CAJ | 학술논문

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。
목적：체외유도화감정만성립세포백혈병-수돌상세포(CML-DC)，병탐토인만성립세포백혈병(CML)총RNA체외전염대기개도적특이성세포독T림파세포(CTL)대만성립세포백혈병세포살상작용적영향。방법분리14례CML환자골수단개핵세포，가입rhIL-4、rhGM-CSF、rhTNF-α유도배양CML-DC。분별우배양제1、3、6、14d용도치현미경진행형태학관찰，류식세포술검측면역학표형，염색체G현대기술검측기염색체핵형，역전록취합매련반응(RT-PCR)검측bcr-abl융합기인。병우CML-DC배양제5d，가입CML세포총RNA지질체전염，혹라총RNA전염，혹불가임하시제계속배양적DC，공삼충CML-DC，분별치민T림파세포，령설이IL-2배양적T림파세포위대조조，비교불동조별적치민T림파세포적살상활성。결과 CML 골수단개핵세포유도전CD1α、CD83표체균재5％이하。이유도성숙적CML-DC세포CD1a、CD83양성표체솔분별위20.13±3.43％、26.76±2.79％，교유도전균명현증고(P<0.01)。CML-DC세포균존재Ph1염색체，병표체bcr-abl융합기인。총RNA지질체전염CML-DC、라총RNA전염CML-DC、단순CML-DC분별치민적T림파세포재효：파비위20：1시대CML단개핵세포적살상효솔분별위75.33±3.11％、37.23±2.92％、29.62±1.61％，균명현고우대조조(9.87±3.43％，P<0.01)。총RNA지질체전염적CML-DC소유도적CTL살상활성최강，여후삼조살상활성균유현저성차이(P<0.001)。결론 CML-DC기구유CML백혈병원성，우구유DC세포적특성，병능유도특이성CTL살상백혈병세포。경지질체전염CML세포총RNA가이제고CML-DC개도적CTL특이성살상만성립세포백혈병세포。
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-α. The morphologic features were observed by inverted microscope at day 1, 3, 8 and 14. CD83 and CD1a 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 cells 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 CD1a 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 CD1a and CD83 in CML-DCs were 20.13 ±3.43% and 26.76 ±2.79%, respectively, higher than the uncultured cells (P<0.01). 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 cells 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 response activated by CML-DC transfected with CML cells total RNA using liposomal was the most active response.