本文選題：SMMC-7721 + 凝集素�。� 參考：《浙江理工大學(xué)》2017年碩士論文

【摘要】：第一部分?jǐn)y帶凝集素基因的重組腺病毒和痘苗病毒對(duì)肝癌細(xì)胞的作用機(jī)制研究癌癥嚴(yán)重威脅人類的生命和健康。肝癌是人類最常見癌癥之一。隨著分子生物學(xué)的發(fā)展和基因技術(shù)的進(jìn)步,靶向基因—病毒聯(lián)合治療已成為肝癌治療新的研究熱點(diǎn)。凝集素是一類專一識(shí)別糖并與之非共價(jià)、可逆地結(jié)合的蛋白質(zhì)或糖蛋白。過(guò)去幾年本課題組應(yīng)用病毒攜帶凝集素基因,通過(guò)病毒感染使凝集素基因在腫瘤細(xì)胞內(nèi)表達(dá),并對(duì)腫瘤細(xì)胞產(chǎn)生的細(xì)胞毒性作了一定的研究。但是,將攜帶不同凝集素的重組腺病毒和痘苗病毒對(duì)體外肝癌細(xì)胞的殺傷機(jī)制還沒(méi)有深入研究。本課題利用已構(gòu)建好的攜帶鱸魚凝集素(Dicentrarchus labrax lectin,DIFBL)、紫海膽凝集素(Strongylocentrotus purpuratus lectin,SPL)、盤鮑魚凝集素(Haliotis discus discus Lectin,HddSBL)和掌葉半夏凝集素(Pinellia pedatisecta agglutinin,PPA)的重組腺病毒Ad-DIFBL、Ad-SPL、Ad-HddSBL和重組溶瘤痘苗病毒OncoPox-PPA、OncoPox-SPL,感染肝癌細(xì)胞,研究凝集素對(duì)細(xì)胞的毒性作用機(jī)制。本課題將人的肝癌細(xì)胞SMMC-7721的肌纖維膜關(guān)聯(lián)蛋白(Sarcolemma associated protein,SLMAP)和Striatin靶點(diǎn)Knock Down,然后用等量感染復(fù)數(shù)的Ad-DIFBL感染細(xì)胞,MTT檢測(cè)以及熒光顯微鏡觀察顯示,Ad-DIFBL對(duì)SLMAP和Striatin Knock Down的SMMC-7721細(xì)胞的毒性作用明顯低于對(duì)照細(xì)胞,說(shuō)明SLMAP和Striatin這兩種基因可能與凝集素的毒性有一定的關(guān)系,但其深入的機(jī)制尚需進(jìn)一步研究。通過(guò)Western blot實(shí)驗(yàn),發(fā)現(xiàn)用Ad-DIFBL和Ad-HddSBL處理后的細(xì)胞中組蛋白Histone H3表達(dá)量下降;用Ad-SPL和Ad-HddSBL處理后的細(xì)胞中ISG15表達(dá)量下降。OncoPox-PPA和OncoPox-SPL相較于對(duì)照病毒OncoPox-pCB,使腫瘤細(xì)胞中C-myc、SLMAP、Beclin 1表達(dá)水平明顯下降;β-catenin微量下降;β-tubulin產(chǎn)生二聚體和三聚體。用OncoPox-PPA、OncoPox-SPL與Onco Pox-pCB分別去感染SMMC-7721腫瘤細(xì)胞,將樣品進(jìn)行轉(zhuǎn)錄組數(shù)據(jù)分析,結(jié)果顯示凝集素對(duì)多種基因水平產(chǎn)生影響。OncoPox-PPA處理與Onco Pox-pCB處理相比,細(xì)胞中Caspase8,FGFR1,MAVS,SIKE1上調(diào);而TMEM173,STRIP1,IFIH1,CTTNBP2NL,Striatin4,FGFR4下調(diào);OncoPox-SPL感染的細(xì)胞與Onco Pox-pCB感染相比,CTTNBP2NL,Striatin4,FGFR2,FGFR3,SIKE1上調(diào);AKT1,Caspase9,FGFR4,PRMT5,STRIP1,FGFR1,MAVS,CERK下調(diào)。綜上所述,Ad-DIFBL對(duì)SLMAP和Striatin Knock Down的SMMC-7721細(xì)胞的毒性相比于對(duì)照細(xì)胞明顯降低,說(shuō)明凝集素DIFBL與SLMAP、Striatin之間存在某種相互作用,具體是什么樣的作用需要后續(xù)深入研究;Western blot,免疫共沉淀,轉(zhuǎn)錄組數(shù)據(jù)分析等結(jié)果顯示,凝集素對(duì)多種基因表達(dá)水平產(chǎn)生影響。本研究為今后將凝集素基因應(yīng)用于肝癌治療研究提供了一定的基礎(chǔ)。第二部分DIFBL、HddSBL凝集素與腺病毒受體連接的融合蛋白的抗腫瘤機(jī)制研究利用已構(gòu)建好的sCAR-DIFBL和sCAR-HddSBL兩種融合凝集素基因,連接到原核表達(dá)載體pQE30上,將質(zhì)粒分別轉(zhuǎn)到M15原核表達(dá)菌株中。通過(guò)IPTG誘導(dǎo)劑成功誘導(dǎo)出兩種融合蛋白的表達(dá),并且是以包涵體的形式表達(dá)。將誘導(dǎo)出的蛋白進(jìn)行Ni離子親和層析純化及包涵體復(fù)性。融合蛋白的sCAR部分是腺病毒受體的膜外片段,能夠和5型腺病毒結(jié)合;融合蛋白的凝集素部分可與腫瘤細(xì)胞膜上的某些糖蛋白結(jié)合。因此,這些融合蛋白可以通過(guò)橋連作用,使腺病毒通過(guò)細(xì)胞膜糖蛋白感染凝集素識(shí)別的腫瘤細(xì)胞。所以本課題利用這兩種融合蛋白作為工具蛋白,和Ad-EGFP聯(lián)合作用感染多種腫瘤細(xì)胞,通過(guò)熒光顯微鏡觀察不同融合蛋白對(duì)Ad-EGFP感染腫瘤細(xì)胞效率的影響,發(fā)現(xiàn)兩種融合蛋白不僅能夠增強(qiáng)Ad-EGFP對(duì)K562/ADR和U87-MG細(xì)胞中的感染。流式檢測(cè)GFP表達(dá)量結(jié)果與熒光顯微鏡下觀察結(jié)果一致,提示凝集素DIFBL和HddSBL能識(shí)別某些腫瘤細(xì)胞膜上的糖蛋白。分別用重組腺病毒Ad-DIFBL和Ad-PPA與融合蛋白s CAR-DIFBL和sCAR-HddSBL單獨(dú)或聯(lián)合作用,MTT和Annexin V-FITC/PI雙標(biāo)記流式結(jié)果表明融合蛋白sCAR-DIFBL能夠與重組病毒協(xié)同作用,增強(qiáng)對(duì)腫瘤細(xì)胞的殺傷效果;sCAR-HddSBL雖然也能增加病毒感染率,卻能夠抑制Ad-DIFBL和Ad-PPA的對(duì)腫瘤細(xì)胞的殺傷作用。Western blot結(jié)果顯示,用重組腺病毒Ad-DIFBL和Ad-PPA與融合蛋白sCAR-DIFBL和sCAR-HddSBL聯(lián)合作用U87-MG細(xì)胞后,p-ERK水平上升,說(shuō)明凝集素DIFBL和HddSBL對(duì)細(xì)胞毒性作用無(wú)關(guān);sCAR-HddSBL無(wú)論是與病毒聯(lián)合還是單獨(dú)作用于U87-MG后,E2F-1的表達(dá)量都呈上升的趨勢(shì),表明HddSBL很可能激活E2F-1的表達(dá),達(dá)到保護(hù)腫瘤細(xì)胞生長(zhǎng)的目的,進(jìn)一步揭示了凝集素DIFBL和HddSBL對(duì)腫瘤細(xì)胞的作用機(jī)制。
