论文标题:树突状细胞的生物学特性及其抗肿瘤作用的实验与临床研究
Experimental and Clinical Study on the Biologic Characteristic of Dendritic Cells and Anti-Tumor Immune Efficacy
论文作者 邬国斌
论文导师 梁安民,论文学位 硕士,论文专业 肿瘤外科学
论文单位 广西医科大学,点击次数 182,论文页数 56页File Size594k
2004-05-01论文网 http://www.lw23.com/lunwen_759962/ 树突状细胞;抗原呈递;细胞免疫;诱导;激活
Dendritic cells; Antigen presentation; Cellular immunity;Induction; Activation
目的：体外诱导培养 DC，探讨 DC 的生物学特性及其抗肿瘤作用。 方法：从人外周全血中分离单个核细胞，用人重组粒细胞／巨噬细胞集落刺激因子（rhGM-CSF）和人重组白细胞介素 4（rhIL-4）诱导扩增 DC。在双相倒置显微镜及电镜下观察 DC 的形态结构，用流式细胞仪检测 DC 细胞表型。体外细胞实验 1：分别以 DC 及巨噬细胞（Mφ）作为刺激细胞，诱导激发混合淋巴细胞反应（MLR），比较两者诱导激发 MLR 的能力。体外细胞实验 2：DC 分别与 BEL-7402 和 Reh 在 20:1、10:1、5:1 三种效靶比下与效应细胞共同培养 18h，检测 DC 在不同效靶比下对两种癌细胞株的杀伤率。体外细胞实验 3：两组效应细胞 CD3AK 细胞+DC 和 CD3AK 细胞分别与 BEL-7402均采用 20：1 与 10：l 两种效靶比共同培养，检测 CD3AK 细胞对 BEL-7402肝癌细胞株的杀伤率。动物实验 1：从 C57BL／6 小鼠骨髓中分离培养 DC 并用 B16 肿瘤抗原肽冲击致敏。取正常 C57BL／6 小鼠 30 只，随机分 3 组分别用抗原肽刺激的 DC、DC 或 HBSS 平衡盐溶液进行小鼠体内免疫。先取每组两只小鼠的脾脏 T 淋巴细胞在体外诱导 CTL 活性，比较每组 CTL 杀伤活性。动物实验 2：用三组余下的小鼠分别皮下接种活的 B16 黑色素瘤细胞，比较观察每组瘤体的生长情况。动物实验 3：制备用 3LL 肿瘤抗原肽冲击致敏的小鼠骨髓 DC。正常 C57BL／6 小鼠 24 只，接种 3LL 细胞均长出肺转移瘤。 2随机分三组分别用经 3LL 肿瘤抗原肽冲击致敏的 DC、DC 或 HBSS 平衡盐溶液治疗，35 天后比较每组小鼠肺重量并计数肺内肿瘤结节。 结果：在双相倒置显微镜观察培养第 7d 的悬浮细胞大部分具有典型 DC形态特征，细胞表面粗糙，有大量皱折和不规则突起。电镜下观察 DC 细胞胞膜伸出长短不一的突起，细胞核染色较深，形态不规则，多偏于一端，胞浆内富含线粒体，较少溶酶体。由 2.9×105DC 前体扩增至 7.1×106 成熟 DC，扩增了 24.5 倍，培养的第 7~15d 增殖最明显，增殖 24.5~37.5 倍。培养至第 15d，DC 纯度达 80%以上。流式细胞仪检测 DC 中度表达 CD1a，高表达 CD86、CD40、HLA-DR 等相对特异性标志。体外细胞实验 1：DC 具有较 Mφ更强的激发 MLR 的能力，二者有显著性差异（P<0.01）。体外细胞实验 2：DC 在20∶1、10∶1、5∶1 三种效靶比下对 BEL-7402 细胞和 Reh 细胞均无明显的直接杀伤作用。体外细胞实验 3：CD3AK+DC 实验组 20∶1 与 10∶l 两种效靶比下对 BEL-7402 细胞的杀伤率显著高于 CD3AK 对照组（P<0.01）。动物实验 1：B16 细胞肿瘤抗原肽致敏的骨髓 DC 比未经致敏的 DC 更有效诱导特异性 CTL 细胞毒活性（P<0.01）。HBSS 处理组不能诱导 CTL 细胞毒活性。动物实验 2：B-16 抗原肽刺激的 DC 免疫的小鼠接种 9 天后均未长出肿瘤，未致敏 DC 免疫小鼠能有效抵抗 B16 肿瘤的生长，但不如肿瘤抗原肽刺激的 DC更有效，二者比较有显著意义（P<0.01）。而 HBSS 对照组所有小鼠均长出肿瘤。动物实验 3：HBSS 对照组小鼠的肺脏重约为 600 mg，肺内肿瘤结节数86.3±21.7，并见多个结节连成块状。未致敏 DC 治疗组小鼠的肺脏重 423mg，肺内肿瘤结节数 67.7±16.1（与 HBSS 对照组比较 P＜0.05）；抗原肽刺激的DC 治疗组肺重平均约为 312mg，肺内肿瘤结节数 23.6±8.7（与 DC 治疗组 3比较 P＜0.01）。结论：1、可从人外周全血中分离单个核细胞，用 rhGM-CSF 和 rhIL-4 联合诱导扩增生成大量 DC。2 DC 具有较 Mφ更强的激发 MLR 的能力，并可辅助增强 CD3AK 细胞的抗肿瘤活性。3、体外培养细胞实验证明 DC 对肿瘤细胞无明显的直接杀伤作用。4、动物实验提示抗原肽刺激的 DC 具有很强的免疫保护作用，能明显增强小鼠体内免疫细胞（如 CTL）抵抗肿瘤发生、生长的过程。5、DC 是一类作用很强的抗原呈递细胞，能呈递肿瘤抗原物质，增强体内、外免疫效应细胞的杀灭肿瘤细胞作用。
Objective: To induce and cultivate DC in vitro and investigate the biologiccharacteristic of DC and anti-tumor immune efficacy. Methods: The mononuclear cells isolated from human peripheral blood werecultured with the supplement of rhGM-CSF and rhIL-4 to generate DC. Themorphological characteristics of those cells were observed and the phenotypefeatures and the nucleio-type were analysed. The first experiment in vitro: DC andMacrophage (Mφ)acted as stimulating cells to activate mixed lymphocyte reaction(MLR) respectively, then observed their ability of activating MLR. The secondexperiment in vitro: DC acted as effector cells had co-cultured with BEL-7402 andReh acted as target cells on three kinds of ratio (20∶1; 10∶1; 5∶1) respectivelyfor 18 hours, to detect the DC killing activity to BEL-7402 cells and Reh cells. Thethird experiment in vitro: DCs were pulsed with BEL-7402 tumor cells antigen.The killing activity on BEL-7402 hepatocarcinoma cells of CD3AK cellscombined with the tumor antigen-pulsed DC was measured in vitro using thelactate dehydrogenase (LDH) release assay. The first animal experiment: DC wereisolated and cultured from C57BL／6 mouse’s marrow and were stimulated by 7impacting B16 tumor antigen. 