转染慢性粒细胞白血病总RNA对树突状细胞介导的抗白血病作用的体外研究  

Effect of chronic myelocytic leukemia total RNA transfection on the anti-leukemia immune response activated by dendritic cells derived from chronic myelocytic leukemia cells

作　　者：饶燕飞[1] 邹湉[2] 杨桂玲[2] 王小中[1] 丁伟荣[3] 

机构地区：[1]南昌大学第二附属医院检验科,江西南昌330006 [2]南昌大学第二附属医院血液科,江西省血液病研究所,江西南昌330006 [3]江西省人民医院,江西南昌330006

出　　处：《实验与检验医学》2013年第6期529-532,共4页Experimental and Laboratory Medicine

基　　金：江西省教育厅科学技术研究资助项目(2006-87)

摘　　要：目的体外诱导和鉴定慢性粒细胞白血病-树突状细胞(CML-DC),并探讨人慢性粒细胞白血病(CML)总RNA体外转染对其介导的特异性细胞毒T淋巴细胞(CTL)对慢性粒细胞白血病细胞杀伤作用的影响。方法分离14例CML患者骨髓单个核细胞,加入rhIL-4、rhGM-CSF、rhTNF-α诱导培养CML-DC。分别于培养第1、3、6、14d用倒置显微镜进行形态学观察,流式细胞术检测免疫学表型,染色体G显带技术检测其染色体核型,逆转录聚合酶链反应(RT-PCR)检测bcr-abl融合基因。并于CML-DC培养第5d,加入CML细胞总RNA脂质体转染,或裸总RNA转染,或不加任何试剂继续培养的DC,共三种CML-DC,分别致敏T淋巴细胞,另设以IL-2培养的T淋巴细胞为对照组,比较不同组别的致敏T淋巴细胞的杀伤活性。结果CML骨髓单个核细胞诱导前CD1α、CD83表达均在5%以下。而诱导成熟的CML-DC细胞CD1a、CD83阳性表达率分别为20.13±3.43%、26.76±2.79%,较诱导前均明显增高(P<0.01)。CML-DC细胞均存在Ph1染色体,并表达bcr-abl融合基因。总RNA脂质体转染CML-DC、裸总RNA转染CML-DC、单纯CML-DC分别致敏的T淋巴细胞在效:靶比为20:1时对CML单个核细胞的杀伤效率分别为75.33±3.11%、37.23±2.92%、29.62±1.61%,均明显高于对照组(9.87±3.43%,P<0.01)。总RNA脂质体转染的CML-DC所诱导的CTL杀伤活性最强,与后三组杀伤活性均有显著性差异(P<0.001)。结论CML-DC既具有CML白血病源性,又具有DC细胞的特性,并能诱导特异性CTL杀伤白血病细胞。经脂质体转染CML细胞总RNA可以提高CML-DC介导的CTL特异性杀伤慢性粒细胞白血病细胞。Abstract： Objective To isolate and identify the chronic myeloid leukemia-dendritic cells （CML-DC）, and to investigate the effect of CML total RNA transfection on the anti-leukemia immune response activated by CML-DC in vitro. Methods Bone marrow mononuclear cells （BMMNCs） were isolated from 14 patients with CML, and co-cultured with rhGM-CSF, rhIL-4 and TNF-ot. The morphologic features were observed by inverted microscope at day 1, 3, 8 and 14. CD83 and CDla expression were assayed by flow cytometry. Karyotypes of CML-DC were assayed by G-banding technique. Bcr-abl fusion gene of CML-DC was assayed by reverse transcription-polymerase chain reaction （RT-PCR）. At day 5, CML-DC cells were transfected with CML ceils total RNA using liposomal, or with the bare total RNA, or without any reagents respectively, then the antigen presenting function of CML-DC were tested by mixed lymphocyte reaction （MLR）. Results The expressions of CDla and CD83 of uncultured CML- BMMNCs were all below 5%. After cultured with cytokines, CML-BMMNCs had the typical morphologic features and immunophentypes of DCs. The positive expression rates of CDla and CD83 in CML-DCs were 20.13±3.43% and 26.76±2.79%, respectively, higher than the uncultured cells （P〈O.01）. CML-DCs had Ph chromosomal and expressed bcr-abl fusion gene, which suggested that CML-DC was derived from CML cells. The cytotoxic rates of T cells induced by CML-DCs transfected with CML cells total RNA using liposomal, CML-DC transfected with CML cells total RNA and CML-DCs transfected without any drug were 75.33± 3.11%,37.23 ±2.92% and 29.62±1.61%, respectively, and all of them were more effective than T cell co-cultured with IL-2 （9.87±3.43%, P〈0.001）. The CTL induced by CML-DCs transfected with CML ceils total RNA using liposomal was more effective than the others （P〈 0.001）. Conculsions The CML-BMMNCs could be induced into CML-DC, which could induce the CTL to get specific anti-leukemia activity in vitro. The anti-leukemia immune respons

关 键 词：树突状细胞 慢性粒细胞白血病 总RNA T淋巴细胞 细胞毒 

分 类 号：R733.72[医药卫生—肿瘤] R730.51[医药卫生—临床医学]