一种新型真核重组蛋白的制备及其对人胶质瘤干细胞的化疗增敏作用

发布时间：2018-03-27 16:13

本文选题：真核重组蛋白(NLS-N80-TAT)　切入点：β-catenin/TCF4转录复合体　出处：《江苏大学》2017年硕士论文

【摘要】：【目的】胶质瘤是人脑最常见的原发性肿瘤,具有极强的增殖和侵袭性,手术切除联合放化疗可显著延长患者生存期,但复发率仍较高,易产生放化疗耐受。关于其机制的研究已日渐深入,其中肿瘤干细胞(TSC)假说变得尤为热门,该假说提出,肿瘤放化疗后的复发和转移可能于其肿瘤干细胞的残存有很大关系,因而对肿瘤干细胞的深入研究或许能为肿瘤的治疗提供新的思路,对研发更有效的抗肿瘤药物具有十分重要的意义。研究发现,在多数实体肿瘤中Wnt/β-catenin通路的表达异常活跃,并参与了肿瘤的侵袭和转移等过程。肿瘤干细胞胞核中β-catenin/TCF4转录复合体表达的异常增高。外部因素刺激后,胞浆中的β-catenin必须穿过细胞膜,进入胞核与转录因子4(TCF4)结合才能发挥对下游靶基因的转录调控。因此,破坏该转录复合体的结合可作为干预肿瘤干细胞形成的一个有效途径。目前,已知β-catenin与TCF4的结合位点位于TCF4 N端D的80个氨基酸。本课题通过将这前80个氨基酸提取出来构建成分子量20左右的蛋白多肽(命名为NLS-N80-TAT),证实该重组蛋白可作为竞争性抑制剂阻断TCF4与β-catenin的结合,并抑制TCF/LEF的转录活性,抑制胶质瘤干细胞的增殖,并诱导其分化,进而恢复胶质瘤干细胞对化疗药物(替莫唑胺)的敏感性。【方法】1、真核重组蛋白NLS-N80-TAT的制备与鉴定(1)NLS-His-FLAG-N80-TAT基因的克隆。以pcDNA/Myc-TCF4载体为模板用PCR扩增试剂盒扩增TCF4的前80个氨基酸的序列,在上下游引物中分别引入His-Flag序列和TAT序列,在N端引入NLS序列,得到初级PCR产物,以所述初级PCR产物为模板扩增最终的NLS-His-FLAG-N80-TAT序列。(2)HEK293细胞的转染与表达,将pcDNA3.1-NLS-His-FLAG-N80-TAT通过脂质体转染试剂转染HEK293细胞48小时后,新霉素加压筛选,构建表达NLS-His-FLAG-N80-TAT基因的HEK293稳转细胞株,通过Western blot检测NLS-His-FLAG-N80-TAT蛋白的表达。(3)NLS-His-FLAG-N80-TAT重组蛋白的分离纯化。经SDS-PAGE电泳、转膜后,通过FLAG单克隆抗体对所述的新型真核重组蛋白进行鉴定。2、免疫荧光双染色实验验证NLS-N80-TAT可否进入细胞核,免疫共沉淀技术证实NLS-N80-TAT与β-catenin的结合。3、免疫共沉淀技术证实NLS-N80-TAT可抑制内源性及外源性β-catenin/TCF4转录复合体的结合,荧光素酶报告基因检测系统检测不同浓度的NLS-N80-TAT作用下,TCF/LEF通路转录的活性。4、RT-PCR检测NLS-N80-TAT对胶质瘤干细胞中标志物CD133表达水平的影响;蛋白质印迹法检测NLS-N80-TAT对胶质瘤干细胞中增殖相关蛋白(c-Myc)以及分化相关蛋白(β-Ⅲtublin)表达水平的影响;免疫荧光双染色检测NLS-N80-TAT对胶质瘤干细胞(β-Ⅲtublin)表达水平的影响【结果】1、NLS-N80-TAT可进入细胞核并与β-catenin相互结合。2、NLS-N80-TAT可作为竞争性抑制剂阻碍β-catenin与TCF4的结合,重组蛋白处理组TCF/LEF通路的转录活性明显下降,且有浓度依赖性。3、NLS-N80-TAT处理组CD133表达明显下调,β-Ⅲtublin表达上调。4、NLS-N80-TAT联合化疗组较单独化疗组的细胞活力显著下降。【结论】NLS-N80-TAT可作为竞争性抑制剂阻断TCF4与β-catenin的结合,并抑制TCF/LEF通路的转录活性,进而促进胶质瘤干细胞对化疗药物(替莫唑胺)的敏感性,对于其在临床上胶质瘤的治疗具有十分广阔的应用前景。
[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.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

【参考文献】