【作者】 王艳；

【导师】 李岩；

【作者基本信息】 中国医科大学 ， 内科学， 2006， 博士

【摘要】 随着分子生物学及基因工程的发展，基因治疗正成为肿瘤防治研究的重点，其中免疫基因治疗因其简单、安全、有效成为颇具临床应用潜力的治疗策略，细胞因子基因治疗即属于免疫基因治疗范畴，是指将编码细胞因子的基因导入肿瘤细胞或免疫效应细胞，可以激活并增强特异性抗肿瘤免疫反应，使肿瘤的生长受到抑制或坏死消退，达到抗肿瘤的目的，同时克服了使用基因重组制剂所产生的严重毒副作用。细胞因子是主要由免疫细胞和部分非免疫细胞合成和分泌的一组多态调节因子，具有广泛的生物学功能，参与肿瘤免疫、自身免疫、移植免疫等过程。已经有部分基因重组细胞因子产品应用于临床，如IL-2(商品名是Proleukin)、TNF(肿瘤坏死因子)、G-CSF(商品名是Neupogen)，但是其直接应用这些制剂大多会产生发热、寒战、恶心、呕吐、头晕、乏力、血小板减少、肝功损伤、毛细血管渗漏等副作用，这些都使其应用受到一定限制。细胞因子之间具有协同作用，有研究已经证实IL-2与IFNβ在很多方面具有协同和增强作用，如果同时将这两个不同的基因分别导入细胞势必会增加基因转染的难度，且很难达到满意的转染效率。据此，我们设计用基因工程的方法，将IL-2与IFNβ连接成为融合基因，一次性转染肿瘤细胞，使其融合蛋白在细胞内表达，同时观察、检测其对肿瘤细胞的作用，期望得到一种具有更强抗肿瘤作用的新的基因重组蛋白。如何提高基因转导的效率和靶向性是基因治疗的关键问题，为了加强转染的基因在胃肠道的特异性表达，我们利用CEA启动子驱动目的基因，增强基因治疗的靶向性，做到有的放矢，从而大大提高肿瘤基因治疗的疗效。研究目的我们设计引物分别获取IL-2与IFNβ基因后，通过基因重组及重组蛋白质新技术将二者通过linker连接，构建融合基因原核表达载体、真核表达载体，使之分别转入大肠杆菌及大肠癌肿瘤细胞中，表达出融合蛋白，并稳定转染。在此基础上进行蛋白表达、抗肿瘤活性的研究。建立裸鼠荷人结肠癌动物模型，以观察融合基因的在体内对肿瘤的作用。研究方法1．PCR、RT-PCR法扩增得到IL-2cDNA、IFNpcDNA、CEA启动子；2．应用原核表达载体pGEX-5X-1，构建出原核表达质粒，并转化至大肠杆菌中诱导融合蛋白的表达，SDS-PAGE电泳、Western-blot方法检测原核表达情况；3．利用真核表达载体pcDNA3.1／HisA、绿色荧光蛋白载体pEGFP-C1，构建出真核表达质粒；4．培养结直肠癌肿瘤细胞株，用脂质体法将真核表达质粒转入结直肠癌细胞株Lovo(CEA高表达)、HT-29(CEA低表达)，转染后分别以流式细胞仪、DNA ladder、荧光显微镜、Western-blot等方法检测肿瘤细胞的凋亡情况及蛋白的表达情况。5．用大肠癌细胞建立裸鼠荷瘤模型，将不同质粒以瘤内注射、尾静脉注射、腹腔注射的形式给药，观察肿瘤的变化。结果1．原核表达质粒可以在大肠杆菌诱导出GST-IL2、GST-IFNβ、GST-IL2／IFNβ融合蛋白表达，大小为40KD、45KD、60KD左右，并经Western-blot证实可同时被IL-2、IFNβ特异性抗体检测出来；2．绿色荧光蛋白载体构建的真核表达质粒pEGFP-IL2／IFNβ瞬时转染Lovo细胞，用荧光显微镜观察，转染效率15-20％左右，能成功表达出绿色荧光蛋白；Western-blot、ELISA检测可被IL-2、IFNβ特异性抗体检测出来；3．流式细胞仪检测转染后细胞，可见细胞周期变化及明显凋亡峰；稳定转染过程中细胞凋亡、坏死明显增加；可见对肿瘤细胞有明显的杀伤作用；4．由CEA启动子调控的融合基因在CEA高表达的Lovo细胞的表达高于在CEA低表达的HT-29细胞的表达。5．由Lovo细胞建立出荷结肠癌裸鼠模型，尾静脉注射较瘤内注射的抗肿瘤效果更为显著、带瘤生存期延长、肿块缩小、肿瘤细胞出现凋亡、坏死现象。结论1．IL-2／IFNβ融合基因可在大肠杆菌可以表达出融合蛋白，分子量大小符合预测，且可被IL-2、IFNβ的抗体用过Western-blot的方法检测出来，说明其保留了IL-2和IFNβ各自的表面结合位点；2．插入绿色荧光蛋白pEGFP-C1载体的融合基因可以在Lovo细胞中表达出融合的绿色荧光蛋白，胞浆表达为主，ELISA法对其细胞裂解蛋白的检测，可见表达出IL-2和IFNβ样物质，具有符合细胞因子的特性；流式细胞仪检测发现细胞周期的改变及凋亡峰的出现，说明其对肿瘤细胞有抑制作用；3．融合基因的真核表达重组质粒可以稳定转染至肿瘤细胞株中；4．由CEA启动子调控的重组质粒在CEA高表达的细胞(Lovo)中的荧光强于在CEA低表达的(HT-29)细胞，说明CEA启动子可靶向性调控融合基因在肿瘤细胞的表达；5．Lovo细胞可成功地在BALB／C-nu／nu小鼠身上建立裸鼠模型，成瘤率可达100％；6．重组质粒尾静脉注射瘤的抑瘤作用强于内注射，带瘤生存期延长、瘤体生长缓慢、可诱发癌细胞凋亡、表达融合蛋白，且没有没明显毒副作用。透射电镜可见明显的凋亡小体，DNA ladder电泳证实凋亡的存在。据此，我们可以推测，所构建的两种具有协同作用的细胞因子的融合基因(IL-2／IFNβ)在原核和真核中都可以成功表达出融合蛋白，该融合蛋白可能具有更强的抗肿瘤作用，在体内、外都可对肿瘤细胞产生抑制作用；同时，用脂质体方法可以完成对细胞的转染。通过软件分析，该融合蛋白的蛋白质二级结构、三级结构基本具有两种细胞因子的结构特点和功能，保留了两种细胞因子的生物活性。这种融合蛋白有望在肿瘤的基因治疗中发挥一定作用。更多还原

