【作者】 王彦刈；

【导师】 郑筱祥；

【作者基本信息】 浙江大学 ， 生物医学工程， 2002， 博士

【摘要】 机体的免疫系统与肿瘤的相互作用是一个多层次、多种物质参与的此消彼长的过程。在正常情况下，机体有强大的免疫系统，能识别和排除肿瘤细胞，但临床上肿瘤病人确屡见不鲜，肿瘤细胞可不停地在体内生长，最后杀死宿主。一般认为这是肿瘤细胞逃避了机体免疫监视。肿瘤逃避免疫监视的机制非常复杂，但总体上可从宿主免疫状态及肿瘤细胞等方面来解释，在宿主免疫状态低下时，肿瘤细胞可以被动地逃避免疫监视，但是肿瘤细胞也能主动地释放一些活性物质，下调免疫杀瘤作用，该作用主要通过两个途径，即对免疫细胞功能的调节和免疫细胞数量的调节。本课题的主要目的是：在体外模拟体内肿瘤的微环境，观察免疫细胞的细胞增殖、凋亡、活性、细胞因子及NO的变化及作用，明确肿瘤对免疫系统的作用及机制。 1．基于目前测定免疫细胞增殖杀伤的方法的局限性，本研究首先建立一种用流式细胞仪测定该指标的新方法。通过对该方法的验证，证明该方法具有：a，不受所测细胞代谢状态的影响：b，在细胞介导的细胞毒性试验中，能区分靶细胞和效应细胞；c，操作步骤少并无放射性；d，具有高灵敏度和可信性。 2．结合以上创建的方法和免疫荧光流式细胞术，用K562细胞株可溶性分泌物(上清)对外周血单个核细胞(PBMC)进行培养以模拟体内微环境，然后分别从细胞增殖、凋亡、坏死、细胞周期、活性、细胞因子和表面抗原表达等方面进行研究，结果发现用肿瘤上清培养的PBMC细胞数量下降明显，但同时对其有激活作用，且呈剂量依赖性；细胞数的下降主要是由细胞坏死和凋亡引起的，肿瘤上清对细胞周期没有阻断作用，反而略有促进作用；T细胞亚群比例增加，并促进表达Th1、Th2细胞因子。这说明K562细胞主要是通过使免疫细胞减少来逃避免疫监视的，这为抗肿瘤研究提供了基础。 3．利用DAF-2DA作为细胞内NO特异性的荧光探针，研究了K562上清对PBMC产生NO的影响，国内未见类似报导，发现肿瘤上清能促进PBMC细胞NO的产生，进一步发现活化的细胞(CD69+)均产生NO，而NO阳性的细胞不一定活化的。说明NO在肿瘤对PBMC的作用中起到一定的作用。 4．利用NO合酶抑制剂L-NAME和NO供体SNP，研究NO在K562上清对PBMC的作用中所发挥的作用。0刀smM的SWh有活化PBMC的作用，大于0．SInM的SNP会大量诱导PBMC的坏死，但对细胞因子、细胞周期、细胞亚群的比例均影响不大。这说明低剂量的NO是PBMC细胞生理活动所必须，而高浓度NO会损害PBMC细胞甚至导致死亡。在K562上清对PBMC作用中，产生的高浓度NO所起的作用也许仅仅是诱导了PBMC的坏死。 本研究结论是K562上清对免疫的抑制是通过减少免疫细胞而起作用的，其中NO起了较大的作用。本研究的创新点在于：l、建立了用流式细胞术定量测定细胞增殖和细胞毒性的直接检测技术；2．发现K562可溶性分泌物剂量依赖性地使PBMC细胞数量减少但活性增加；3、K562可溶性分泌物能诱导PBMC产生NO，但是NO的作用并不等同于肿瘤上清的全部作用。4、为抗肿瘤药物筛选提供了一个体外模型，并明确了一些筛选指标。更多还原

【Abstract】 The interaction between tumor cells and immune system is very complex. In normal situation,the immune system can recognize and eliminate the tumor cells. However,there are a lot of patients with tumor in clinic. The reason is the tumor cells escape the immunosurveillance whose mechanism is very complex. In general,the explanation is based on two aspects,one is that the tumor cells can passively escape the host immunosurveillance when the host immnue state is low and another is tumor cells can actively secrete factors to down-regulate immune function. The purpose of this paper is to study the mechanism of immunosuppression by tumor cells using a model rnimicing in vivo microinviroment.1, Due to the limitation of existing assay for evaluation of cell proliferation and cytotoxicity,in this study we developed a novel method. The characteristics of this method are:a,directly counting cell number without the influence of the metabolic state of the cells;b,discrimination of target cells from effector cells in cell-mediated cytotoxicity assay;c,less treatment step,and free-radioactivity;d,high sensitivity and reliability.2, Using the above assay,immunofluorescent labeled technique,and flow cytometry,the PBMC proliferation,apoptosis,necrosis,cell cycle,activation,cytokines and membrane marker were detected. The results showed that the number of PBMC reduced,but the activity of PBMC increased dose-dependently;the reduction of cell number resulted from necrosis and apoptosis;the supernatant of K562 cell lines were not able to block the cell cycle,but to promote it;the ratio of T cell subset and the expression of Thl and Th2 cytokines increased. These suggested the immunosuppression by K562 cell lines is mediated by reduction of the number of immunocytes.3, The effect of K562 cell lines supernatant on NO production by PBMC was investigated using the specific immunofluorescence probe DAF-2DA. The results showed that the K562 cell lines supernatant could promote the NO production byPBMC. Further study found all the activated PBMC(CD69+) produced NO,while not all the NO positive PBMC were activated. These suggested that NO plays a part role in the action of K562 cell lines supernatant to PBMC.4,The role of NO was investigated using NO inhibitor (L-NAME) and NO donor (SNP). O.OSmM of SNP could activate the PBMC,while above 0.5 mM of SNP could significantly induce PBMC necrosis,nevertheless,the higher concentration of SNP could not induce significant change of cytokine production,cell cycle,and ratio of PBMC subsets. These results indicated low concentration of NO is necessary for cell physiological activities,whereas high concentration of NO will impare cell physiological activities and even result in cell necrosis. The high concentration of NO production induced by K562 cell lines supernatant may only play the role of induction of PBMC necrosis.The conclusion of this study is that the immunosuppression by K562 cell lines supernatant is through reduction of the PBMC number. In the immunosuppression,NO plays a part but significant role. The originalities of this paper are:1,development of a a flow cytometry-based assay for quantitative analysis of cellular proliferation and cytotoxicity in vitro;2,the soluble secretion of K.562 cell lines reduce the number of PBMC,but promote the activity of PBMC in dose-dependent manner;3,soluble secretion of K562 cell lines can induce the NO production by PBMC,but NO only plays a part of the role of soluble secretion of K562 cell lines;4,establishing a in vitro model and giving some parameters for sreening and appraising anti-tumor medicine.更多还原