人急性髓系白血病HL-60来源的树突样细胞诱导的CTL体内外作用研究

【作者】 周剑芳；

【导师】 杨锡强；

【作者基本信息】 重庆医科大学 ， 内科学， 2001， 博士

【摘要】 白血病是儿童最常见的恶性肿瘤，常用治疗措施包括化疗及放疗，可获得临床缓解，但易复发，再次化疗反应较差。残存微小病灶及患者免疫力降低是白血病复发的主要原因。血液肿瘤与抗原提呈细胞同源于造血干细胞，却具有分化障碍、无限增殖、低抗原提呈状态的特点。为探讨免疫支持治疗在抗白血病复发及缓解后治疗中的可行性，本研究采用PKC激活剂佛波酯（PMA）及能增强抗原提呈细胞免疫活性的细胞因子（GM-CSF、IL-4、TNF-α）在体外诱导培养HL-60细胞，获得形态、细胞表型与树突状细胞（dendritic cell）相近，即髓系白血病HL-60来源的树突样细胞（myeloid leukemia-derived DC-like Cells，HL-60DC），观察其对细胞毒性T淋巴细胞（cytotoxic T lymphocyte，CTL）体外诱导作用，并进一步探讨急性髓系血液肿瘤向树突样细胞分化调节的胞内信号转导机制。同时为更真实模拟临床血液肿瘤演进过程，选用SCID小鼠在放射处理和不经放射处理后以静脉及腹腔两种途径荷瘤，建立人白血病HL-60/SCID小鼠异种移植模型。结合血液肿瘤HL-60在这种异种移植模型成瘤和生长特点，观察人急性髓系白血病HL-60来源的树突样细胞诱导的CTL的体内抗瘤作用。主要结果及结论如下： 1．HL-60细胞呈球形、大小不一、悬浮生长。在细胞因子GM-CSF+IL-4+TNF-α诱导下向DC样细胞分化，在诱导后约一周呈现不规则形状、非贴壁生长，细胞表面分化标志CD86、CDla在第13天出现弱阳性表达，诱导15-16天后，CD86、CDla表达增加，分别约为34％和14％，但CD80 重庆医科大学博士研究主论文 中、英文摘要 仍为阴性。联合 PMA，调整应用次序诱导，作用更明显，在诱导 7刁 天，CD阳性率约为 20％，CD86、CD80约为 72％、19％。诱导分化 过程中，长时间低剂量TNF－仁的诱导作用强于短时高剂量诱导。 2．分化过程中PMA，TNF－口抑制HL十0的增殖，并启动凋亡，GM CSF＋TNF－a＋IL－4也能诱导 HL－60凋亡及坏死。 3．以PMA及GM－CSF、TNF－口诱导获得的HL－60DC，在体外有诱导产 生具有较高杀伤活性的CTL细胞，其杀伤效应明显高于灭活的HL千0肿 瘤细胞所诱导的 CTL的溶瘤作用，最佳效靶在 10：1以上。实验中观察 到CTL粘附于靶细胞，受损伤的肿瘤细胞胞膜内陷，胞核出现空泡等征 象。 4．未观察到细胞因子、PMA短时间作用的髓系血液肿瘤HL60胞内 钙明显变化，联合细胞因子及 PMA诱导 3天后，HL千0细胞内的＊a‘＊ i明 显降低。 5．在未束激的讥－60细胞中未检狈到 ReV呷－。B亚单位Re1B。RNA 表达，经PMA、GM｛SF汀NF－口诱导后表达，在诱导后期表达又减弱。 6．经 GM－CSF＋TNF－a－PMA作用，HL－60细胞中他／NF－。B亚单位 P65蛋白表达增强，并发生核易位，并在诱导过程中稳定表达，而受诱 导的细胞其凋亡不可逆，持续发生。 7．在严重联合免疫缺陷小鼠体内，通过腹腔或静脉接种人急性髓系 白血病HL乃0细胞建立异种移植模型。接种的HL亿0细胞在放射线处理 或未处理的SCID小鼠体内均能生长，发生全身性白血病，3周时外周血 即可见白血病细胞浸润。放射线处理后给瘤的小鼠病情变化较急速，存 活期约40天。静脉接种SX106HL干0细胞川。鼠，受累脾脏增生期细胞 比例增加。腹腔接种瘤细胞IX106、 ＊小鼠，受累肝、脾增生期细 胞比例增加。骨髓CD33＋细胞率与肝、脾增生期细胞比例间有明显正相 关，说明这三项指标均可作为该模型小鼠病情观察指标。 5 重庆医科大学博士研究生论文 中、英文摘要 8．以腹腔接种瘤细胞 IX106l ＊小鼠，病程约 50天，骨髓细胞CD33 阳性率差异不大，说明采用腹腔接种方式，接种瘤细胞量（IX10刁 ＊ 小鼠）对病情、病程影响不明显。 9．腹腔荷瘤 IXI 06川鼠，腹腔回输 HL乃0DC活化的 CTL乃：1效 ． 靶比）治疗，治疗组小鼠对治疗反应有差别，治疗组 4飓小鼠存活。 这些差异可能同治疗小鼠个体、回输治疗细胞状态等因素有关。治疗组 小鼠存活时间明显延长、瘤块重量、肝、脾、骨髓受肿瘤浸润程度明显 低于未?更多还原

