【作者】 王亮；

【导师】 崔满华；

【作者基本信息】 吉林大学 ， 妇产科学， 2020， 博士

【摘要】 研究背景:卵巢癌是妇科三大恶性肿瘤中发病率最低,病死率最高的肿瘤类型,具有起病隐匿、进展迅速、早期转移、预后较差等特点。晚期卵巢癌发生盆腹腔广泛种植转移非常常见,往往伴有大量腹水,给临床治疗带来巨大挑战。尽管近年来除传统肿瘤细胞减灭术和化学治疗等方法外,出现了很多新型治疗方法,但临床应用的效果并未显著改善疾病生存率,寻找新的治疗方法尤其关键。免疫治疗,基于细胞免疫和体液免疫的基本原理发展起来,包括细胞因子、单抗隆抗体、肿瘤疫苗、免疫细胞输注、免疫检查点阻断等多项手段。其中,在免疫细胞治疗领域中,嵌合抗原受体T细胞(CAR-T)逐渐受到关注,其基本原理是特异性识别肿瘤细胞表面的靶抗原并通过胞内信号的激活形成对肿瘤细胞的特异性杀伤。CAR-T细胞在血液肿瘤中的疗效非常显著,抗CD19-CAR-T细胞已有多项产品获得FDA批准,部分患者可达痊愈。但是CAR-T细胞在以卵巢癌为代表的实体瘤上的治疗尚无突破性进展,主要难题和实体瘤的特征有着密切关系,例如,实体瘤微环境复杂,免疫细胞的功能经常受到抑制;实体瘤的肿瘤实质具有高度异质性,免疫靶点的选择存在局限;实体瘤是致密的团块组织,常规免疫细胞难以突破实体瘤的物理屏障。简言之,探究新的卵巢癌免疫治疗方法是非常必要的。同时根本上,免疫治疗的研究也应立足于卵巢癌的疾病特征。尿激酶型纤溶酶原激活剂受体(uPAR)是一种糖基磷脂酰肌醇(GPI)锚定膜蛋白,属于纤溶系统的成员之一,在多种恶性肿瘤中,uPAR呈现显著性高表达,通过与尿激酶型纤溶酶原激活剂(uPA)特异性结合启动纤溶酶原活化并参与细胞外基质降解,影响肿瘤的增殖、转移、侵袭、耐药及复发等病理过程,是逐渐得到重视的肿瘤标志物之一。现有的研究显示uPAR在卵巢癌组织中特异性高表达,不仅表达在卵巢癌实质细胞上而且表达在肿瘤基质细胞上。那么,不同于传统的肿瘤治疗靶点,靶向uPAR的治疗方法有望同时攻克卵巢癌的基质成分和抑制卵巢癌的实质成分。本研究以uPAR为卵巢癌治疗的靶点,首先验证uPAR对于卵巢癌细胞转移能力的影响,并结合动物模型验证uPAR促进卵巢癌的腹腔种植转移和腹水形成的能力;结合uPAR在卵巢癌中促进卵巢癌进展的功能特点和存在特异性分布的表达特点,设计靶向uPAR的CAR-T细胞用于卵巢癌细胞的杀伤,验证该细胞的杀伤效率和细胞因子的分泌。实验目的:分别构建uPAR基因过表达和基因敲除的稳定转染卵巢癌细胞系;研究uPAR对于卵巢癌细胞转移能力的影响;研究uPAR影响卵巢癌转移过程中上皮间质转化过程各指标的变化情况;建立裸鼠卵巢癌腹腔种植瘤模型,探究uPAR对于卵巢癌腹腔种植和腹水形成的影响;构建靶向uPAR的CAR-T细胞。验证靶向uPAR的CAR-T细胞对于卵巢癌细胞的杀伤能力,为临床上卵巢癌的细胞治疗方法提供新的思路和策略。实验方法:第1部分:uPAR增强卵巢癌的转移能力的体外实验1、通过流式细胞术检测体外培养的卵巢癌细胞系上uPAR蛋白表达情况;2、设计构建uPAR基因过表达质粒载体,转染uPAR阴性细胞系A2780;设计构建靶向uPAR基因的shRNA慢病毒载体,转导uPAR强阳性细胞系ES-2;分别构建并鉴定uPAR基因过表达和敲除的稳定转染细胞系;3、通过细胞划痕实验和Transwell小室实验探究uPAR对于卵巢癌细胞的转移能力的影响;4、利用蛋白质免疫印迹实验检测uPAR变化对uPA、PAI-1的影响和促进卵巢癌转移能力的机制中上皮间质转化过程各指标的变化情况。第2部分:uPAR促进卵巢癌腹腔种植转移和腹水形成的体内实验1、利用ES-2对照细胞系及其uPAR基因敲除的细胞系构建裸鼠腹腔种植瘤模型,验证uPAR对于卵巢癌腹腔种植转移的影响;2、通过测量裸鼠体重及腹围研究uPAR对于裸鼠腹水形成的影响;3、通过HE染色和免疫组化染色的方法探究uPAR基因敲除对于卵巢癌裸鼠的肿瘤病灶及肝、脾等器官的影响。第3部分:靶向uPAR-CAR-T细胞的构建与制备1、利用第三代CAR-T细胞的基本框架设计靶向uPAR的CAR-T细胞,创新性选择靶向uPAR的自然结合片段作为胞外识别区;2、利用三质粒慢病毒包装系统构建CAR慢病毒;3、利用密度梯度离心法提取外周血T淋巴细胞,慢病毒转导法构建CAR-T细胞,并用流式细胞术检测T细胞CD3、CD4、CD8的表达情况。第4部分:靶向uPAR-CAR-T细胞对卵巢癌细胞的杀伤1、利用靶向uPAR-CAR-T细胞对uPAR阳性卵巢癌细胞系、uPAR阴性卵巢癌细胞系进行效应细胞、靶细胞共孵育实验;2、利用LDH kit方法检测效靶实验的肿瘤裂解率,寻找最佳效靶比;3、在最佳效靶比下,利用CBA kit结合流式细胞仪对于效靶实验的Th细胞因子IL-2、IFN-γ、TNF、IL-4、IL-5、IL-10进行检测;4、利用ELISA方法检测效靶实验下颗粒酶B的分泌,确定CAR-T发挥细胞毒性的基本规律。实验结果:第1部分:1、流式细胞术显示在不同病理类型的5种卵巢癌细胞系中,有4种细胞高表达uPAR;ES-2细胞是uPAR强阳性细胞系,表达率为95.2%;A2780是uPAR阴性细胞系;2、稳定转染过表达uPAR的A2780细胞及其对照细胞构建完成,过表达uPAR上调uPA、PAI-1的表达;靶向uPAR基因敲除的ES-2细胞及其对照细胞构建完成。稳转细胞系的uPAR蛋白、mRNA水平与对照组相比均存在显著统计学差异。3、划痕实验和Transwell小室实验表明,uPAR的过表达促进卵巢癌细胞的迁移;相反,敲除uPAR则抑制卵巢癌细胞的转移能力。4、uPAR增强卵巢癌细胞的转移能力,上调间质细胞表型蛋白N-cadherin和vimentin并下调上皮细胞表型蛋白E-cadherin,促进上皮间质转化过程。第2部分:1、在裸鼠腹腔种植瘤模型中,基因敲除uPAR后,卵巢癌细胞系sh-ES-2形成腹腔种植瘤和产生腹水的能力均降低;2、基因敲除uPAR的ES-2细胞形成裸鼠卵巢癌模型后,其uPAR阴性表达的特征成功保留。HE染色显示uPAR阴性细胞形成的肿瘤病灶中细胞排列紧密、胞核深染、细胞形态呈卵圆形;uPAR阳性细胞形成的肿瘤病灶中细胞排列紊乱、细胞形态呈梭形。