發(fā)布時(shí)間：2018-11-08 20:14

【摘要】：目的:1.研究micro RNA-146a對(duì)人神經(jīng)膠質(zhì)瘤U251細(xì)胞耐藥株的作用。2.探討Notch1基因與micro RNA-146a在人膠質(zhì)瘤耐藥性中的相關(guān)性。方法:1.通過(guò)慢病毒包裝、轉(zhuǎn)染、篩選,分別獲得高表達(dá)NICD的U251細(xì)胞株及其空載plvx的U251細(xì)胞株,并通過(guò)Western Blotting及免疫熒光染色進(jìn)行鑒定。2.micro RNA芯片檢測(cè)和篩選NICD高表達(dá)和空載plvx的U251細(xì)胞及對(duì)照U251細(xì)胞中差異性表達(dá)的micro RNAs。3.RT-q PCR檢測(cè)鑒定NICD高表達(dá)和空載plvx的U251細(xì)胞及對(duì)照U251細(xì)胞中mi RNA-146a的表達(dá)情況。4.懫用替莫唑胺(TMZ)濃度遞增的作用方式誘導(dǎo)U251細(xì)胞,初始誘導(dǎo)劑量0.25μg/ml(0.2875μM),最高誘導(dǎo)劑量為20μg/ml(103.0μM),獲得穩(wěn)定的對(duì)TMZ耐藥的U251細(xì)胞,命名為U251TR,用CCK-8法檢測(cè)U251,U251TR的細(xì)胞增殖抑制率,計(jì)算半數(shù)抑制濃度IC50及耐藥指數(shù)(RF)。5.RT-q PCR檢測(cè)U251TR及其對(duì)照U251細(xì)胞中mi RNA-146a的表達(dá)情況。6.用脂質(zhì)體介導(dǎo)的轉(zhuǎn)染方法將mi R-146a-mimics轉(zhuǎn)入U(xiǎn)251TR,稱為轉(zhuǎn)染組細(xì)胞(U251TR-mimics),熒光顯微法檢測(cè)轉(zhuǎn)染組轉(zhuǎn)染效率,RT-q PCR檢測(cè)U251TR及U251TR-mimics中mi R-146a的表達(dá)情況,CCK-8法檢測(cè)U251TR-mimics細(xì)胞增殖抑制率,計(jì)算IC50及RF。7.Western Blotting檢測(cè)U251,U251TR及U251TR-mimics細(xì)胞中Notch1基因的表達(dá)情況。 8.通過(guò)生物信息學(xué)預(yù)測(cè)靶向調(diào)控Notch1的mi RNA-146a,然后通過(guò)雙熒光素酶報(bào)告基因?qū)嶒?yàn)及Western blotting實(shí)驗(yàn)對(duì)mi R-146a進(jìn)行靶標(biāo)的驗(yàn)證。結(jié)果:1.成功建立了高表達(dá)NICD和空載plvx的U251細(xì)胞株。2.Micro RNA芯片結(jié)果顯示NICD高表達(dá)的U251細(xì)胞中mi RNA-146a與對(duì)照組相比明顯降低(P0.05)。3.成功建立了對(duì)TMZ耐藥的U251TR,U251TR細(xì)胞IC50(320.68μM)是未經(jīng)誘導(dǎo)U251細(xì)胞IC50(30.54μM)的10.5倍(P0.05),其耐藥指數(shù)(RF)約為11。4.RT-q PCR結(jié)果顯示,mi R-146a在NICD高表達(dá)組細(xì)胞及其對(duì)照組細(xì)胞中的表達(dá)情況與micro RNA芯片結(jié)果一致;RT-q PCR顯示耐藥株U251TR中mi R-146a相對(duì)表達(dá)量低于U251(P0.05)。5.轉(zhuǎn)染組mi R-146a-mimics的轉(zhuǎn)染效率在90%以上;轉(zhuǎn)染組細(xì)胞U251TR-mimics IC50(160.79μM)是未經(jīng)誘導(dǎo)的U251細(xì)胞IC50(30.54μM)的5.26倍(P0.05),其耐藥指數(shù)(RF)約為5。6.雙熒光素酶報(bào)告基因?qū)嶒?yàn)和Western blotting實(shí)驗(yàn)均顯示mi R-146a-5p作用后,熒光素酶活性及Notch1下調(diào)明顯(P0.05)。結(jié)論:成功構(gòu)建了對(duì)TMZ耐藥的U251TR細(xì)胞,其耐藥指數(shù)(RF)約為10,U251TR細(xì)胞中mi R-146a相對(duì)表達(dá)量低于U251細(xì)胞;將mi R-146a-mimics轉(zhuǎn)入U(xiǎn)251TR細(xì)胞后,其耐藥指數(shù)下降。雙熒光素酶報(bào)告基因和Western blotting結(jié)果均顯示mi R-146a靶向調(diào)控Notch1。mi RNA-146a可能通過(guò)Notch1信號(hào)通路在人膠質(zhì)瘤細(xì)胞U251耐藥形成機(jī)制中發(fā)揮一定的作用。
[Abstract]:Objective: 1. To study the effect of micro RNA-146a on human glioma cell line U251. 2. To investigate the relationship between Notch1 gene and micro RNA-146a in human glioma resistance. Methods: 1. U251 cell lines with high expression of NICD and U251 cell lines with no plvx were obtained by packaging, transfection and screening of lentivirus. Western Blotting and immunofluorescence staining were used to identify the high expression of NICD in U251 cells with high NICD expression and non-loaded U251 cells and U251 cells without plvx and control U251 cells. Micro RNAs.3.RT-q PCR detection was used to identify the high expression of NICD in U251 cells. The expression of mi RNA-146a