本文選題：腺病毒 + CTTNBP2NL　； 參考：《浙江理工大學》2017年碩士論文

【摘要】：Ⅰ.隨著生活壓力增大,環(huán)境污染加劇,惡性腫瘤的發(fā)病率越來越高,所以尋找一種高效治療方法已成為近年的研究重點。CTTNBP2NL(CTTNBP2N-terminal like)編碼的蛋白為N末端樣皮肌動蛋白結合蛋白2,與CTTNBP2同源,在神經系統(tǒng)的發(fā)生過程中起重要作用。又因為腺病毒在病毒療法中具有穩(wěn)定性好,宿主范圍廣,使用安全的優(yōu)點。我們將CTTNBP2NL基因插入到腺病毒載體上,構建了非復制型重組腺病毒Ad-CTTNBP2NL。利用MTT法檢測重組病毒對不同癌細胞系增殖有抑制作用。結晶紫實驗觀察到重組腺病毒對癌細胞有很好的殺傷效果。通過Hoechst33258染色和流式細胞術實驗分析攜帶CTTNBP2NL基因的重組腺病毒可以誘導細胞凋亡。轉染pEGFP-LC3-C1到癌細胞后,感染重組病毒檢測癌細胞沒有自噬。Western blot檢測細胞內凋亡相關蛋白及自噬相關蛋白的表達量,證明CTTNBP2NL基因通過凋亡途徑誘導癌細胞死亡。本實驗為將CTTNBP2NL基因運用到癌癥的治療上提供了一定的理論基礎。Ⅱ.在海洋自然資源中,海洋凝集素在治療癌癥方面具有很大的潛力。其中UPL1(Ulva pertusa lectin 1)能夠對紅細胞起凝集作用,具有研究價值。本實驗在已有的研究基礎上對UPL1和細胞信號通路之間相互作用的關系做了進一步的探究。通過IP檢測UPL1與PRMT5(精氨酸N端甲基化轉移酶5)、β-Tubulin、β-Actin的相互作用。利用激光共聚焦顯微鏡觀察UPL1和MEP50WD(WDR77)的亞細胞定位。Western blot分別檢測感染了復制缺陷型腺病毒Ad-UPL1的BEL-7404、Huh7細胞系中ERK、p-ERK、Akt、p38、p-p38、STAT、p-STAT、ISG15的表達量,以及自噬相關蛋白Beclin1、LC3-II的表達。EBSS饑餓處理誘導凋亡后,檢測UPL1對癌細胞的存活率的影響。利用U0126(MEK抑制劑)、SB203580(p38抑制劑)、LY294002(PI3K抑制劑)分別處理癌細胞后,檢測到UPL1可以導致癌細胞有更低的存活率。Western blot分析UPL1對β-Tubulin、Akt、PARP、caspase3蛋白有影響。UPL1在癌細胞中與MEK、p38、PI3K等信號通路存在關聯(lián)。其在腫瘤治療方面有廣泛的應用前景。
[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.
【學位授予單位】：浙江理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R730.5

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