携带CTTNBP2NL基因的重组腺病毒的抗癌研究

发布时间：2018-05-14 10:20

本文选题：腺病毒 + CTTNBP2NL　；参考：《浙江理工大学》2017年硕士论文

【摘要】：Ⅰ.随着生活压力增大,环境污染加剧,恶性肿瘤的发病率越来越高,所以寻找一种高效治疗方法已成为近年的研究重点。CTTNBP2NL(CTTNBP2N-terminal like)编码的蛋白为N末端样皮肌动蛋白结合蛋白2,与CTTNBP2同源,在神经系统的发生过程中起重要作用。又因为腺病毒在病毒疗法中具有稳定性好,宿主范围广,使用安全的优点。我们将CTTNBP2NL基因插入到腺病毒载体上,构建了非复制型重组腺病毒Ad-CTTNBP2NL。利用MTT法检测重组病毒对不同癌细胞系增殖有抑制作用。结晶紫实验观察到重组腺病毒对癌细胞有很好的杀伤效果。通过Hoechst33258染色和流式细胞术实验分析携带CTTNBP2NL基因的重组腺病毒可以诱导细胞凋亡。转染pEGFP-LC3-C1到癌细胞后,感染重组病毒检测癌细胞没有自噬。Western blot检测细胞内凋亡相关蛋白及自噬相关蛋白的表达量,证明CTTNBP2NL基因通过凋亡途径诱导癌细胞死亡。本实验为将CTTNBP2NL基因运用到癌症的治疗上提供了一定的理论基础。Ⅱ.在海洋自然资源中,海洋凝集素在治疗癌症方面具有很大的潜力。其中UPL1(Ulva pertusa lectin 1)能够对红细胞起凝集作用,具有研究价值。本实验在已有的研究基础上对UPL1和细胞信号通路之间相互作用的关系做了进一步的探究。通过IP检测UPL1与PRMT5(精氨酸N端甲基化转移酶5)、β-Tubulin、β-Actin的相互作用。利用激光共聚焦显微镜观察UPL1和MEP50WD(WDR77)的亚细胞定位。Western blot分别检测感染了复制缺陷型腺病毒Ad-UPL1的BEL-7404、Huh7细胞系中ERK、p-ERK、Akt、p38、p-p38、STAT、p-STAT、ISG15的表达量,以及自噬相关蛋白Beclin1、LC3-II的表达。EBSS饥饿处理诱导凋亡后,检测UPL1对癌细胞的存活率的影响。利用U0126(MEK抑制剂)、SB203580(p38抑制剂)、LY294002(PI3K抑制剂)分别处理癌细胞后,检测到UPL1可以导致癌细胞有更低的存活率。Western blot分析UPL1对β-Tubulin、Akt、PARP、caspase3蛋白有影响。UPL1在癌细胞中与MEK、p38、PI3K等信号通路存在关联。其在肿瘤治疗方面有广泛的应用前景。
[Abstract]:I. With the increase of life pressure and environmental pollution, the incidence of malignant tumors is increasing. Therefore, finding an efficient treatment method has become the research focus in recent years. CTTNBP2NL- CTTNBP2N-terminal like (CTTNBP2NL- CTTNBP2N-terminal like) encodes N-terminal actin binding protein 2, which is homologous to CTTNBP2 and plays an important role in the development of nervous system. Adenovirus has the advantages of good stability, wide range of hosts and safe use in viral therapy. We inserted CTTNBP2NL gene into adenovirus vector and constructed non-replicating recombinant adenovirus Ad-CTTNBP2NL. MTT assay was used to detect the inhibitory effect of recombinant virus on the proliferation of different cancer cell lines. Crystal violet experiment showed that recombinant adenovirus had a good killing effect on cancer cells. Hoechst33258 staining and flow cytometry analysis showed that recombinant adenovirus carrying CTTNBP2NL gene could induce apoptosis. After transfection of pEGFP-LC3-C1 into cancer cells, the expression of apoptosis-related proteins and autophagy related proteins in cancer cells was detected by CTTNBP2NL gene. The results showed that CTTNBP2NL gene induced cancer cell death by apoptosis pathway. This study provides a theoretical basis for the application of CTTNBP2NL gene to the treatment of cancer. Among marine natural resources, marine lectins have great potential for cancer treatment. Among them, UPL1(Ulva pertusa lectin 1) can agglutinate red blood cells, so it has research value. The relationship between UPL1 and cellular signaling pathway is further explored in this study. The interaction between UPL1 and PRMT5 (arginine N-terminal methyltransferase 5, 尾 -Tubulin, 尾 -Actin) was detected by IP. The subcellular localization of UPL1 and MEP50WDD-WDR77) was observed by laser confocal microscopy. Western blot was used to detect the expression of ERKPP-ERKP38-p38-STATP-STATISG15 in BEL-7404 Huh7 cell line infected with replication-deficient adenovirus Ad-UPL1, and the expression of the autophagy associated protein Beclin1LC3-II induced apoptosis. The effect of UPL1 on the survival rate of cancer cells was detected. After treated with U0126(MEK inhibitor SB203580 / p38 (LY294002PI3K), it was found that UPL1 could lead to lower survival rate of cancer cells. Western blot analysis showed that UPL1 had an effect on 尾 -Tubulinine AktPARPnase 3 protein. UPL1 was associated with MEKP38-PI3K signaling pathway in cancer cells. It has a wide application prospect in tumor therapy.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.5

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