【作者】 程峰；

【导师】 王立新；

【作者基本信息】 东南大学 ， 临床检验诊断学， 2015， 硕士

【摘要】 细胞自噬作为一个进化上十分保守的物质降解过程,对于维持细胞代谢、能量循环、细胞器更新起到重要的调控作用。研究发现自噬参与了一系列生理及病理过程并且与很多人类疾病的发生和发展有关,尤其是白噬在肿瘤的发生和发展中起到重要作用。然而细胞内自噬水平并不是一成不变的,很多情况如轻度氧化应激和长时间营养剥夺会上调细胞内自噬水平,新近研究发现自噬活性在多种肿瘤细胞中发生了变化,从而使自噬在肿瘤方面的研究也成为国际肿瘤治疗的研究热点。本实验团队前期研究已经证实了肿瘤细胞来源的自噬小体中包含了多种损伤相关分子模式(DAMPs)分子如高迁移率族蛋白1(HMGB1)和热休克蛋白90(HSP90)。自噬小体中的这些模式子可以作为天然佐剂在免疫应答过程中发挥重要作用。众所周知,B细胞的主要功能是产生抗体、提呈抗原、分泌细胞因子。然而近年来研究发现,一种全新的B细胞亚群—调节性B细胞(Regulatory B cell, Breg)在一些感染性疾病、自身免疫性疾病和肿瘤的发生、发展和转归过程中发挥重要的免疫调节作用。Breg抑制免疫应答的方式有许多种,其中分泌IL-10是Breg介导免疫应答的主要方式,因此分泌IL-10也是Breg的主要特征。已有文献报道,Breg在肿瘤免疫中发挥了重要作用。一方面,Breg可以通过产生IL-10来抑制CD8+T细胞分泌IFN-γ,从而促进肿瘤的发展。另一方面,调节性B细胞也可以通过募集Treg来促进肿瘤细胞的肺转移。虽然Breg在肿瘤的发生发展中发挥重要的调节作用,然而对于Breg在荷瘤小鼠及肿瘤患者体内是如何产生的仍需要进一步的研究。本实验团队前期研究发现,肿瘤细胞在某些条件下释放的自噬小体可以诱导小鼠B细胞活化、分泌抗体和细胞因子(IL-6,IL-10)。另外,肿瘤细胞来源的自噬小体(Tumor cell-derived autophagosome, TDAP)能够在体内和体外诱导小鼠B细胞为具有免疫抑制功能的IL-10+B细胞且TDAP上的DAMP分子HMGB1在TDAP诱导B细胞成为IL-10+B细胞过程中发挥了重要作用,所以本课题想要在动物实验的基础上进一步探讨肿瘤病人来源的自噬小体能否诱导人B细胞成为IL-10+B细胞及其诱导产生的IL-10+B细胞具有何种免疫调节功能。由于在肿瘤形成阶段经历的低氧、低血供的营养匮乏过程,促使实体瘤中产生丰富的自噬现象并且多种肿瘤的病程中会产生含有大量肿瘤细胞的胸水或者腹水。所以我们猜想肿瘤病人恶性胸／腹水中可能也存在自噬小体,鉴于恶性胸／腹水标本容易获得,所以本课题将以恶性胸腹水来源的自噬小体(Malignant effusions-derived autophagosome, MedAP)为研究对象,结合本课题组前期研究结果探讨MedAP对人外周血B细胞的影响。目的：本课题以临床肿瘤患者恶性胸腹水为研究对象,主要探讨以下问题：1)临床肿瘤患者恶性胸腹水中是否存在自噬小体-MedAP? 2) MedAP能否诱导人B细胞成为IL-10+B细胞?3)MedAP诱导IL-10+B细胞对T细胞应答是否具有调节功能?4) MedAP诱导IL-10+B细胞是否与MedAP上HMGB1的含量具有相关性?方法：1.收集肿瘤患者恶性胸腹水,通过高速离心法,得到提取物,并对提取物进行蛋白定量,根据胸腹水体积统一将提取物的含量换算成mg/ml恶性胸腹水。2. 通过流式细胞术检测提取物上LC3表达量、Western Blot检测提取物LC3V1I蛋白、透射电镜检测提取物的超微结构来鉴定提取物中是否含有自噬小体。3.将MedAP和健康人外周血PBMC共孵育3天,流式细胞术检测IL-10+B的比例。4.经MedAP诱导3天的B细胞和CFSE标记的PBMC在包被有抗CD3单抗的48孔板中共孵育4天,流式细胞术检测CD4+T和CD8+T细胞增殖情况。5.流式细胞术检测MedAP上HMGB1的含量,分析MedAP诱导产生的IL-10+B细胞比例与MedAP上HMGB1的含量是否具有相关性。结果：1：临床肿瘤患者恶性胸腹水中存在自噬小体。1)流式细胞术检测发现提取物高表达LC3,可高达90%以上。2) Western Blot检测到提取物上含有LC3Ⅰ/Ⅱ。3)透射电镜检查发现提取物中存在大量双层膜结构的自噬小体。2：健康人外周血PBMC体外与MedAP共孵育3天后,流式细胞术检测发现IL-10+B的比例(5%-8%)较未刺激组(1%-2%)明显升高,差异具有明显的统计学意义。3：MedAP诱导的B细胞能够抑制T细胞的增殖, CD4+T和CD8+T细胞的增殖比例较未加入B细胞组明显降低,仅为33.1%和45.3%,未加入B细胞组则分别为49.9%和61.1%。4：流式细胞术检测发现不同患者来源的MedAP上HMGB1的含量相差很大,并且不同患者来源的MedAP诱导产生的IL-10+B细胞比例与MedAP上HMGB1的含量具有正相关性。结论：1.临床肿瘤患者恶性胸腹水中存在自噬小体；2.MedAP能够诱导健康人外周血B细胞成为IL-10+B细胞并且诱导产生的IL-10+B细胞具有免疫抑制功能,能够抑制T细胞增殖；3.MedAP诱导产生的IL-10+B细胞比例与MedAP上HMGB1的含量具有正相关性。更多还原