第3部分:1、靶向uPAR-CAR-T细胞的胞外识别区采用了uPAR的自然结合片段ATF,命名为ATF-CAR-T细胞。并采用了CD28-CD137-CD3ζ作为胞内区;2、利用外源性添加CD3、CD28抗体、IL-2因子的方法体外扩增、活化人外周血来源的淋巴细胞,CD3阳性比例为99.0%,且CD8阳性的比例明显升高;3、CAR慢病毒的对于T淋巴细胞转导效率为61.0%。第4部分:1、CAR-T细胞对uPAR阳性卵巢癌细胞具有显著杀伤作用,对于uPAR阴性卵巢癌细胞则不明显,说明CAR-T细胞发挥作用具有靶抗原特异性;采用不同梯度的效靶比1:1、5:1、10:1、20:1时发现,CAR-T的杀伤作用具有效应细胞剂量依赖性,随着效靶比的升高杀伤作用逐渐增强。在效靶比为10:1时,ATF-CAR-T细胞对ES-2的裂解率为65.2%±2.7%;在效靶比为20:1时,ATF-CAR-T细胞对ES-2的裂解率可达到70.6%±1.7%。考虑到成本和效果的平衡,10:1为最适宜的效靶比例。2、CAR-T细胞对uPAR阳性细胞产生特异性杀伤时,IL-2、IFN-γ、TNF细胞因子的分泌及颗粒酶B的释放显著升高。实验结论:1、分别成功构建了uPAR稳定过表达、敲除的卵巢癌细胞系;确定了过表达uPAR促进了uPA的上调;过表达uPAR基因增强卵巢癌细胞的转移能力,反之,敲除uPAR基因导致卵巢癌的转移能力降低;2、uPAR通过影响上皮间质转化过程促进了卵巢癌细胞发生转移。uPAR过表达促进间质细胞表型的产生,抑制上皮细胞表型的产生;反之,敲除uPAR基因具有相反作用;3、在裸鼠腹腔卵巢癌模型中,卵巢癌uPAR基因敲除导致腹腔病灶减少、腹水形成减少;4、效靶杀伤实验显示,CAR-T细胞对于卵巢癌细胞具有杀伤作用,且该作用具有uPAR靶抗原特异性和CAR-T细胞剂量依赖性,为临床试验的开展奠定了基础;5、CAR-T细胞发挥杀伤作用时,造成IFN-γ、TNF、IL-2和颗粒酶B释放显著升高。证实了靶向uPAR-CAR-T细胞治疗uPAR阳性卵巢癌的有效性。更多还原

【Abstract】 Background:Ovarian cancer has the lowest incidence and the highest mortality rate among the three major gynecological tumors.Ovarian cancer has the characteristics of insidious onset,rapid progression,early metastasis and poor prognosis.Ovarian cancer with pelvic and abdominal cavity extensive implantation and metastasis is very common,often accompanied by a large amount of ascites,which brings great challenges to clinical treatment.Although in recent years,in addition to traditional tumor cytoreductive surgery and chemotherapy,many new treatment methods have appeared,but the actual clinical application has not significantly improved the prognosis.Finding new treatment methods is particularly critical.Immunotherapy is developed based on the basic principles of cellular immunity and humoral immunity,including cytokines,monoclonal antibodies,tumor vaccines,immune cell infusion,and immune checkpoint blocking.Among them,in the field of immune cell therapy,chimeric antigen receptor T cells(CAR-T)have gradually attracted attention,whose basic principle is to specifically recognize the target antigen on the surface of tumor cells and form specific cytotoxicity against tumor cells through the activation of intracellular signals.The efficacy of CAR-T cells in hematological tumors is very significant.Many anti-CD19 CAR-T cell methods have been approved by the FDA,and some patients can be cured totally.However,CAR-T cells have not made any breakthrough in the treatment of ovarian cancer.The main problems are closely related to the characteristics of solid tumors.For example,the microenvironment of solid tumors is complex,and the function of immune cells is often inhibited.The tumors of solid tumors are essentially highly