论文标题:超抗原联合树突状细胞诱导抗肺癌免疫反应的实验研究
The Experiment Research of Anti-lung Cancer Immune Response Induced by Superantigen Pulsed Dendritic Cells
论文作者
论文导师 杨达宽；洪志鹏,论文学位 博士,论文专业 外科学
论文单位 昆明医学院,点击次数 87,论文页数 115页File Size9410K
2007-05-01论文网 http://www.lw23.com/lunwen_235093767/
Dendritic cell; High agglomerative;Staphylococcin (HAS); Lung cancer;Immunotherapy
目前，以LAK细胞、TIL、CIK、CD3AK等为代表的继承性细胞免疫治疗是肿瘤免疫治疗的主要方法，对肿瘤有一定的疗效，已成功的应用于某些肿瘤临床治疗。这几种方法均属于非主动性的细胞免疫治疗方法。动物实验和临床经验证明，自身的免疫系统虽然不能根除肿瘤，但却可以识别和杀伤具有抗原性的肿瘤细胞。近年的研究表明，机体之所以缺乏强有力的抗肿瘤免疫应答，一个可能的原因是肿瘤细胞的抗原性弱，而且患者抗原提呈细胞的功能低下，使肿瘤抗原不能有效的提呈给淋巴细胞。树突状细胞(dendritic cell，DC)是目前已知的体内抗原提呈功能最强的专职抗原提呈细胞(APC)，是免疫反应的启动因子和调节因子，是T、B淋巴细胞的有效刺激因子。所以，以DC为基础的主动特异性肿瘤免疫治疗逐渐成为肿瘤免疫治疗的主要研究方向。将DC体外培养扩增，并用肿瘤抗原致敏，然后回输到体内，也就是说将DC作为一种细胞型免疫佐剂用于制备肿瘤疫苗，是其中的一个基本研究思路。高聚金萄素(HAS)是一种超抗原抗癌生物制剂，具有刺激T细胞增殖，增强免疫功能等生物学活性。 我们建立了体外培养扩增DC的方法，并将其与超抗原HAS活化的淋巴细胞结合起来，诱导产生了高效特异性的抗肿瘤免疫应答能力。主要研究内容有3部分。 1、树突状细胞的体外培养和鉴定 常规分离外周血单个核细胞(PBMC)，用含10％小牛血清的RPM1 1640悬浮，细胞加入6孔板，贴壁细胞加完全1640培养液，用GM-CSF及IL-4联合刺激诱导DC分化，再用TNF-α和PGE_2促进DC成熟。DC培养至第10天，收集细胞，直接或间接免疫荧光染色后，用流式细胞仪分析。经此方法培养的细胞，相差显微镜下可看到细胞表面有大量毛刺状突起，CD1a的阳性率71％，CD86为64.43％，CD14阳性率低于10％，并高表达HLA-Ⅰ、HLA-Ⅱ分子和CD45，为典型的DC形态和表型特征。同时用原子力显微镜对树突状细胞表面结构进行观察，得到了清晰的树突状细胞的AFM图像，树突状细胞表面的树突状纤维呈山脊状，较为扁平，走行弯曲或呈轻度迂曲状，自细胞伪足部细胞膜发出，并可在细胞体上观察到少量的深度达到孔洞周边总厚度1／3致1／2，直径2um左右的“孔洞”样细胞膜凹陷。激光共聚焦显微镜对树突状细胞骨架的观察则显示树突状细胞内的F-actin结构清晰，荧光强度较强，在胞浆内纤维较粗大，主要分布在细胞膜附近和细胞质中，较集中地分布于细胞核周围，呈现沿细胞长轴直线走行的现象。对树突状细胞的超微结构进行透射电镜形态学观察，则见细胞表面粗糙，有大量皱褶和不规则突起，还可见伸展的突起，胞浆中富含线粒体，较少溶酶体，为典型的树突状细胞超微结构。同种异体混合淋巴细胞反应则提示少量DC即可强烈激发同种T淋巴细胞的增殖。肺癌患者和健康对照者DC于收获日(7d)对照分析其表型特点无差异。 2、效应细胞的培养及体外细胞毒实验 PBMC中的非贴壁细胞，调浓度至1×10~8／ml，加HAS培养，每4天换液1次，即为HASL。DC用GLC-82细胞粗提抗原致敏。第7天，将致敏DC与经或未经HAS刺激的淋巴细胞混合培养，分别称混合培养细胞为DC-HASL和DC-L。分别以HASL、DC-HASL和DC-L为效应细胞，以肺癌细胞A549、GLC-82、大肠癌细胞moser和乳腺癌细胞MCF-7为靶细胞，以效靶比为60∶1放入96孔板中。用MTT实验测定效应细胞对各肿瘤细胞的杀伤作用。结果，DC-HASL对GLC-82细胞具有高效而特异性的杀伤作用(杀伤率100％)；对另一肺癌细胞A549细胞的杀伤率也达94.2％；对moser和MCF-7的杀伤率分别为73.5％和71.2％。而HASL和DC-L对A549、GLC-82、moser和MCF-7细胞的杀伤率则分别为48％、63％、60％、45％和42.1％、46.4％、39％、25.6％。 3、裸鼠肺癌模型的建立和肿瘤生长抑制实验 裸鼠股部皮下接种GLC-82细胞5×10~5个，随机分为4组：(1)生理盐水对照组；(2)DC-HASL组；(3)DC-L组；(4)HASL组。分别于接种瘤的第4，10，15日向肿瘤局部注射效应细胞，对照组注射生理盐水。至第20日结束实验，测量各组肿瘤体积并杀鼠取瘤。肿瘤局部大体观察可见，治疗组瘤块较小、较光滑、较局限，对照组肿瘤块较大，形状不规则。DC-HASL治疗组竟有一只裸鼠未见肿瘤块。对照组、DC-HASL治疗组、DC-L组和HASL组的肿瘤体积分别为298.40±77.58、18.5±14.2、43.15±19.44和70.67±70.51 mm~3。与对照组相比，三种治疗组肿瘤抑制率分别达93.8％、85.5％和76.3％。免疫组织化学C-erbB-2癌蛋白的表达结果显示，三组治疗组的蛋白表达指数均较对照组明显降低(P＜0.05)，且以DC-HASL治疗效果明显。应用流式细胞技术表明三个治疗组GLC-82细胞均有明显的G_0／G_1期阻滞效应和凋亡峰出现。 研究结果表明，HAS在体外可维持淋巴细胞的活性并使其获得一定程度的增殖，而且外周血淋巴细胞经HAS刺激后，具有一定的杀伤肿瘤的作用。将DC与HASL结合可诱导产生高效特异性的抗肿瘤免疫活性细胞，为利用同种异体效应细胞进行肿瘤免疫治疗提供了依据。 本研究首次将DC介导的主动特异性细胞疗法与超抗原活化的淋巴细胞结合，在体外实验及裸鼠移植瘤生长抑制实验中获得非常令人振奋的结果。所以，将DC介导的主动特异性细胞疗法和非特异性免疫疗法相结合是一种非常有应用前景的新的肿瘤免疫治疗方法。
There are many outstanding issues in cancer immunotherapy. LAK, TIL, CDC, et al are all adoptive immunotherapy and have certain effect. Dendritic cells (DC) are considered the most potent antigen-presenting cells (APC) and are thus promising new tools for the immunotherapy of cancer. DC can stimulate the primary activation of T cells due to their enhanced capacity of presenting immunogenic peptides in association with self-major histocompatibility complex I and II molecules, DC can also process both exogenous proteins and intracellular protein to T cells and can directly modulate B - cell growth and differentiation. To use DC as "nature"s adjuvants" for the potentation of immune reactivity against tumors, these cells are modified by pulsing them with tumor lysates, tumor proteins, tumor