【Abstract】 Construction of prokaryotic and eukaryon expression system of cytokine fusion gene driven by CEA promoter and identification of the recombinant protein antitumor effect on colorectal carcinoma vivo and invitroGene therapy is becoming a hotspot in neoplasm disease by the development of molecular and cytology. Cytokine gene therapy belongs to the immunological gene therapy. Use technological tansgene methods to insert the target cytokine gene into the tumor cells, it’s product can activate the immunosyterm of the body and cause the death of the tumor cells. The endogenesis cytokines can avoid the side-effects in direct cytokine regent usage.Cytokines are a group of immuno regulators secreted by most immuno cells, they are concerned with a lot of immunological reactions such as tumor-immuno、self-immuno、plant-immuno. Some kinds of gene recombinant cytokines have been used in clinic, such as IL-2(Proleukin), TNF(tumor necrosis factors), G-CSF (Neupogen). But in there using phase, we see many sideeffects: fever、chill、nausea、vomit, swirl, vapidity, platelet decrease, hypohepatic, capillary vessel leakage, etal. They limit recombinant cytokines’ clinicle usage. When it comes to gene therapy,we can avoid this problem.Some study have revealed that IL-2 and IFNβhave cooperate effects in many aspects,and they both are small moleculars easy to transfect into cells,so if they are united, perhaps we may see a more significant antitumor effect. We hope to gained a new kind of fusion protain which has higher antitumor effect and both the character of IL-2 and IFNβ.How to make it more targetable and improve the efficiency is a key problem in gene transfer. To receive a differential transfer, increase the target probability, some regulators such as promoter can be used. CEA is a high expressed protein in many gastrointestinal tumors. CEA promoter was designed to regulate the IL-2/IFNβfusion gene expressing in E. coli and colorectal tumor cell lines.ObjectiveA fusion gene of IL-2 and IFNβwas designed and constructed by DNA engineering through a linker. The fusion gene driven by CEA promotor was insert prokaryotic and eukaryon expression vectors: pGEX-5X-1, pcDNA3. 1A, pEGFP-C1. Then were transferd into E. colli and colorectal cell lines. In this experiment, we construct nude mice bearing human colorectal carcinoma to see the vivo effect of the fusion gene.Methods1. CEA promotor、IL-2cDNA and IFNβcDNA were obtained by PCR and RT-PCR.2. Prokaryotic expression vector of pGEX-5-1 was used to construct the Prokaryotic expression system, pGEX-IL2, pGEX-IFNβ, pGEX-IL2/ IFNβwere transfected to BL21 to express GST-fusion protein; SDS-PAGE and Western-blot was used to examine the results;3. Eukaryon expression vector pcDNA3. 