【Abstract】 Autophagy is an evolutionarily conserved process of degradation of intracellular substances and plays an important role in regulating the cell metabolism, energy cycle, organelles renewal. Autophagy involves in a number of physiological and pathological processes and it is related to the development and progression of many human diseases, especially cancer.However, the level of autophagy in cells is not static and it will rise in many cases such as mild oxidative stress and nutrient deprivation over a long period. Recent studies have revealed that the activity of autophagy changes in a variety of tumor cells and it makes the study of autophagy in tumors become a hot topic in the treatment of cancer. Our group have confirmed that tumor cell-derived autophagosome contained a variety of damage-associated molecular patterns (DAMPs) molecules such as high mobility group box protein 1(HMGB1) and heat shock protein 90(HSP90)and they play an important role as a natural adjuvant in the immune response.It is well known that the main functions of B cells are antibodies production, antigen presentation and cytokine secretion. However, recent studies have found that a new B cell subset-regulatory B cells (Breg) plays an important role in the immune regulation of the occurrence, development and prognosis of some infectious diseases, autoimmune diseases and cancer. The secretion of IL-10 is one of the most important ways of Breg to suppress immune responses, and therefore the secretion of IL-10 is the main feature of Breg. It has been reported that Breg is vital in the regulation of tumor immunity. On the one hand, Breg produce IL-10 to suppress the IFN-y secretion of CD8+T cells, thereby promoting the development of tumors; On the other hand, Breg can also lead to tumor cell metastasis to the lung by recruiting Tregs Although Breg is important in tumor immunity, its exact mechanism needs to further study.Our previous study have found that autophagosome released from tumor cells under certain conditions could induce mouse B cell activation and secretion of antibody and cytokines (IL-6, IL-10). In addition, we found that the tumor cell-derived autophagosome(TDAP) could induce murine B cells to become an immunosuppressive IL-10+B cell both in vivo and in vitro. Interestingly, the DAMP molecules HMGB1 in TDAP play an important role in the induction of IL-10+ B cells. Therefore, this study wanted to further investigate whether the autophagosome derived from clinical cancer patients could induce human B cells to become IL-10+ B cells on the basis of animal experiments. Many solid tumors could produce malignant effusions that contain large of tumor cells and the level of autophagosome in these cells will rise due to hypoxia and nutrient deprivation. We suspect that there may be autophagosome in malignant effusions of tumor patient. Therefore, the aim of this study wanted to explore the effect of malignant effusions-derived autophagosome (MedAP) on human peripheral blood B cells.Objective:In this study, we use autophagosome derived from malignant effusions (MedAP) to explore the follow issues:1) whether malignant effusions contain autophagosome 2) The production and immunomodulatory functions of IL-10+B cells induced by MedAP 3) The relationship between the proportion of IL-10+B cells induced by MedAP and the levels of HMGB1in MedAP.Methods:1. Collect malignant effusions and obtain extracts via high-speed centrifugation. Quantify total protein levels of the extracts and converted its content into mg/ml according to the volume of malignant effusions.2. Detection expression of LC3 by flow cytometryx, LC3I/II by Western Blot and ultrastructural by TEM to confirm whether the extracts contain autophagosome.3. The MedAP and PBMC from healthy donors were incubated in vitro for three days and then, the proportion of IL-10+B was evaluated by flow cytometry.4. B cells induced by MedAP was incubated with CFSE labeled PBMC in a 48-well plate coated with anti-CD3 antibody for four days and than using flow cytometry to detect the proliferation of CD4+and CD8+T cells.5. Examine the levels of HMGB1 in MedAP by flow cytometryx and analyze the relationship between the proportion of IL-10+ B cells induced by MedAP and the levels of HMGB1 in MedAP.Results:1. Malignant effusions contain autophagosome.1) A high expression of LC3 in extracts was found by flow cytometryx, and the expression of LC3 in extracts was as high as 90%.2) The extracts contain LC3II protein detected by western blot.3) TEM examination revealed that there were a lot of autophagosome with bilayers structure in extracts.2. The proportion of IL-10+B cells was as high as 5%-8% in PBMC after co-culture with MedAP in vitro for 3 days and it was significantly higher than that in unstimulated PBMC (1%-2%).3. Compared with the control group, the rate of cell division of CD4+T and CD8+T cells from PBMC concultured with B cells which was induced by MedAP for three days was significantly reduced from 49.9% and 61.1% to 33.1% and 45.3%.4. The level of HMGB1 in MedAP from different patient varied widely and there was a positive correlation between the proportion of IL-10+B cells induced by MedAP and the level of HMGB1in MedAP.Conclusions:1. Malignant effusions collected from tumor patient contain autophagosome.2. MedAP induce B cells to become IL-10+B cells and the IL-10+B cells can inhibit the proliferation of T cells.3. The proportion of IL-10+ B cells induced by MedAP is positive correlation with the levels of HMGB1in MedAP.更多还原