heterogeneous,there are very limited choices of immune targets;solid tumors are dense masses of tissues,and it is difficult for conventional immune cells to break through the physical barriers of solid tumors.In short,it is necessary to explore new treatments for ovarian cancer.At the same time,fundamentally,immunotherapy methods must also be based on the disease characteristics of ovarian cancer.Urokinase-type plasminogen activator receptor(uPAR)is a GPI-anchored membrane protein,which belongs to one of the members of the plasmin system.In a variety of malignant tumors,significant high expression of uPAR has been found,which can bind urokinase-type plasminogen activator(uPA)to initiate plasminogen activation and participate in the degradation of extracellular matrix,affecting pathological processes such as tumor proliferation,metastasis,invasion,drug resistance and recurrence,and is one of the tumor markers that have gradually received attention.Existing studies show that uPAR is specifically and highly expressed in ovarian cancer tissues,not only on ovarian cancer parenchymal cells but also on tumor stromal cells.Then,unlike traditional tumor treatment targets,uPAR-targeted therapies are expected to simultaneously capture the stromal components of ovarian cancer and the substantial components that inhibit ovarian cancer.In this study,uPAR was used as a therapeutic target for ovarian cancer.Firstly,the effect of uPAR on the metastatic ability of ovarian cancer cells was verified,and the animal model was used to verify the ability of uPAR to promote the peritoneal implantation and ascites formation of ovarian cancer.Considering characteristics of progression function and specific expression of uPAR in ovarian cancer,CAR-T cells targeting uPAR were designed to kill ovarian cancer cells,and the efficiency of cytotoxicity and cytokine secretion were verified.Objective:Construct stable transfection ovarian cancer cell lines with uPAR gene overexpression and gene knockout,respectively;study the effect of uPAR on ovarian cancer cell metastasis ability;study uPAR influence on the changes of various parameters of epithelial-mesenchymal transition during ovarian cancer metastasis;Mouse ovarian cancer intraperitoneal implant tumor model was used to investigate the effects of uPAR on ovarian cancer intraperitoneal implantation and ascites formation.CAR-T cells targeting uPAR were constructed.Validate the cytotoxicity ability of uPAR-targeted CAR-T cells to ovarian cancer cells,and provide new ideas and strategies for clinical cell therapy of ovarian cancer.Methods: Part 1: In vitro experiments with uPAR to