1A、green fluorescence protein vector pEGFP-C1 were all used to construct the Eukaryon expression system;4. pcDNA-IL2, pcDNA-112/ IFNβ, pEGFP-IL2/ IFNβwere transfected to CEA high expression cell line Lovo and CEA low expression cell line HT29 by use of lipofectamine 2000. The apoptosis of cells was monitored using flow cytometry and DNA ladder; fusion protein expression was examined by flurescence microscope and Western-blotting. 5. Lovo cells were uese to constitute nude mice model bearing colorectal carcinoma. Through FCM method、DNA gel electrophoresis and transmission electron microscope, we found that the outstanding effect was related to cell apoptosis.Results1. IL2/ IFNβfusion gene can expressed in E. coli, the fusion protein product is about 40KD, it can be detected by SDS-PAGE and Western-blot;2. IL2/ IFNβfusion gene inserted into green fluorescence protein vector pEGFP-C1 and can be transferd into Lovo cell and express green fluorescence fusion protein. When in instant transfer,it’ s efficiency is about 15-20%,when in stable transfer, fusion protein expression can be examined by flurescence microscope;3. The product are all can be detected by Western-blot, The apoptosis of cells was monitored using flow cytometry and DNA ladder. In FCM we can see significant change in cellular cycle.4. The expression is more obvious in CEA high expressed cells (Lovo) than in CEA low expressed cells (HT-29). The fusion protein has significant targetable effect.5. Strong inhibitory effect both on tumor growth and metastasis of human colon cancer implanted in nude mice. Tail-vein injection has more notable effect than direct inject into tumor.Conclusions1. The fusion gene can express the targated fusion protein in E. Coli systerm. It’ s molecular accord with forecast and can be detected by Western-blot.2. IL2/ IFNβfusion gene can be inserted into green fluorescence protein vector pEGFP-C1 and transferd inton Lovo cell and can express green fluorescence fusion protein. It’s orientated in cytoplasm. It can be detected by IL-2 and IFNβantibody through ELISA,FCM can see significant apoptic peak. 3. The fusion gene can be stable transferd to Lovo cell lines.4. The expression in CEA high expression cell lines (Lovo) is more evident than in CEA low expressed cells (HT-29). It show that CEA promoter can targated regulate the fusion gene to express.5. The Lovo cell lines can be used successfully to build colorectal tumor model in BALB/C-nu/nu node mice.6. Strong inhibitory effect both on tumor growth and metastasis of human colon cancer implanted in nude mice. Tail-vein injection has more notable effect than direct inject into tumors.更多还原