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目的探讨重组腺相关病毒(rAAV)介导癌胚抗原(CEA)基因转染DC对结直肠癌的治疗作用。方法构建rAAV/CEA质粒,制备病毒。采用密度梯度离心法分离外周血单个核细胞(PBMC)来源的DC前体细胞,经rAAV/CEA转染DC后,采用PCR、Southern blot、流式细胞仪测定DC中CEA的表达,并检测rAAV/CEA的染色体整合情况及转染效率。加入GM-CSF、IL-4和TNF-α诱导DC前体细胞至成熟DC。将成熟DC和T淋巴细胞混合,加入IL-2和IL-7培养,获得细胞毒性T淋巴细胞(CTL)。采用体外51铬(51Cr)释放实验测定CTL对恶性肿瘤细胞的杀伤作用,3H掺入法检测rAAV/CEA转染DC对被激活T淋巴细胞增殖能力的影响。结果rAAV成功介导了CEA基因转染DC,且在DC内高表达,超过90%的DC前体细胞表达CEA蛋白,且rAAV/CEA病毒基因成功地整合入DC基因组内。rAAV/CEA转染的DC可以诱导特异性CTL,有效地识别并杀伤CEA阳性的结直肠癌细胞株LoVo,并且具有MHCⅠ类限制性,而对CEA阴性的肿瘤细胞无杀伤作用。结论以rAAV/CEA转染DC疫苗能够诱导抗肿瘤活性,为以人CEA作为靶抗原的结直肠癌的基因治疗奠定了基础。
Objective To investigate the therapeutic effect of recombinant adeno-associated virus (rAAV)-mediated transfection of CEA gene on colorectal cancer. Methods The rAAV/CEA plasmid was constructed and the virus was prepared. DCs derived from peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and DCs were transfected with rAAV/CEA. The expression of CEA in DC was measured by PCR, Southern blot, and flow cytometry, and rAAV was detected. /CEA chromosome integration and transfection efficiency. Addition of GM-CSF, IL-4 and TNF-α induces DC precursor cells to mature DCs. Mature DCs and T lymphocytes were mixed and IL-2 and IL-7 were added to culture to obtain cytotoxic T lymphocytes (CTLs). In vitro Cr(51Cr) release assay was used to determine the killing effect of CTL on malignant cells. The effect of rAAV/CEA transfected DCs on the proliferation of activated T lymphocytes was detected by 3H incorporation assay. RESULTS rAAV successfully transduced CEA gene into DC and was highly expressed in DC. More than 90% of DC precursor cells expressed CEA protein, and rAAV/CEA virus gene was successfully integrated into DC genome. DCs transfected with rAAV/CEA can induce specific CTLs, effectively recognize and kill CEA-positive colorectal cancer cell line LoVo, and have MHC class I-restriction, but have no killing effect on CEA-negative tumor cells. Conclusion The transfection of DC vaccine with rAAV/CEA can induce antitumor activity and lay a foundation for gene therapy of colorectal cancer with human CEA as target antigen.