in U251 cells and control U251 cells without plvx. 4. U251 cells were induced by the increasing concentration of temozolidomide (TMZ). The initial induction dose was 0.25 渭 g/ml (0.2875 渭 M),) and the maximum inducing dose was 20 渭 g/ml (103.0 渭 M),) to obtain stable TMZ resistant U251 cells named U251 TR. CCK-8 assay was used to detect the proliferation inhibition rate of U251TR cells. The expression of mi RNA-146a in U251TR and its control U251 cells was detected by calculating the half inhibitory concentration (IC50) and drug resistance index (RF). 5.RT-q PCR). The transfection efficiency of mi R-146a-mimics was detected by fluorescence microscopy, and the expression of mi R-146a in U251TR and U251TR-mimics was detected by RT-q PCR. CCK-8 assay was used to detect the proliferation inhibition rate of U251TR-mimics cells, and the expression of Notch1 gene in U251TR and U251TR-mimics cells was calculated by IC50 and RF.7.Western Blotting. 8. Mi RNA-146a, targeting Notch1 was predicted by bioinformatics. Then the target of mi R-146a was verified by double luciferase reporter gene experiment and Western blotting experiment. The result is 1: 1. U251 cell lines with high expression of NICD and no plvx were successfully established. The results of 2.Micro RNA microarray showed that mi RNA-146a in U251 cells with high NICD expression was significantly lower than that in control group (P0.05). The IC50 (320.68 渭 M) of U251 TR-U251TR cells resistant to TMZ was 10. 5 times as much as that of uninduced U251 cells IC50 (30.54 渭 M) (P0.05). The drug resistance index (RF) of U251 TR- U251TR cells was about the same as that of 11.4.RT-q PCR. The expression of mi R-146a in the cells with high expression of NICD and its control group was consistent with the results of micro RNA microarray. RT-q PCR showed that the relative expression of mi R-146a in U251TR was lower than that in U251 (P0.05). The transfection efficiency of mi R-146a-mimics in transfection group was more than 90%, and the transfection group U251TR-mimics IC50 (160.79 渭 M) was 5.26 times as high as the uninduced U251 cell IC50 (30.54 渭 M) (P0.05), and its drug resistance index (RF) was about 5.6. Double luciferase reporter gene experiment and Western blotting assay showed that luciferase activity and Notch1 decreased significantly after mi R-146a-5p treatment (P0.05). Conclusion: U251TR cells resistant to TMZ were successfully constructed, and the relative expression of mi R-146a in U251TR cells was lower than that in U251 cells, and the drug resistance index decreased after mi R-146a-mimics was transferred to U251TR cells. The results of double luciferase reporter gene and Western blotting showed that mi R-146a could play a role in the formation of drug resistance in human glioma cell line U251 through Notch1 signaling pathway.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R739.41