[Abstract]:The first part carries the lectin gene recombinant adenovirus and vaccinia virus cancer mechanism research on liver cancer cells a serious threat to human life and health. Liver cancer is the most common human cancers. With the development of molecular biology and gene technology, targeting gene virus therapy has become a new research hotspot of lectin treatment of hepatocellular carcinoma. Is a kind of specific sugar and non covalent, reversibly binding proteins or glycoproteins. Over the past few years, the research group used virus carrying lectin gene, the expression in tumor cells by lectin gene in virus infection, and produce cytotoxicity to tumor cells was studied. However, the mechanism of killing recombinant adenovirus and vaccinia virus carrying different lectins on hepatocellular carcinoma cells in vitro has not yet in-depth study. This topic has been carried by the sea bass built condensate Set in (Dicentrarchus labrax lectin, DIFBL), purple sea urchin (Strongylocentrotus purpuratus lectin SPL agglutinin lectin (Haliotis), discus disk abalone discus Lectin, HddSBL) and Pinellia pedatisecta agglutinin (Pinellia pedatisecta agglutinin, PPA) of the recombinant adenovirus Ad-DIFBL, Ad-SPL, Ad-HddSBL and recombinant oncolytic vaccinia virus OncoPox-PPA, OncoPox-SPL infection, liver cancer cell toxicity mechanism of lectin on cells. The muscle fiber membrane associated protein of human hepatocellular carcinoma cell line SMMC-7721 (Sarcolemma associated protein, SLMAP) and Striatin Knock Down target, then with the same amount of multiplicity of infection in Ad-DIFBL infected cells, MTT assay and fluorescence microscopy showed that the toxic effects of Ad-DIFBL on SLMAP and Striatin Knock Down of SMMC-7721 cells was significantly lower than that of control cells, indicating that these two genes SLMAP and Striatin may be related to coagulation There is a certain relationship in pigment toxicity, but its deep mechanism needs further study. Through the Western blot experiment, found by Ad-DIFBL and Ad-HddSBL of the cells treated with histone Histone H3 expression decreased; with the decreasing expression of ISG15.OncoPox-PPA and OncoPox-SPL compared with control virus OncoPox-pCB and Ad-SPL cells after Ad-HddSBL treatment, the C-myc, SLMAP in tumor cells, the expression of Beclin 1 was significantly decreased; -catenin beta trace decreased; beta -tubulin two dimer and trimer. OncoPox-PPA, OncoPox-SPL and Onco Pox-pCB respectively to SMMC-7721 infection of tumor cells, the samples of transcriptome data, results show that the influence of.OncoPox-PPA lectin treatment and Onco treatment compared to Pox-pCB a variety of genes, cells in Caspase8, FGFR1, MAVS, SIKE1 and TMEM173, STRIP1, up-regulated; IFIH1, CTTNBP2NL, Striatin4, FGFR4 by OncoP; Pox-pCB ox-SPL cells and Onco infection compared to CTTNBP2NL, Striatin4, FGFR2, FGFR3, AKT1, Caspase9, SIKE1 increased; FGFR4, PRMT5, STRIP1, FGFR1, MAVS, CERK