低温冻存对脐血树突状细胞介导的食管癌疫苗的影响-消化内科学专业论文.docx

汕头大学医学院硕士学位论文 汕头大学医学院硕士学位论文 PAGE PAGE VI Abstract Purpose To study the biological characteristics and specific cytotoxic activity of the vaccine of esophageal cacinoma cells fused with dendritic cells (DCs) that derived from the umbilical cord blood after cryopreservation. Methods First, the CD34+ hematopoietic stem cells were isolated from mononuclear cells, which were prepared with Ficoll-Hypaque density-gradients centrifugation from human cord blood by magnetic cell sorting system (MACS) with CD34+Progenitor Cell Isolation Kit. Second, CD34+ hematopoietic stem cell were expanded in RPMI 1640 medium containing 10% fetal calf serum, 300ng/ml rhGM-CSF (human Granulocyte-Macrophage Colony-Stimuting Factor), 150u/ml rhTNF-α (human Tumor Neucrosis Factor α) and 200ng/ml rhSCF (human Stem Cell Factor) at 37℃, 5%CO2 for 2 weeks. Third, CD34+ hematopoietic stem cell could differentiate into mature DCs in 2 weeks. Then, the mature DCs were fused with esophageal carcinoma cell line by polyethyleneglycol (PEG-3600) at the ratio of 5:1. Selecting by MACS, the cells marked with HLA-DR MicroBeads were the EC109-DC that acted as tumor vaccine. Fourth, the fused cells were stored at -80℃ without rate-controlled freezing, in which DMSO and glucose were the main cryoprotective agents (CPA). The recovery rates and viability of the fused cells after thawing were tested with trypan blue staining. Morphology, immunophenotypes of EC109-DC before/after cryopreservation were observed by light microscope, flow cytometry and technique of cell culture in vitro. T cell subset activated by the fusion cell before and after cryopreservation was observed by flow cytometry .The cytotoxic T lymphocytes (CTL) cytotoxicity of EC109-DC before/after cryopreservation was examined with MTT assay. Results ①Percentages of CD34+ hematopoietic stem cells purified from cord blood monocyte by magnetic cell sorting system (MACS) were 0.8%-1.10%. The purified CD34+ hematopoietic stem cells were suspende