發(fā)布時(shí)間：2018-03-27 16:13

  本文選題：真核重組蛋白(NLS-N80-TAT)　切入點(diǎn)：β-catenin/TCF4轉(zhuǎn)錄復(fù)合體　出處：《江蘇大學(xué)》2017年碩士論文

【摘要】：【目的】膠質(zhì)瘤是人腦最常見的原發(fā)性腫瘤,具有極強(qiáng)的增殖和侵襲性,手術(shù)切除聯(lián)合放化療可顯著延長患者生存期,但復(fù)發(fā)率仍較高,易產(chǎn)生放化療耐受。關(guān)于其機(jī)制的研究已日漸深入,其中腫瘤干細(xì)胞(TSC)假說變得尤為熱門,該假說提出,腫瘤放化療后的復(fù)發(fā)和轉(zhuǎn)移可能于其腫瘤干細(xì)胞的殘存有很大關(guān)系,因而對(duì)腫瘤干細(xì)胞的深入研究或許能為腫瘤的治療提供新的思路,對(duì)研發(fā)更有效的抗腫瘤藥物具有十分重要的意義。研究發(fā)現(xiàn),在多數(shù)實(shí)體腫瘤中Wnt/β-catenin通路的表達(dá)異�；钴S,并參與了腫瘤的侵襲和轉(zhuǎn)移等過程。腫瘤干細(xì)胞胞核中β-catenin/TCF4轉(zhuǎn)錄復(fù)合體表達(dá)的異常增高。外部因素刺激后,胞漿中的β-catenin必須穿過細(xì)胞膜,進(jìn)入胞核與轉(zhuǎn)錄因子4(TCF4)結(jié)合才能發(fā)揮對(duì)下游靶基因的轉(zhuǎn)錄調(diào)控。因此,破壞該轉(zhuǎn)錄復(fù)合體的結(jié)合可作為干預(yù)腫瘤干細(xì)胞形成的一個(gè)有效途徑。目前,已知β-catenin與TCF4的結(jié)合位點(diǎn)位于TCF4 N端D的80個(gè)氨基酸。本課題通過將這前80個(gè)氨基酸提取出來構(gòu)建成分子量20左右的蛋白多肽(命名為NLS-N80-TAT),證實(shí)該重組蛋白可作為競爭性抑制劑阻斷TCF4與β-catenin的結(jié)合,并抑制TCF/LEF的轉(zhuǎn)錄活性,抑制膠質(zhì)瘤干細(xì)胞的增殖,并誘導(dǎo)其分化,進(jìn)而恢復(fù)膠質(zhì)瘤干細(xì)胞對(duì)化療藥物(替莫唑胺)的敏感性�！痉椒ā�1、真核重組蛋白NLS-N80-TAT的制備與鑒定(1)NLS-His-FLAG-N80-TAT基因的克隆。以pcDNA/Myc-TCF4載體為模板用PCR擴(kuò)增試劑盒擴(kuò)增TCF4的前80個(gè)氨基酸的序列,在上下游引物中分別引入His-Flag序列和TAT序列,在N端引入NLS序列,得到初級(jí)PCR產(chǎn)物,以所述初級(jí)PCR產(chǎn)物為模板擴(kuò)增最終的NLS-His-FLAG-N80-TAT序列。(2)HEK293細(xì)胞的轉(zhuǎn)染與表達(dá),將pcDNA3.1-NLS-His-FLAG-N80-TAT通過脂質(zhì)體轉(zhuǎn)染試劑轉(zhuǎn)染HEK293細(xì)胞48小時(shí)后,新霉素加壓篩選,構(gòu)建表達(dá)NLS-His-FLAG-N80-TAT基因的HEK293穩(wěn)轉(zhuǎn)細(xì)胞株,通過Western blot檢測NLS-His-FLAG-N80-TAT蛋白的表達(dá)。(3)NLS-His-FLAG-N80-TAT重組蛋白的分離純化。經(jīng)SDS-PAGE電泳、轉(zhuǎn)膜后,通過FLAG單克隆抗體對(duì)所述的新型真核重組蛋白進(jìn)行鑒定。2、免疫熒光雙染色實(shí)驗(yàn)驗(yàn)證NLS-N80-TAT可否進(jìn)入細(xì)胞核,免疫共沉淀技術(shù)證實(shí)NLS-N80-TAT與β-catenin的結(jié)合。3、免疫共沉淀技術(shù)證實(shí)NLS-N80-TAT可抑制內(nèi)源性及外源性β-catenin/TCF4轉(zhuǎn)錄復(fù)合體的結(jié)合,熒光素酶報(bào)告基因檢測系統(tǒng)檢測不同濃度的NLS-N80-TAT作用下,TCF/LEF通路轉(zhuǎn)錄的活性。4、RT-PCR檢測NLS-N80-TAT對(duì)膠質(zhì)瘤干細(xì)胞中標(biāo)志物CD133表達(dá)水平的影響;蛋白質(zhì)印跡法檢測NLS-N80-TAT對(duì)膠質(zhì)瘤干細(xì)胞中增殖相關(guān)蛋白(c-Myc)以及分化相關(guān)蛋白(β-Ⅲtublin)表達(dá)水平的影響;免疫熒光雙染色檢測NLS-N80-TAT對(duì)膠質(zhì)瘤干細(xì)胞(β-Ⅲtublin)表達(dá)水平的影響【結(jié)果】1、NLS-N80-TAT可進(jìn)入細(xì)胞核并與β-catenin相互結(jié)合。2、NLS-N80-TAT可作為競爭性抑制劑阻礙β-catenin與TCF4的結(jié)合,重組蛋白處理組TCF/LEF通路的轉(zhuǎn)錄活性明顯下降,且有濃度依賴性。3、NLS-N80-TAT處理組CD133表達(dá)明顯下調(diào),β-Ⅲtublin表達(dá)上調(diào)。4、NLS-N80-TAT聯(lián)合化療組較單獨(dú)化療組的細(xì)胞活力顯著下降�！窘Y(jié)論】NLS-N80-TAT可作為競爭性抑制劑阻斷TCF4與β-catenin的結(jié)合,并抑制TCF/LEF通路的轉(zhuǎn)錄活性,進(jìn)而促進(jìn)膠質(zhì)瘤干細(xì)胞對(duì)化療藥物(替莫唑胺)的敏感性,對(duì)于其在臨床上膠質(zhì)瘤的治療具有十分廣闊的應(yīng)用前景。
[Abstract]:[Objective] brain glioma is the most common primary tumor with strong proliferation and invasion, surgical resection combined with radiotherapy and chemotherapy can significantly prolong the survival time of the patients, the recurrence rate is still high, easy to produce the chemotherapy tolerance. Research on its mechanism has been gradually deepening, the cancer stem cell (TSC) the hypothesis has become particularly popular, the hypothesis proposed that after radiotherapy and chemotherapy of tumor recurrence and metastasis in the residual tumor stem cells have a great relationship, thus further study of tumor stem cells may provide a new way for cancer treatment, has very important significance for the development of more effective anticancer drugs. The study found in most solid tumors, the expression of Wnt/ beta -catenin pathway is active, and is involved in tumor invasion and metastasis. Tumor stem abnormal expression of beta -catenin/TCF4 transcription complex in the nucleus of the cell. The external Factors after stimulation in the cytoplasm of beta -catenin must pass through the cell membrane into the nucleus, and transcription factor 4 (TCF4) in order to play with the transcriptional regulation of downstream target genes. Therefore, combined with the