The Generation Of AFP-cDNA Priming Dendritic Cell Vaccine And Immunotherapeutic Activity Against Hepatocellular Carcinoma Cell Line HepG2

Hepatocellular carcinoma (HCC) is a common malignancy with a very poor prognosis.Only a minority of patients is eligible for surgical therapies due to advanced tumors or extrahepatic disease at primary diagnosis. 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) .One possible target for an HCC-specific vaccination is the oncofetal alpha-fetoprotein (AFP) .AFP is usually expressed in high concentrations in- 7 -the fetal liver , the gastrointestinal tract , and the yolk sack and transcriptionally down-regulated after birth. AFP is frequently reexpressed in HCCs and therefore used as a diagnostic marker for this tumor. As a TAA, AFP may be a target for HCCs immunotherapy and gene therapy. Dendritic cells (DC) are considered the most potent antigen-presenting cells (APC) and are thus promising new tools for the immunotherapy of cancer. DC can stimulate the primary activation of T cells due to their enhanced capacity of presenting immunogenic peptides in association with self-major histocompatibility complex I and II molecules. The immunogen can be TAA, TSA, tumor lysates, DNA, mRNA, etc.OBJECTIVE: To construct human AFP eukaryotic expression vector, therefore transfect AFP gene into DCs, and discuss the feasibility of immunotherapeutic activity of AFP gene priming dendritic cell vaccine to hepatocellular carcinoma cells expressing AFP antigen molecule.METHODS: AFP-cDNA eukaryotic expression vector was constructed and transfected into immature dendritic cells with liposome. The specific CTL was induced ex vivo with the constructed AFP-DC vaccine and detected its targeting cytotoxic functions to hepatocellular carcinoma cells expressing AFP.- 8 -RESULTS: The induced cells presented the typical features of dendritic cell, the pricking and dendritic eminences on the cells surface could be observed through microscope and scan electron microscope, as well as lysosomes> mitochondria and rough endoplasmic reticulums through the transmission electron microscope. The expressions of CD?GDI Ic was 47.1%? 78.49% respectively , the expressions of MHC molecule and co-stimulators HLA-DR, CD8(K CD86 were 66.09% ?51.37%? 55.83% respectively. AFP-DC could secrete AFP antigen molecule, there was 0.8805 lU/ml AFP antigen detected in the supernatant of AFP-DC, compared with the control groups, the differences were significant (P<0.05) .It also could be detected through immunofluorescence staining. AFP-DC activated CTL presented the specific killer ability to the hepatocellular carcinoma cells expressing AFP, the killer ability rate was 84.05%, compared with the control groups, the differences were significant (P<0.05) .CONCLUSION: In our experiment, we constructed the re-combined human AFP eukaryotic expression vector successfully, which provides experimental data for gene therapy and immunotherapy of hepatocellular carcinoma of mankind. Maybe we can use it as a target of human HCC for treatment. We obtained DCs from hematopoietic cells stimulated by GM-CSF and IL-4 in vitro then transfected AFP gene into DCs. The established AFP-DC vaccine may be a- 9 -tool of the hepatocellular carcinoma immunotherapy, and it will be foundation of future clinical use of DC vaccine 劇鯡ND-END ■- 10 - 第四军医大学硕士学位论文 研究内容＿ 月 ～ 肝细胞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。直接作用于肿瘤特。异性抗原或相关抗原的预防性疫苗是目前研究的热点。甲胎蛋白(AFP)通＿常在胎肝,胃肠道,卵黄囊中高表达,出生后表达量下降。但是在部分肝细＿胞癌患者,APP的表达量异常增高,临床上可作为肝癌的一个诊断和预后指－标。因此,制备AFP特异性的疫苗,诱导针对AFP的特异性的兔疫反应,可作为肝细胞癌治疗的一种新思路。DC是体内功能强大的专职性抗原提呈细...