【Abstract】 Leukemia is a lethal disease to children. Standard treatments for leukemia ,such as chemotherapy and radiation, often achieve a clinical remission ,but it is frequent recurrence and second-line chemotherapies have a poor response rate. The leukemic cell minimal residue and host immunocompromise are mainly the causes of relapse. Because leukemie cells share the ontogeny with professional antigen presenting cells (APC), enhancing the capacity of leukemic cells to present endogenously expressed tumor-associated antigen directly to T cells has recently been intensive focus. Evidences suggest specific cytokines or other agents can enhance APC func-t ion. To clarify the feasibility of DC-based immunotherapy in leukemia, we therefore investigated the biological characteristic of human acute promyelocytic cell line HL-60 responding to protein kinase C activator-phorbol ester(PMA), granulocyte- macrophage colony stimulating factor(GM-C SF), tumor necrosis factor- alpha (TNF- a ) and intcrleukin-4QL-4}.The inyeloid leukemia-derived dendritic-like cells were induced by these factors. Phenotypic DC characteristics of HL-60 dendritic-like cell (HL-6ODC) include morphologic feature, surface antigens CD1a+/CD86+/CD8O+ and RelB expression as well. The anti-tumor effect of cytotoxic T lymphocyte (CTL) induced by HL-6ODC was tested in vitro and in HL-60/SCID mouse xenograft model. Main results and conclusions were as follows. 1. HL-60 shows a homogeneous population of different size, round, suspension growing cells. About one week after culturing with GM-6 CSF , TNF-6Q and IL-4, some fractions appeared to be morphological manifestations of dendritic cell. The surface antigens CD1a and co stimulatory molecule CD86 expressed on the 13th day. On the 15th or 16th day, CD1a and CD86 expression on the co-cultured HL-60 were up to about 14% and 34% respectively, but not CD8O expression. Combined PMA and GM-CSF plus TNF-6Q , the development of HL-60 DC was more pronounced than those without PMA treated. CD1a reached about 20 percent. CD86 and CD8O was about 72% and 19% correspondingly on culture day 7-61. 2.HL-60 proliferation was suppressed and apoptosis was triggered by PMA and TNF-6a. 3.The specific tumor-lyses capability in vitro of CTL activated by HL-6ODC induced by PMA and GM-CSF plus TNF-6a increased significantly. The effective ratio of effectors to target cells is above 10:1.And the vacuolation in nucleus or indentation of cell membrane was found in the injured HL-60 cells. 4.The level of [Ca2+]i decreased significantly in differentiated HL-60 treated with GM-CSF plus TNF-6a then PMA for 3 days. PMA and the tested cytokines alone show no obvious effect on [Ca2+]i in short time. 5. Re1B mRNA was not detectable in HL-60 and could be induced by the stimulations including cytokines as well as PMA and GM-6 CSF plus TNF-6a. Combined PMA with GM-CSF plus TNF, the expression of Re1B mRNA increased and then declined. 6. Constitutive expression of p65, a subunit of Rel/NF-6-6B, was found in HL-60 cells which was up-regulated by PMA within 24h or GM-CSF plus TNF then PMA within 72h , plateaued through about one更多还原