉瘤相關。(2)U20S、Saos2、MG63骨肉瘤細胞系中NRSN2的mRNA和蛋白表達水平均高于hFOB1.19組,NRSN2與骨肉瘤細胞增殖、分化相關,差異有統(tǒng)計學意義(均P0.01)。U20S和Saos2骨肉瘤細胞系相對高于MG63骨肉瘤細胞系,差異有統(tǒng)計學意義(P0.05),U20S和Saos2骨肉瘤細胞系間差異無統(tǒng)計學意義(P0.05)。(3)沉默NRSN2明顯抑制骨肉瘤細胞系U2OS、Saos2的增殖、分化能力(P0.01),重新上調NRSN2生物學作用后重新提高U2OS、Saos2骨肉瘤細胞系增殖、分化能力(P0.01),沉默NRSN2組克隆細胞數(shù)少于對照組(均P0.01),沉默NRSN2組裸鼠皮下形成的瘤體重量小于對照組(P0.01),未轉染和重新上調NRSN2組作為對照組。(4)過表達NRSN2使MG63骨肉瘤細胞系增殖、分化能力增強(P0.01),重新下調NRSN2生物學作用后MG63骨肉瘤細胞系增殖、分化能力下降(P0.01),過表達NRSN2組克隆細胞數(shù)多于對照組(均P0.01),過表達NRSN2組裸鼠皮下形成的瘤體重量大于對照組(P0.01),空質粒和重新下調NRSN2組作為對照組。(5)沉默NRSN2的U2OS骨肉瘤細胞系中p-Akt、p-m TOR、p-GSK3β、nu-β-catenin的蛋白表達水平,CCND1及c-myc的mRNA表達水平均低于對照組(P0.01),未轉染組和重新上調NRSN2組作為對照組。(6)過表達NRSN2的MG63骨肉瘤細胞系中p-Akt、p-mTOR、p-GSK3β、nu-β-catenin的蛋白表達水平,CCND1及c-myc的mRNA表達水平均高于對照組(P0.01),加入β-catenin信號通路抑制劑IWR-1-endo后,MG63骨肉瘤細胞系增殖、分化能力比對照組明顯受到抑制(P0.01),空質粒組作為對照組。結論:(1)通過檢測骨肉瘤組織及U20S、Saos2、MG63骨肉瘤細胞系中NRSN2的mRNA和蛋白表達水平,確定NRSN2與骨肉瘤之間的相關性。(2)通過沉默/過表達NRSN2骨肉瘤細胞系的體內體外實驗研究,進一步證實NRSN2對骨肉瘤細胞增殖、分化有促進作用。(3)通過NRSN2的分子機制研究,闡明NRSN2調控PI3k/AKT/mTOR及Wnt/β-catenin信號通路促進骨肉瘤細胞增值、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.

【學位授予單位】：新疆醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R738

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