reduced. In summary, compared to SLMAP and Striatin Ad-DIFBL Knock Down SMMC-7721 cytotoxicity in control cells decreased significantly, indicating DIFBL and SLMAP lectin and there is some interaction between Striatin, specifically what kind of role needs further research; Western blot, CO immunoprecipitation, transcriptome data analysis showed that influence on a variety of radicals produced by lectin expression. This study will be applied to the treatment of hepatocellular carcinoma agglutinin gene provides a basis. The second part DIFBL study on the anti tumor mechanism of the fusion protein HddSBL lectin connected with adenovirus receptor by using constructed sCAR-DIFBL and sCAR-HddSBL two kinds of lectin based fusion Because, connected to the prokaryotic expression vector pQE30, the plasmid M15 respectively to prokaryotic expression strains. Inducer successfully induced two fusion protein expression by IPTG, and is expressed in the form of inclusion bodies. The protein induced by Ni ion purification and renaturation sCAR affinity chromatography. The fusion protein is adenovirus receptor extracellular fragment, capable of binding and adenovirus type 5 glycoprotein lectin; some part of the fusion protein with tumor cell membrane binding. Therefore, these fusion proteins can be bridged by the effect of adenovirus infection lectin recognition of tumor cells by cell membrane glycoprotein. So this topic the use of these two kinds of fusion protein as a tool for protein, and the combined effect of Ad-EGFP infection of tumor cells by fluorescence microscopy on different fusion protein on Ad-EGFP infection efficiency of tumor cell, hair The two kinds of fusion protein can not only enhance Ad-EGFP infection of K562/ADR and U87-MG cells. Flow cytometry to detect the expression of GFP and the results were observed under fluorescence microscope results, suggesting that DIFBL and HddSBL can identify lectin glycoprotein of some tumor cell membrane. With the recombinant adenovirus Ad-DIFBL and Ad-PPA and CAR-DIFBL fusion protein S and sCAR-HddSBL alone or the joint effect of MTT and Annexin V-FITC/PI double labeled flow cytometry showed that sCAR-DIFBL fusion protein can act synergistically with recombinant virus, enhance the killing effect on cancer cells; although sCAR-HddSBL can also increase the infection rate of the virus, but can inhibit Ad-DIFBL and Ad-PPA on tumor cell killing effect of.Western blot showed that the recombinant adenovirus Ad-DIFBL and the fusion of Ad-PPA and U87-MG cells with sCAR-DIFBL protein interacting with sCAR-HddSBL, p-ERK levels increased, indicating lectin DI Independent role of FBL and HddSBL on cell toxicity; sCAR-HddSBL either alone or combined with the virus in U87-MG, the expression of E2F-1 was increased, showed that the expression of HddSBL may activate E2F-1, to protect the growth of tumor cells to further reveal the mechanism of lectin DIFBL and HddSBL on tumor cells.

【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)】：R735.7

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