發(fā)布時(shí)間：2018-05-14 10:20

  本文選題：腺病毒 + CTTNBP2NL　； 參考：《浙江理工大學(xué)》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(lái)越高,所以尋找一種高效治療方法已成為近年的研究重點(diǎn)。CTTNBP2NL(CTTNBP2N-terminal like)編碼的蛋白為N末端樣皮肌動(dòng)蛋白結(jié)合蛋白2,與CTTNBP2同源,在神經(jīng)系統(tǒng)的發(fā)生過(guò)程中起重要作用。又因?yàn)橄俨《驹诓《警煼ㄖ芯哂蟹�(wěn)定性好,宿主范圍廣,使用安全的優(yōu)點(diǎn)。我們將CTTNBP2NL基因插入到腺病毒載體上,構(gòu)建了非復(fù)制型重組腺病毒Ad-CTTNBP2NL。利用MTT法檢測(cè)重組病毒對(duì)不同癌細(xì)胞系增殖有抑制作用。結(jié)晶紫實(shí)驗(yàn)觀察到重組腺病毒對(duì)癌細(xì)胞有很好的殺傷效果。通過(guò)Hoechst33258染色和流式細(xì)胞術(shù)實(shí)驗(yàn)分析攜帶CTTNBP2NL基因的重組腺病毒可以誘導(dǎo)細(xì)胞凋亡。轉(zhuǎn)染pEGFP-LC3-C1到癌細(xì)胞后,感染重組病毒檢測(cè)癌細(xì)胞沒(méi)有自噬。Western blot檢測(cè)細(xì)胞內(nèi)凋亡相關(guān)蛋白及自噬相關(guān)蛋白的表達(dá)量,證明CTTNBP2NL基因通過(guò)凋亡途徑誘導(dǎo)癌細(xì)胞死亡。本實(shí)驗(yàn)為將CTTNBP2NL基因運(yùn)用到癌癥的治療上提供了一定的理論基礎(chǔ)。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?qū)t細(xì)胞起凝集作用,具有研究?jī)r(jià)值。本實(shí)驗(yàn)在已有的研究基礎(chǔ)上對(duì)UPL1和細(xì)胞信號(hào)通路之間相互作用的關(guān)系做了進(jìn)一步的探究。通過(guò)IP檢測(cè)UPL1與PRMT5(精氨酸N端甲基化轉(zhuǎn)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(xì)胞定位。Western blot分別檢測(cè)感染了復(fù)制缺陷型腺病毒Ad-UPL1的BEL-7404、Huh7細(xì)胞系中ERK、p-ERK、Akt、p38、p-p38、STAT、p-STAT、ISG15的表達(dá)量,以及自噬相關(guān)蛋白Beclin1、LC3-II的表達(dá)。EBSS饑餓處理誘導(dǎo)凋亡后,檢測(cè)UPL1對(duì)癌細(xì)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(xì)胞后,檢測(cè)到UPL1可以導(dǎo)致癌細(xì)胞有更低的存活率。Western blot分析UPL1對(duì)β-Tubulin、Akt、PARP、caspase3蛋白有影響。UPL1在癌細(xì)胞中與MEK、p38、PI3K等信號(hào)通路存在關(guān)聯(lián)。其在腫瘤治療方面有廣泛的應(yīng)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(xué)位授予單位】：浙江理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.5

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本文編號(hào)：1887467