本文選題：miRNA　切入點(diǎn)：奧沙利鉑耐藥　出處：《鄭州大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：背景結(jié)直腸癌是第三類常見癌癥,也是世界范圍內(nèi)癌癥死亡的第三大原因。在過去的10年中,已發(fā)現(xiàn)一類非編碼微小RNA分子(mi RNA)在癌癥發(fā)展中具有抑癌基因和癌基因的作用。已證實(shí)在結(jié)直腸癌中許多mi RNA的表達(dá)發(fā)生了變化,并且有些變化可能與通過靶向腫瘤相關(guān)基因的調(diào)控誘導(dǎo)腫瘤發(fā)生有關(guān)。目的研究mi RNA在人結(jié)腸癌細(xì)胞(SW480、HCT-15)和對奧沙利鉑耐藥的結(jié)腸癌細(xì)胞(SW480/OXA、HCT-15/OXA)中的差異化表達(dá),對差異表達(dá)顯著的mi RNA在耐藥中的調(diào)控機(jī)制進(jìn)行初步研究,為進(jìn)一步探索有效的逆轉(zhuǎn)腫瘤耐藥的方法開拓新領(lǐng)域。方法(1)采用藥物持續(xù)作用和大劑量藥物間歇誘導(dǎo)相結(jié)合的方法,建立耐奧沙利鉑的人結(jié)腸癌細(xì)胞系SW480/OXA、HCT-15/OXA。(2)利用基因芯片技術(shù)檢測親代與耐藥結(jié)腸癌細(xì)胞中mi RNA的表達(dá)差異,并采用實(shí)時(shí)熒光定量PCR技術(shù)進(jìn)行確證,將差異表達(dá)mi RNA的反義核苷酸和模擬物轉(zhuǎn)染親代細(xì)胞,測定其對耐藥的影響。(3)采用生物信息學(xué)對耐藥細(xì)胞中顯著低表達(dá)的hsa-mi R-137的靶基因進(jìn)行預(yù)測,利用熒光報(bào)告載體實(shí)驗(yàn)來驗(yàn)證預(yù)測靶基因,Western blot技術(shù)檢測靶基因蛋白表達(dá)水平。Real-time PCR檢測親代細(xì)胞轉(zhuǎn)染hsa-mi R-137-ASO后靶基因m RNA轉(zhuǎn)錄水平,Western blot檢測蛋白水平,驗(yàn)證mi RNA-137對靶基因的調(diào)控作用。(4)MTT法測定結(jié)腸癌組織原代細(xì)胞對奧沙利鉑耐藥性,并利用實(shí)時(shí)熒光定量PCR和免疫組化法進(jìn)一步研究耐藥性、has-mi R-137的m RNA水平與靶基因YB-l蛋白表達(dá)水平之間的關(guān)系。結(jié)果(1)成功建立了耐受OXA的SW480/OXA和HCT-15/OXA模型,與親代細(xì)胞相比,兩株耐藥細(xì)胞均產(chǎn)生了中度耐藥,對作用機(jī)理不同的化療藥物也產(chǎn)生了一定的交叉耐藥。(2)基因芯片檢測和熒光PCR驗(yàn)證顯示,在人結(jié)腸癌親代細(xì)胞與耐藥細(xì)胞之間,hsa-mi R-93、hsa-mi R-21、hsa-mi R-96、hsa-mi R-22、hsa-mi R-191、hsa-mi R-192、hsa-mi R-137存在明顯差異性表達(dá)。MTT試驗(yàn)結(jié)果顯示hsa-mi R-93、hsa-mi R-137、hsa-mi R-21和hsa-mi R-22具有調(diào)節(jié)OXA殺傷結(jié)腸癌細(xì)胞作用的功能。(3)mi RNA預(yù)測軟件綜合測定YB-l為hsa-mi R-137直接作用的候選靶基因。綠色熒光蛋白報(bào)告載體實(shí)驗(yàn)和實(shí)時(shí)熒光定量PCR證實(shí),結(jié)腸癌細(xì)胞中抑制hsa-mi R-137m RNA的表達(dá)后,內(nèi)源性YB-l的m RNA表達(dá)升高。Western Blot實(shí)驗(yàn)證實(shí)結(jié)腸癌細(xì)胞中hsa-mi R-137對YB-l蛋白表達(dá)具有負(fù)性調(diào)節(jié)作用。(4)real-time PCR顯示hsa-mi R-137m RNA表達(dá)水平與結(jié)腸癌組織對OXA的敏感性密切相關(guān),表達(dá)越高,敏感性越強(qiáng)。免疫組化顯示,OXA敏感結(jié)腸癌組織中YB-l蛋白高表達(dá)比例較低,而耐藥組織中高表達(dá)比例較高。結(jié)論(1)人結(jié)腸癌細(xì)胞OXA耐藥與多種mi RNA的異常表達(dá)相關(guān)。(2)人結(jié)腸癌細(xì)胞中has-mi R-137的靶基因?yàn)閅B-l,并具有負(fù)性調(diào)節(jié)作用。(3)has-mi R-137可通過靶基因YB-l調(diào)控人結(jié)腸癌細(xì)胞對OXA的敏感性。(4)結(jié)腸癌組織耐藥標(biāo)本has-mi R-137表達(dá)降低,YB-l蛋白增加,OXA耐藥與has-mi R-137和靶基因YB-l表達(dá)密切相關(guān)。
[Abstract]:Background Colorectal cancer is the third most common type of cancer and the third leading cause of cancer deaths worldwide. A class of noncoding minute RNA RNAs have been found to play a role in tumor suppressor genes and oncogenes in the development of cancer. It has been proved that the expression of mi RNA has changed in colorectal cancer. Some of the changes may be related to the induction of tumorigenesis by targeting oncogene. Objective to study the differential expression of mi RNA in human colon cancer cell line SW480, HCT-15) and oxaliplatin resistant colon cancer cell line SW480 / OXA- 15 / OXA. A preliminary study on the regulatory mechanism of the differentially expressed mi RNA in drug resistance was carried out. In order to further explore effective methods to reverse drug resistance in cancer and explore new fields. Methods 1) the combination of drug persistence and intermittent induction of large doses of drugs was used. A human colon cancer cell line SW480 / OXAHHCT-15 / OXA.2was established to detect the