重组人p53腺病毒转染淋巴瘤源性树突状细胞的抗肿瘤免疫效应

Anti-tumor Immunse Response of Dendritic Cells Derived from Lymphoma Cells Transducted with Recombinant Adenovirs Encoding Human P53

本研究旨在探讨重组人p53腺病毒转染淋巴瘤源性树突状细胞的抗肿瘤免疫效应。采集初诊弥漫性大B细胞淋巴瘤(DLBCL)患者肿大的淋巴结和外周血,分别分离单个核细胞,进行树突状细胞(DC)体外诱导培养,均分为实验组(rAd-p53-DC)、对照组(rAd-DC)和空白对照组。用重组人p53腺病毒(rAd-p53)转染2种来源的DC,流式细胞术检测DC免疫表型,Western blot鉴定P53蛋白的表达,ELISA法检测上清中的细胞因子IL-12的含量,用混合淋巴细胞反应(MLR)测定DC刺激同种异体淋巴细胞增殖能力,用乳酸脱氢酶(LDH)释放法检测经rAd-p53转染的两种来源DC的细胞毒性T淋巴细胞(CTL)反应。结果表明,实验组DC的表型(CD1a除外)CD83、CD80、CD86和HLA-DR表达均较对照组及空白对照组明显增高(P<0.05)。实验组P53蛋白的表达上调,培养上清液中IL-12分泌水平较对照组及空白对照组明显增高(P<0.05)。实验组明显刺激自体淋巴细胞增殖,且刺激能力随rAd-p53-DC与淋巴细胞比例的增加而升高。对照组及空白对照组也能刺激同种自体淋巴细胞增殖,但较实验组差(P<0.05)。对靶细胞的细胞毒作用(CTL效应)检测显示,实验组介导同种异体的淋巴细胞杀伤率显著高于对照组及空白对照组(P<0.05),且淋巴结来源DC的CTL效应明显高于外周血来源DC(P<0.05)。结论:rAd-p53-DC为基础的肿瘤疫苗有可能在解决淋巴瘤的微小残留病、DC免疫耐受等问题方面发挥作用。

This study was aimed to investigate the immunological effect of modified dendritic cells（DC） which inducing cytotoxic T cells（CTL） against lymphoma cells.The DC were isolated from the lymph node and periphroal blood of patients with diffuse large B cell lymphoma（DLBCL）.DC were transfected with recombinant adenovirus vector carrying human p53 gene（rAd-p53-DC）.The expression of p53 gene was detected by flow cytometry.Western-blot was used to detect the expression of P53.ELISA was used to detect IL-12 level in supernatant.The mixed lymphocyte reaction（MLR） was used to detect the proliferative ability of auto-lymphocyte stimulated by DC.The lactate dehydrogenase（LDH） release test was used to determine the cytotoxicity of CTL.The results indicates that the expressions of DC surface molecule（except for CD1a）such as CD83,CD80,CD86 and HLA-DR were significantly higher in experiment group than that in control group and blank control group.The secretion of IL-12 in supernatant was higher in experiment group than that in control group.The autologous T lymphocyte proliferation and cytotoxic activity against the same kind of DLBL-cells increased in experiment group as compared with control group and blank control group（P〈0.05）.The ability to stimulate T lymphocyte proliferation increased with the rising of the ratio of DC and T lymphocyte.However,there was statistically significant difference between rAd-p53-DC derived from Lymph node and peripheral blood（P〈0.05）.It is concluded that rAd-p53-transfected DC can induce CTL response in vitro against lymphoma cells.