携带凝集素基因的重组腺病毒和痘苗病毒对肝癌细胞的作用机制研究

发布时间：2018-04-18 21:17

本文选题：SMMC-7721 + 凝集素　；参考：《浙江理工大学》2017年硕士论文

【摘要】：第一部分携带凝集素基因的重组腺病毒和痘苗病毒对肝癌细胞的作用机制研究癌症严重威胁人类的生命和健康。肝癌是人类最常见癌症之一。随着分子生物学的发展和基因技术的进步,靶向基因—病毒联合治疗已成为肝癌治疗新的研究热点。凝集素是一类专一识别糖并与之非共价、可逆地结合的蛋白质或糖蛋白。过去几年本课题组应用病毒携带凝集素基因,通过病毒感染使凝集素基因在肿瘤细胞内表达,并对肿瘤细胞产生的细胞毒性作了一定的研究。但是,将携带不同凝集素的重组腺病毒和痘苗病毒对体外肝癌细胞的杀伤机制还没有深入研究。本课题利用已构建好的携带鲈鱼凝集素(Dicentrarchus labrax lectin,DIFBL)、紫海胆凝集素(Strongylocentrotus purpuratus lectin,SPL)、盘鲍鱼凝集素(Haliotis discus discus Lectin,HddSBL)和掌叶半夏凝集素(Pinellia pedatisecta agglutinin,PPA)的重组腺病毒Ad-DIFBL、Ad-SPL、Ad-HddSBL和重组溶瘤痘苗病毒OncoPox-PPA、OncoPox-SPL,感染肝癌细胞,研究凝集素对细胞的毒性作用机制。本课题将人的肝癌细胞SMMC-7721的肌纤维膜关联蛋白(Sarcolemma associated protein,SLMAP)和Striatin靶点Knock Down,然后用等量感染复数的Ad-DIFBL感染细胞,MTT检测以及荧光显微镜观察显示,Ad-DIFBL对SLMAP和Striatin Knock Down的SMMC-7721细胞的毒性作用明显低于对照细胞,说明SLMAP和Striatin这两种基因可能与凝集素的毒性有一定的关系,但其深入的机制尚需进一步研究。通过Western blot实验,发现用Ad-DIFBL和Ad-HddSBL处理后的细胞中组蛋白Histone H3表达量下降;用Ad-SPL和Ad-HddSBL处理后的细胞中ISG15表达量下降。OncoPox-PPA和OncoPox-SPL相较于对照病毒OncoPox-pCB,使肿瘤细胞中C-myc、SLMAP、Beclin 1表达水平明显下降;β-catenin微量下降;β-tubulin产生二聚体和三聚体。用OncoPox-PPA、OncoPox-SPL与Onco Pox-pCB分别去感染SMMC-7721肿瘤细胞,将样品进行转录组数据分析,结果显示凝集素对多种基因水平产生影响。OncoPox-PPA处理与Onco Pox-pCB处理相比,细胞中Caspase8,FGFR1,MAVS,SIKE1上调;而TMEM173,STRIP1,IFIH1,CTTNBP2NL,Striatin4,FGFR4下调;OncoPox-SPL感染的细胞与Onco Pox-pCB感染相比,CTTNBP2NL,Striatin4,FGFR2,FGFR3,SIKE1上调;AKT1,Caspase9,FGFR4,PRMT5,STRIP1,FGFR1,MAVS,CERK下调。综上所述,Ad-DIFBL对SLMAP和Striatin Knock Down的SMMC-7721细胞的毒性相比于对照细胞明显降低,说明凝集素DIFBL与SLMAP、Striatin之间存在某种相互作用,具体是什么样的作用需要后续深入研究;Western blot,免疫共沉淀,转录组数据分析等结果显示,凝集素对多种基因表达水平产生影响。本研究为今后将凝集素基因应用于肝癌治疗研究提供了一定的基础。第二部分DIFBL、HddSBL凝集素与腺病毒受体连接的融合蛋白的抗肿瘤机制研究利用已构建好的sCAR-DIFBL和sCAR-HddSBL两种融合凝集素基因,连接到原核表达载体pQE30上,将质粒分别转到M15原核表达菌株中。通过IPTG诱导剂成功诱导出两种融合蛋白的表达,并且是以包涵体的形式表达。将诱导出的蛋白进行Ni离子亲和层析纯化及包涵体复性。融合蛋白的sCAR部分是腺病毒受体的膜外片段,能够和5型腺病毒结合;融合蛋白的凝集素部分可与肿瘤细胞膜上的某些糖蛋白结合。因此,这些融合蛋白可以通过桥连作用,使腺病毒通过细胞膜糖蛋白感染凝集素识别的肿瘤细胞。所以本课题利用这两种融合蛋白作为工具蛋白,和Ad-EGFP联合作用感染多种肿瘤细胞,通过荧光显微镜观察不同融合蛋白对Ad-EGFP感染肿瘤细胞效率的影响,发现两种融合蛋白不仅能够增强Ad-EGFP对K562/ADR和U87-MG细胞中的感染。流式检测GFP表达量结果与荧光显微镜下观察结果一致,提示凝集素DIFBL和HddSBL能识别某些肿瘤细胞膜上的糖蛋白。分别用重组腺病毒Ad-DIFBL和Ad-PPA与融合蛋白s CAR-DIFBL和sCAR-HddSBL单独或联合作用,MTT和Annexin V-FITC/PI双标记流式结果表明融合蛋白sCAR-DIFBL能够与重组病毒协同作用,增强对肿瘤细胞的杀伤效果;sCAR-HddSBL虽然也能增加病毒感染率,却能够抑制Ad-DIFBL和Ad-PPA的对肿瘤细胞的杀伤作用。Western blot结果显示,用重组腺病毒Ad-DIFBL和Ad-PPA与融合蛋白sCAR-DIFBL和sCAR-HddSBL联合作用U87-MG细胞后,p-ERK水平上升,说明凝集素DIFBL和HddSBL对细胞毒性作用无关;sCAR-HddSBL无论是与病毒联合还是单独作用于U87-MG后,E2F-1的表达量都呈上升的趋势,表明HddSBL很可能激活E2F-1的表达,达到保护肿瘤细胞生长的目的,进一步揭示了凝集素DIFBL和HddSBL对肿瘤细胞的作用机制。
[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.

【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】：R735.7

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