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張麗;夏德雨;李國(guó)輝;蔣姍姍;谷芳娜;梁英民;;Notch信號(hào)在急性早幼粒細(xì)胞白血病患者骨髓中的表達(dá)[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2009年24期

2 姜萌;何奔;;Notch信號(hào)對(duì)血管新生調(diào)控作用的研究進(jìn)展[J];上海交通大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2010年05期

3 ;Effect of Trastuzumab on Notch-1 Signaling Pathway in Breast Cancer SK-BR3 Cells[J];Chinese Journal of Cancer Research;2012年03期

4 魯茁壯,王立生,吳祖澤;Notch信號(hào)通路研究進(jìn)展[J];生理科學(xué)進(jìn)展;2004年02期

5 張謙慎;Notch信號(hào)與肺發(fā)育[J];中國(guó)當(dāng)代兒科雜志;2004年02期

6 孫力,詹啟敏,章文華;Notch信號(hào)通路與腫瘤[J];國(guó)外醫(yī)學(xué)(腫瘤學(xué)分冊(cè));2004年09期

7 張謙慎,常立文,劉漢楚,容志惠,祝華平,陳紅兵,李文斌;Notch信號(hào)與大鼠肺發(fā)育的關(guān)系研究[J];華中科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2004年03期

8 張謙慎,常立文,劉漢楚,容志惠,陳紅兵;TEMPORAL EXPRESSION OF NOTCH RECEPTORS DURING LUNG DEVELOPMENT IN RAT[J];Journal of Shanghai Second Medical University;2005年02期

9 吳穎亞;王季石;;Notch及其配體在血液系統(tǒng)中的作用[J];國(guó)外醫(yī)學(xué).輸血及血液學(xué)分冊(cè);2005年05期

10 張謙慎,常立文,劉漢楚,容志惠,陳紅兵;Relationship between Notch Receptors and Hyperoxia-induced Lung Injury in Newborn Rats[J];華中科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)英德文版);2005年02期

相關(guān)會(huì)議論文 前10條

1 張麗;梁英民;夏德雨;李國(guó)輝;蔣姍姍;谷芳娜;;Notch信號(hào)在急性早幼粒細(xì)胞白血病患者骨髓中的表達(dá)研究[A];第12屆全國(guó)實(shí)驗(yàn)血液學(xué)會(huì)議論文摘要[C];2009年

2 ;Expression profile of Notch-related genes in multi-drug resistant K562/A02 cells compared with parental K562 cells[A];第12屆全國(guó)實(shí)驗(yàn)血液學(xué)會(huì)議論文摘要[C];2009年

3 ;Notch signaling in lymphopoiesis[A];第10屆全國(guó)實(shí)驗(yàn)血液學(xué)會(huì)議論文摘要匯編[C];2005年

4 韓驊;;Notch信號(hào)途徑的功能和作用機(jī)理的研究[A];全面建設(shè)小康社會(huì)：中國(guó)科技工作者的歷史責(zé)任——中國(guó)科協(xié)2003年學(xué)術(shù)年會(huì)論文集（下）[C];2003年

5 趙昱;侯麗宏;馬磊;朱正華;朱蕭玲;王強(qiáng);呂巖;陳紹洋;;Electroacupuncture pretreatment induces the tolerance against focal cerebral ischemia through activation of Notch pathway[A];中華醫(yī)學(xué)會(huì)第五次全國(guó)重癥醫(yī)學(xué)大會(huì)論文匯編[C];2011年

6 黃佳圓;王銳;陳龍邦;;Expression of Notch-1 and Its Clinical Significance in Different Histological Subtypes of Human Lung Adenocarcinoma[A];2013華東胸部腫瘤論壇暨第六屆浙江省胸部腫瘤論壇論文集[C];2013年

7 ;Aberrant expression profile of Notch signaling pathway involved in immune thrombocytopenic purpura patients[A];第12屆全國(guó)實(shí)驗(yàn)血液學(xué)會(huì)議論文摘要[C];2009年

8 ;Notch induced protein degradation in lymphoid development[A];第六屆全國(guó)免疫學(xué)學(xué)術(shù)大會(huì)論文集[C];2008年

9 ;The functions of ptrl in Notch and Integrin pathways[A];2012全國(guó)發(fā)育生物學(xué)大會(huì)摘要集[C];2012年

10 Yaochun Wang;Guorui Dou;Lin Wang;Hua Han;;Notch signaling:licenses and limits cellular responses to extrinsic stimulations?[A];中國(guó)生物化學(xué)與分子生物學(xué)會(huì)第十屆會(huì)員代表大會(huì)暨全國(guó)學(xué)術(shù)會(huì)議摘要集[C];2010年

相關(guān)重要報(bào)紙文章 前1條

1 ;為什么心在左肝在右？[N];新華每日電訊;2004年

相關(guān)博士學(xué)位論文 前10條

1 王凱;Notch信號(hào)在小鼠創(chuàng)傷性腦損傷后神經(jīng)元死亡中的作用及機(jī)制研究[D];第四軍醫(yī)大學(xué);2015年

2 陳娟娟;Notch重組配體D1R在促進(jìn)造血重建中的作用與機(jī)理[D];第四軍醫(yī)大學(xué);2015年

3 梁燕;Notch3對(duì)低氧誘導(dǎo)的人肺動(dòng)脈平滑肌細(xì)胞增殖的作用機(jī)制探討[D];首都醫(yī)科大學(xué);2014年

4 孫建華;肺癌患者在乏氧條件下VEGF和Dll4/Notch通路分子的異常表達(dá)和相互關(guān)系[D];山東大學(xué);2015年

5 付偉;Notch信號(hào)在慢性粒細(xì)胞白血病細(xì)胞增殖和伊馬替尼耐藥的調(diào)控作用研究[D];第四軍醫(yī)大學(xué);2014年

6 柏振江;Notch配體DLL4在手足口病患兒中表達(dá)、臨床意義和功能分析[D];蘇州大學(xué);2016年

7 田z，

本文編號(hào)：2319563