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脂质体RNA负载慢性粒细胞白血病来源树突状细胞的性能比较[J]. 肿瘤防治研究, 2010, 37(01): 47-51. DOI: 10.3971/j.issn.1000-8578.2010.01.013
引用本文: 脂质体RNA负载慢性粒细胞白血病来源树突状细胞的性能比较[J]. 肿瘤防治研究, 2010, 37(01): 47-51. DOI: 10.3971/j.issn.1000-8578.2010.01.013
Anti-Chronic Myelocytic Leukemia Induced by Dentritic Cells Transfected with RNA-lipofectamin[J]. Cancer Research on Prevention and Treatment, 2010, 37(01): 47-51. DOI: 10.3971/j.issn.1000-8578.2010.01.013
Citation: Anti-Chronic Myelocytic Leukemia Induced by Dentritic Cells Transfected with RNA-lipofectamin[J]. Cancer Research on Prevention and Treatment, 2010, 37(01): 47-51. DOI: 10.3971/j.issn.1000-8578.2010.01.013

脂质体RNA负载慢性粒细胞白血病来源树突状细胞的性能比较

Anti-Chronic Myelocytic Leukemia Induced by Dentritic Cells Transfected with RNA-lipofectamin

  • 摘要: 目的 研究人慢性粒细胞白血病(CML)总RNA经过脂质体负载后体外转染自体慢性粒细胞白血病-树突状细胞(CML-DC),并诱导特异性细胞毒T淋巴细胞(CTL)免疫反应。 方法 CML患者骨髓单个核细胞(CML-BMMNC)在rhIL-4、rhGM-CSF、rhTNF-α细胞因子联合培养诱导出CML-DC。采用Trizol法提取CML-BMMNC的总RNA;反复冻融法制备CML-BMMNC抗原。于CML-DC培养第5天,加入脂质体转染的CML总RNA、CML总RNA、CML肿瘤冻融抗原及不加抗原。倒置显微镜观察DC 形态学变化;流式细胞仪器检测免疫表型变化;染色体G显带技术检测其染色体核型及逆转录聚合酶链反应(RT-PCR)检测Bcr-abl融合基因的表达;用MTT法检测CTL作用。 结果 CML BMMNC诱导成CML-DC, CD、CD83表型表达较诱导前明显上调,形态学有典型的DC形态,且均存在Bcr-abl基因带和Ph1染色体,提示CML-DC为白血病源性。总RNA经脂质体转染CML-DC 、CML肿瘤冻融抗原负载CML-DC、总RNA不经脂质体转染CML-DC、未负载抗原CML-DC分别致敏的CTL及IL-2培养的T淋巴细胞在效:靶比为20∶1时的杀伤效率依次降低。 结论 CML BMMNC来源的 DC既具有CML白血病源性,又具有DC细胞的特性,能诱导特异性CTL杀伤作用;总RNA经脂质体转染的CML-DC诱导的CTL杀伤性对CML细胞的杀伤作用最强。

     

    Abstract: Objective To investigate the specific cytotoxic T lymphocyte(CTL) immunoreaction of choronic myelocytic leukemia-dendritic cells(CML-DC) transfected with CML total RNA in vitro, and to provide a theoretic basis of CML vaccine in clinical study. Methods Bone marrow mononuclear cells (BMMNCs)from 10 cases of chronic myeloid leukemia were used. BMMNCs were incubated with recombined human interleukin-4 (rhIL-4), recombined with human granulocyte/ macrophage colony-stimulating-factor(rhGM-CSF) and tumor necrosis factor–α(TNF-α).Using Trizol method was used to extract CML-BMMNCs total RNA, and CML-BMMNCs were applied to prepare tumor frozen-thawed antigen. At the 5th day of culture, CML-DCs were divided into four groups: CML-DCs pulsed with total RNA-lipofectamin; CML-DCs pulsed with total RNA; CML-DCs pulsed with tumor frozen-thawed antigen and CML-DCs pulsed with nothing. All of them could induce T cells into specific CTL. T cells cultured with IL-2 served as normal control group. The morphological features of DCs were observed under an inverted microscope. The CD and CD83 were analyzed by flow cytometry. Karyotypes of CML-DC were tested by G-banding technique. The Bcr-abl expression in CML-DCs was detected by reversed transcription polymerase chain reaction (RT-PCR). MTT assay was used to test the capacity of CML-DCs for mixed leukocyte reaction(MLR). Results CML-BMMNCs could be induced into CML-DCs. The expression levels of CD and CD83 in CML-DCs were increased obviously as compared with those in uncultured CML-BMMNCs. Bcr-abl and Ph chromosome all exsisted in cultured CML-DCs and uncultured CML-BMMNCs. It was suggested that CML-DCs were from CML cells. The cytotoxic rate of CML-DCs pulsed with total RNA-lipofectamin, CML-DCs pulsed with total RNA, CML-DCs pulsed with tumor frozen-thawed antigen, CML-DCs pulsed with nothing, and T cells cultured with IL-2 was decreased in turn. Conclusion CML-BMMNCs-derived DCs Being incubated recombined with IL-4,GM-CSF and TNF-α, CML-BMMNCs could be induced into CML-DC. CD and CD83 pressions all increased obviously. The CML-DCs could induce the CTL to get specific anti-tumor activity in vitroThe cytotoxicity of CTL induced by CML-DCs pulsed with total RNA-lipofectamin was the most significant.

     

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