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

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,CD1α、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。

Objective To investigate the specific cytotoxic T lymphocyte（CTL） irnmunoreaction of choronic myeloeytie leukemia-dendritic cells（CMLq3C） transfected with CIVIL total RNA in vitro, and to provide a theoretic basis of CML vaccine in clinical study. Metheds 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 micmscope. The CD1a and CD83 were analyzed by flow eytometry. 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, a and CD83 in CML-DCs were increased obviously as com- pared 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-IX2s were from CML cells. The cytotoxie 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 could induce the CTL to get specific anti-tumor activity in vitro. The eytotoxieity of CTL induced by CML-DCs pulsed with total RNA-lipofectamin was the most significant.