destruction of the transcription complex can be used as an effective way of intervention of cancer stem cell formation. At present, 80 sites are located in TCF4 N end D combined with known beta -catenin and TCF4. This paper will be the first 80 amino acids extracted from the protein polypeptide to construct a molecular weight of about 20 (named NLS-N80-TAT), confirmed that the recombinant protein can be used as a competitive inhibitor with TCF4 and beta -catenin, and inhibit TCF/LEF transcriptional activity, inhibition the proliferation of glioma stem cells, and induce differentiation and recovery of glioma stem cells to chemotherapeutic drugs (temozolomide) sensitivity. [Methods] 1, preparation and identification of recombinant eukaryotic protein NLS-N80-TAT system (1) NLS-His-FL Cloning of AG-N80-TAT gene in pcDNA/Myc-TCF4 vector. For the first 80 amino acid sequence template for PCR amplification kit amplification of TCF4, His-Flag and TAT sequences were introduced in the primers, using NLS sequence in the N terminal, to obtain the primary product of PCR, with the primary PCR product was amplified NLS-His-FLAG-N80-TAT sequence of the final. (2) transfection and expression of HEK293 cells, pcDNA3.1-NLS-His-FLAG-N80-TAT by liposome transfection reagent 48 hours after transfection into HEK293 cells, using neomycin selection, construction and expression of NLS-His-FLAG-N80-TAT gene in HEK293 stably transfected cell lines, the expression of NLS-His-FLAG-N80-TAT protein detected Western blot. (3) separation and purification of NLS-His-FLAG-N80-TAT recombinant protein. By SDS-PAGE electrophoresis,. After the film, were identified by.2 model eukaryotic recombinant protein FLAG monoclonal antibody on the double immunofluorescence staining experiments. Verify NLS-N80-TAT can enter the nucleus, combined with.3 and NLS-N80-TAT technology confirmed that beta -catenin immunoprecipitation technology confirmed that NLS-N80-TAT can inhibit the binding of endogenous and exogenous beta -catenin/TCF4 transcription complex co immunoprecipitation, NLS-N80-TAT luciferase reporter gene assay system test under different concentrations, the activity of.4 TCF/LEF transcription pathway, detection of RT-PCR NLS-N80-TAT on cell in the marker CD133 expression of glioma stem; Western blotting of NLS-N80-TAT on glioma stem cell proliferation related protein (c-Myc) and differentiation related protein (beta tublin) on the expression of NLS-N80-TAT; double staining immunofluorescence detection of glioma stem cells (beta tublin) expression level effect of [results] 1, NLS-N80-TAT can enter the nucleus and beta -catenin combination of.2, NLS-N80-TAT can be used for competitive inhibition Combining -catenin and TCF4 beta blocking agent, the recombinant protein TCF/LEF pathway transcription activity in treatment group decreased significantly, and there is a concentration dependent.3, NLS-N80-TAT treatment group CD133 was significantly reduced, beta III tublin expression of.4, NLS-N80-TAT combined with chemotherapy group compared with chemotherapy alone group cell activity decreased significantly. [Conclusion] NLS-N80-TAT as a competitive inhibitor with TCF4 and beta -catenin, and inhibit the transcriptional activity of the TCF/LEF pathway, thereby promoting glioma stem cells to chemotherapeutic drugs (temozolomide) sensitivity, has very broad application prospects for the treatment of gliomas in clinical tumor.

【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.41

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Sarah Heiler;Zhe Wang;Margot Z?ller;;Pancreatic cancer stem cell markers and exosomes- the incentive push[J];World Journal of Gastroenterology;2016年26期

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本文編號(hào)：1672257