difference in the expression of mi RNA between parental and drug-resistant colon cancer cells by gene chip technique, and was confirmed by real-time fluorescence quantitative PCR. The antisense nucleotides and analogue of differentially expressed mi RNA were transfected into parental cells to determine their effect on drug resistance. Bioinformatics was used to predict the target gene of hsa-mi R-137, which was significantly underexpressed in drug resistant cells. The expression level of target gene protein was detected by Western blot technique. Real-time PCR was used to detect the transcription level of target gene m RNA after transfection of hsa-mi R-137-ASO. Western blot was used to detect the protein level. To verify the regulatory effect of mi RNA-137 on target gene. MTT assay was used to determine the resistance of primary colon cancer cells to oxaliplatin. The relationship between the expression of target gene YB-l protein and the level of m RNA of drug-resistant OXA R-137 was further studied by real-time fluorescent quantitative PCR and immunohistochemistry. Results 1) the SW480/OXA and HCT-15/OXA models of OXA tolerance were successfully established, compared with those of parental cells. Two strains of drug-resistant cells developed moderate drug resistance, and also produced a certain cross-resistance to chemotherapeutic drugs with different mechanisms. The microarray analysis and fluorescence PCR verification showed that the two cell lines were resistant to drugs. Hsa-mi R-93hsa-mi R-137hsa-mi and hsa-mi R-22 hsa-mi R-192 hsa-mi R-192 hsa-mi R-192 hsa-mi R-192 hsa-mi R-137 showed that hsa-mi R-93hsa-mi R-137hsa-mi R-21 and hsa-mi R-22 had the function of regulating the function of OXA in killing colon cancer cells. YB-l was identified as a candidate target gene for direct action of hsa-mi R-137. Green fluorescent protein report vector experiment and real-time fluorescence quantitative PCR were used. After inhibiting the expression of hsa-mi R-137m RNA in colon cancer cells, The increase of m RNA expression of endogenous YB-l. Western Blot assay confirmed that hsa-mi R-137 negatively regulated the expression of YB-l protein in colon cancer cells. The expression of hsa-mi R-137m RNA was closely related to the sensitivity of colon cancer tissue to OXA, and the higher the expression was, the higher the expression of hsa-mi R-137m RNA was. The higher the sensitivity, the lower the expression of YB-l protein in OXA-sensitive colon cancer tissues. Conclusion the drug resistance of human colon cancer cell line OXA is related to the abnormal expression of various mi RNA.) the target gene of has-mi R-137 in human colon cancer cells is YB-land has a negative regulatory effect. [conclusion] the drug resistance of human colon cancer cell line OXA is related to the abnormal expression of a variety of mi RNA, and the target gene of has-mi R-137 can pass through the target base. Because YB-l regulates the sensitivity of human colon cancer cells to OXA, the expression of has-mi R-137 in drug-resistant colon cancer tissues decreases and the increase of OXA-resistance is closely related to the expression of has-mi R-137 and target gene YB-l.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.35

