【作者】 薛晓荣；

【导师】 汤华；

【作者基本信息】 天津医科大学 ， 病原生物学， 2006， 硕士

【摘要】 背景与目的:原发性肝细胞肝癌是一种常见的恶性肿瘤,适合手术治疗的患者只占一少部分,具有较高的复发率,预后很差,严重威胁着人类的健康。人类肿瘤相关性抗原以及肿瘤抗原基因的发现,为肿瘤的免疫治疗尤其是主动性免疫治疗提供了新的手段。直接作用于肿瘤特异性抗原或相关抗原的预防性瘤苗是目前研究的热点。胰岛素样生长因子Ⅰ受体(IGFlR)在各种肿瘤细胞中高水平表达,尤其是在肝癌细胞,而在正常的肝细胞则低表达。因此,制备IGF1R特异性瘤苗,可作为肝癌治疗的一种新思路。树突状细胞(DC)是体内功能强大的专职性抗原提呈细胞,它能够高效地摄取、处理抗原,并将处理后的多肽呈递给静息型T细胞,引起针对该抗原的特异性免疫反应。DC应用于肿瘤免疫洽疗是目前的研究热点,许多研究证实,肿瘤患者树突状细胞的质量及功能均减弱,MHC分子和共刺激分子CD80、CD86的表达量下降。因此,体外诱导功能正常的树突状细胞,经修饰的肿瘤相关性抗原或特异性抗原,作为疫苗免疫肿瘤患者,是肿瘤治疗的一个发展方向。制备DC瘤苗的方法有肿瘤特异性或相关性抗原、肿瘤细胞裂解物与DC共培养,肿瘤细胞与DC融合、肿瘤细胞总mRNA转染DC等。相关性抗原与DC共孵是常用的方法。本研究拟构建IGF1RαcDNA真核表达载体,体外表达IGF1Rα蛋白片段,与DC细胞共孵,诱导针对表达IGF1R的HepG2特异性免疫反应,寻找新的治疗肝癌的途径,并初步探讨肝癌相关性抗原IGF1R作为肝癌靶向治疗的可行性。方法:1.提取HepG2的RNA,RT-PCR扩增IGF1Rα蛋白片段序列,PCR产物与pGEM—Teasy载体的连接,经过扩增测序确证后,利用pcDNA3/HA载体构建了IGF1Rα段编码区与载体HA的融合真核表达重组子,并在HEK293细胞中进行瞬时表达,转染72小时通过Western印迹检测2.取健康供者外周血单个核细胞,Ficoll密度梯度离心法分离单个核细胞,贴壁法进一步纯化细胞,收集非贴壁细胞(淋巴细胞)。贴壁细胞在细胞因子rhGM-CSF(1000u/ml),rhIL-4(100ng/ml)作用下,体外诱导DC。收集培养4d的DC,将IGF1Rα蛋白加入DC中,另设不加抗原对照组和无关抗原对照组,倒置显微镜观察细胞形态,流式细胞仪检测表型,收集各组DC,与淋巴细胞按5:1比例混合接种于6孔板,培养7d,收集的细胞作为效应细胞。选择表达IGF1R肝癌细胞株HepG2细胞作为靶细胞,按效靶比20:1、10:1混合接种培养48h,同时设单一靶细胞组、单一效应细胞组。以MTT法检测效应细胞对靶细胞的杀伤率。结果:1.成功构建了IGF1α-pGEMT重组载体,测序后经NCBI同源性比较,结果与IGF1R mRNA(NM 000875)一致。进一步构建IGF1Rα-pcDNA3/HA融合真核表达载体,用EcoRI/XhoI双酶切确定。转染HEK293蛋白表达,经Western检测,可检测到分子量约为34KD的蛋白条带,与预期的蛋白分子量一致。2.诱导的DC光镜下,细胞周围可见毛刺状突起和树枝状突起,IGF1Rα蛋白刺激的DC比未经抗原刺激的DC的CD86、CD83、CD80、CD40都有所升高,表达量分别为87.9%,69.7%,88.69%,90.1%;而CD14水平下降,表达量仅为11.8%。说明IGF1Rα蛋白刺激的DC比未经抗原刺激的DC进一步成熟。MTT检测对照组为未经致敏的DC细胞激活的T细胞,空质粒转染得到的蛋白致敏的DC细胞激活的T细胞,试验组为目的基因真核转染得到的目的蛋白致敏的DC细胞激活的T细胞,试验组与对照组比有明显的杀伤HepG2细胞的作用(P＜0.05),其活化的CTL对表达IGF1R的肝癌细胞株HepG2在效靶比为20:1时杀伤率为30.1%。对照组之间没有明显差异。在20:1和10:1情况下,目的蛋白致敏的DC细胞激活的T淋巴细胞对HepG2细胞与NIH3T3细胞的杀伤作用有明显的差异(P＜0.05)。结论:我们的实验证实构建表达的IGF1Rα蛋白片段具有良好的免疫原性,可以刺激DC细胞进一步成熟,致敏的DC可有效诱导CTL克隆的生成。提示了IGF1Rα蛋白片段在DC免疫治疗作用。因此,IGF1R作为靶点治疗肝癌具有可行性。更多还原