30 C57BL／6 mouse were divided into three groupsrandomly. DC stimulated by B16 tumor antigen, DC not stimulated by tumorantigen and HBSS acted as comparison were immunized three-group mouserespectively, then compared with the killing activity of CTL that induced formthree groups mouse’s spleen T lymphocytes in vitro which obtained from twomouse’s spleen of each group. The second animal experiment: Three-group othermouse were inoculated B16 cells respectively and observed the growth of tumor.The third animal experiment: DC were isolated and cultured from C57BL／6mouse marrow and were stimulated by impacting 3LL tumor antigen. 24 C57BL／6 mouse which were inoculated by 3LL cells and all had come out pulmonarymetastasis were divided to three groups randomly. Three-group mouse were treatedwith DC stimulated by 3LL tumor antigen, DC not stimulated by tumor antigenand HBSS treated respectively, then weighed and counted their lung metastasistumors. Results: We could observe a lot of suspension cells with the typical DCmorphologic characteristics, which observed cell surface being showed rough andwrinkle by inverted microscope and observed cell membrane stretching outdifferent-length apophysis, irregularity cellular nucleus shape, deep karyotin, morechondrosome and less lysosome in intracytoplasm by electron microscope. DCpresented moderately mark such as CD1 and presented highly mark such as CD86,CD40, HLA-DR by flow cytometry. The first experiments in vitro: DC was moreeffective on activating mixed lymphocyte reaction (MLR) than Mφ(P<0.01). The 8second experiments in vitro: DC was not able to kill BEL7402 cells and Reh cellsdirectly on three kind of ratios (20∶1; 10∶1; 5∶1). The third experiments invitro: CD3AK cells combined with DC were more effective on killing BEL-7402cells than CD3AK cells without DC(P<0.01) on two kind of ratios((20∶1; 10∶l).The fist animal experiment: the killing activity of CTL induced by DC whichstimulated by B16 tumor antigen was more effective on killing B16 cells than thatof DC which wasn’t stimulated by B16 tumor antigen(P<0.01) and the CTLdisposed by HBSS could not kill B16 cells. The second animal experiments: Thegroup of mice immunized by DC pulsing with B-16 tumor antigen hadn’t tumorgrowth after injecting B16 cells on hypodermis for 9 days. The group of miceimmunized by DC not pulsing with B-16 tumor antigen could resist the B-16tumor growth but less effective than the group immunized DC pulsing with B-16tumor antigen(P<0.01). The group treated with HBSS all had grown tumor. Thethird animal experiments: Compared the weight and the quantities of lungmetast
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