peptides, or are transfected with cDNA encoding tumor antigens. Various studies in animal models have clearly shown that DC pulsed with tumor antigens in vitro and then re-injected in vivo induce immune responses that lead either to protection against lethal tumor challenge or to regression of established tumors. Clinical trials with modified DC have confirmed the value of this approach. This might hold true especially in the treatment of minimal residual disease after therapy for primary tumors. We obtain DC from PBMC stimulated by GM-CSF, IL-4, TNF-αand PGE_2 in vitro. High potent and specific anti-tumor effect is induced using DC and high agglomerative staphylococcin (HAS), a kind of superantigen. 1. In vitro generation of dendritic cells PBMC were isolated from leukocyte-enriched buffy coats by standard density gradient centrifugation on Ficoll-Paque, resuspended in complete RPMI 1640 medium. PBMC were allowed to adhere in cell culture flasks. Nonadherent cells were removed and adherent cells were cultured in medium containing GM-CSF and IL-4. After 5-6 day of culture, cells were washed and recultured in medium containing TNF-αand PGE2. Cells were analyzed for surface antigen by FACS. The cultured cells exhibit the typical morphological features of DC and express high levels of HLA - I, HLA - II and CD45 molecules. Positive rates of CD1a and CD86 are 71% and 64.43%, respectively. 2. Effector cells culture and cytotoxicity assay in vitro Nonadherent PBMC were resuspended in complete medium supplemented with HAS and cultured at 37℃and 5% CO_2(named HASL). Culture medium were exchanged every 4 days. GLC-82 antigen pulsed DC and HASL were cocultured and the obtained cells were named DC-HASL. DC-L was DC concultured with lymphocytes. Cytotoxicity of HASL, DC - L and DC - HASL were determined by MTT assay. In brief, effector cells (HASL, DC - L, DC - HASL) and target cells (GLC - 82, A549, moser and MCF—7) were seeded in 96 - well microtiter plates at an appropriate ratio. Cytotoxic rates of DC - HASL to GLC - 82 cells reached 100% and the cytotoxic rates to other cancer cell lines A549, moser and MCF - 7 were 94.2%, 73.5% and 71.2%, respectively; Cytotoxic rates of HASL and DC - L to the above four cell lines were 48%, 63%, 60%, 45% and 42.1%,46.4% , 39%, 25.6%, respectively. 3. Growth inhibition test of lung cancer nude mice model Every nude mouse was inoculated with 5×10~5 GLC - 82 cells at thigh. All mice were divided into 4 groups:①control group,②DC-HASL therapy group,③DC-L therapy group,④HASL therapy group. Nude mice in 3 therapy groups were injected 1×10~7 effector cells on day 4,10,15 after tumor inoculation, respectiverly. Nude mice were killed on day 20 and tumor tissues were resected. The tumor volume of four groups were 298.40 + 77.58, 18.5 + 14.2, 43.15 + 19.44 and 70.67 + 70.51 mm3, respectively. Tumor inhibition rates of 3 therapy groups compared with control group is 93.8%, 85.5% and 76.3%, respectively. Our research showed lymphocyte can proliferate to a certain extent stimulated by HAS in vitro and HASL can lyse tumor cells. High potent and specific antitumor immune response can be induced when combining DC with HASL. We combine DC mediated tumor active immunotherapy with super antigen mediated immunotherapy in our research for the first time. The results are very exhilarating. So it may become a new promising scheme for tumor immunotherapy.
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