【Abstract】 Background and objective:Hepatocellular carcinoma(HCC) is a common malignancy with a very poor prognosis.Only a minority of patients are eligible for surgical therapies.Therefore,novel strategies to prevent proliferation of malignant cells are urgently needed.A promising approach may be the prophylactic vaccination directed against a tumor-associated antigen(TAA)or tumor-special antigen(TSA).IGF1Rαis usually expressed in high concentrations in the liver cancer,however, low expression in the normal liver cells.IGF1Rαmay be a target for special tumor vaccine against IGF1R in the special immunotherapy and gene therapy.Dendritic cells(DC) are considered the most potent antigen-presenting cells(APC) because they may incept and manage the antigen,and stimulate the primary activation of T cells causing to special immune reaction against the antigen.So DCs are promising new tools for the immunotherapy of cancer.Many research approved the DCs of the cancer patients get worse,and MHC、CD80、CD86 of DC expression decline.It may be a development aspect that TAA or TSA stimulating normal DC cells immune cancer patient as vaccine.The immunogen can be TAA,TSA,tumor lysates, DNA,mRNA,etc.The coculture of DC and TSA antigen is common method.Methods:1.distill RNA of HepG2,enlarge IGF1Rαantigen using RT-PCR, connect aim gene and pGEM-Teasy carrier,construct PcDNA3/HA and IGF1Rαrecombining carrier,instantaneous expression in the HER293 cells, inspect using Western Blot in the 72 hours.2.we induced peripheral blood mononuclear cell into DC cells with rhGM-CSF(1000 u/ml).rhIL-4(100 ng/ml). After 4 days of culture,DCs were harvested pulsing of autologous DC with IGF1Rα.Modality of DCs were observed bythe microscope.FACS analyses the phenotype of DC.After coculture of DC and T cells in 7days by 1:5, to measure cytotoxicity of CTL in MTT assays.Results:1.we obtain IGF1Rα-pGEMT carrier,and IGF1Rα-pcDNA3/HA expression carrier.We find the protein which molecular weight is 34KD after transfecting HEK293 cells.2.The matured DCs sticked out many pseudopods.By FCM analysis,the matured DCs’ surface marker were upregulated compared with immature DCs,including CD86 87.9%、CD83 69.7%、CD80 88.69%、CD40 90.1%,but CD14 were downregulated which numerical value is only 11.8%.MTT assay revealed that the cytokine-driven DCs with antigen induced a strong specific CTL cytotoxicity to HepG2 campare with comparison groups(P＜0.05),The cytotoxicity rate 30.1%when the ratio of the effect cell to target cell was 20:1.The comparison groups has no distinct difference.The CTL cytotoxicity with IGF1Rαprotein loading DCs to HepG2 was distinct difference compare with to NIH3T3.Conclusion:The recombined human IGF1Rαprotein was expressed successfully in mammalian cells producing mature DC cells with its stimulation can induce specific HepG2 cells CTL.The results revealed that IGF1Rαis important for immunotherapy of HCC of human.更多还原