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 胡平,琚立華,徐元鼎;結(jié)腸癌細(xì)胞核DNA含量測量及形態(tài)分析[J];復(fù)旦學(xué)報(bào)(醫(yī)學(xué)版);2003年05期

2 王春暉;歐陽欽;唐承薇;黃明慧;李獻(xiàn);;選擇性及非選擇性COX-2抑制劑對結(jié)腸癌細(xì)胞生長的影響[J];四川大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2006年04期

3 韓忠寶;李洪祥;楊翊研;;生長抑素聯(lián)合阿霉素體外抑制結(jié)腸癌細(xì)胞的實(shí)驗(yàn)研究[J];吉林醫(yī)學(xué);2006年09期

4 張朝陽;徐克森;楊廣運(yùn);王金申;高穎;劉炎峰;帥晶;王家勇;李少燕;壽楠海;牛軍;;反義整合素β6基因?qū)Y(jié)腸癌細(xì)胞作用的研究[J];中華醫(yī)學(xué)雜志;2007年37期

5 沈丹;鄧長生;;過氧化物酶體增殖物激活受體γ在結(jié)腸癌中的表達(dá)及其激動(dòng)劑對結(jié)腸癌細(xì)胞的生長抑制作用[J];臨床內(nèi)科雜志;2008年01期

6 田永剛;許軍;劉昶;;人工氣腹對結(jié)腸癌細(xì)胞生物學(xué)行為的影響[J];腹腔鏡外科雜志;2009年10期

7 高喜廉，，傅志民，姜宗源;結(jié)腸癌細(xì)胞腸壁縱向浸潤與臨床意義[J];中國醫(yī)科大學(xué)學(xué)報(bào);1994年03期

8 鄭平菊,黃威權(quán),王瑞安,孫嵐,高平;5-羥色胺及其受體在結(jié)腸癌細(xì)胞的免疫組織化學(xué)定位[J];科學(xué)通報(bào);1995年21期

9 林潔;黃瓊;來茂德;;5-氮-2′-脫氧胞苷對結(jié)腸癌細(xì)胞生物學(xué)行為的影響[J];臨床與實(shí)驗(yàn)病理學(xué)雜志;2008年04期

10 王德力;齊東麗;;光鑷?yán)庾V技術(shù)檢測結(jié)腸癌細(xì)胞的研究[J];長春大學(xué)學(xué)報(bào);2009年04期

相關(guān)會(huì)議論文 前10條

1 許峰;王杉;葉穎江;崔志榮;;腫瘤浸潤淋巴細(xì)胞在結(jié)腸癌細(xì)胞殺傷中的作用及機(jī)制[A];中華醫(yī)學(xué)會(huì)第七次全國消化病學(xué)術(shù)會(huì)議論文匯編（下冊）[C];2007年

2 王貴玉;王錫山;;激光捕獲微切割技術(shù)分離結(jié)腸癌細(xì)胞用于蛋白組學(xué)研究[A];中國蛋白質(zhì)組學(xué)第三屆學(xué)術(shù)大會(huì)論文摘要[C];2005年

3 沈丹;鄧長生;;過氧化物酶體增殖物激活受體γ在結(jié)腸癌中的表達(dá)及其激動(dòng)劑對結(jié)腸癌細(xì)胞的生長抑制作用[A];中華醫(yī)學(xué)會(huì)第七次全國消化病學(xué)術(shù)會(huì)議論文匯編（下冊）[C];2007年

4 郭俊明;賴依峰;肖丙秀;劉瓊;;大豆異黃酮對體外培養(yǎng)的結(jié)腸癌細(xì)胞生長和細(xì)胞周期的影響[A];華東六省一市生物化學(xué)與分子生物學(xué)會(huì)2003年學(xué)術(shù)交流會(huì)論文摘要集[C];2003年

5 李本輝;楊賢子;張文杰;;人類結(jié)腸癌細(xì)胞缺陷型Stat6~(null)活化表型與癌細(xì)胞高凋亡率及低侵襲轉(zhuǎn)移力相關(guān)[A];湖北省抗癌協(xié)會(huì)青年委員會(huì)成立大會(huì)暨第一屆青年學(xué)術(shù)論壇資料匯編[C];2009年

6 王國強(qiáng);方m錁

本文編號(hào)：1620077