enhance metastatic capacity of ovarian cancer1.Detection of uPAR protein expression in ovarian cancer cell lines cultured in vitro by flow cytometry;2.Design and construct a uPAR gene overexpression plasmid vector and transfect the uPAR negative cell line A2780;design and construct a shRNA lentiviral vector targeting the uPAR gene to transduce the uPAR strong positive cell line ES-2;construct and identify the uPAR gene stably transfected cell lines at the overexpression and knockout sites;3.To explore the effect of uPAR on the metastatic ability of ovarian cancer cells through cell scratch experiments and Transwell chamber experiments;4.The changes of various indexes in epithelial-mesenchymal transition and the expression of uPA and PAI-1 were detected by Western blotting.Part 2: In vivo experiments uPAR promotes ovarian cancer metastasis and ascites formation1.Establish a mouse abdominal cavity implant tumor model using ES-2 cell line and its uPAR gene knockout cell line to verify the effect of uPAR on abdominal cavity metastasis of ovarian cancer;2.Measure the effect of uPAR on the formation of ascites in mice by measuring the weight and abdominal circumference of mice;3.To investigate the effects of uPAR gene knockout on tumor lesions,liver and spleen organs of ovarian cancer mice by HE staining and immunohistochemical staining.Part 3: Construction and preparation of targeted uPAR-CAR-T cells1.Utilize the basic framework of the third generation CAR-T cells to design X CAR-T cells that target uPAR,and creatively select natural binding fragments that target uPAR as extracellular recognition regions;2.Construct a CAR lentivirus using a three-plasmid lentivirus packaging system;3.T lymphocytes in peripheral blood were extracted by density gradient centrifugation,CAR-T cells were constructed by lentiviral transduction method,and the expression of CD3,CD4,CD8 of T cells was detected by flow cytometry.Part 4: Targeting uPAR-CAR-T cells to kill ovarian cancer cells1.Using anti-uPAR-CAR-T cells,uPAR positive ovarian cancer cell lines,and uPAR negative ovarian cancer cell lines for effector cell and target cell incubation experiments;2.Use the LDH kit method to detect the tumor lysis rates of the efficacy target experiment and find the best efficacy target ratio;3.Under the best effect target ratio,use CBA kit combined with flow cytometry to detect Th cytokines IL-2,IFN-γ,TNF,IL-4,IL-5,IL-10 in the effect target experiment;4.Use the ELISA method to detect granzyme B in the efficacy target experiment to determine the basic law of CAR-T cytotoxicity.Results: Part 1:1.Flow cytometry showed that among 5 ovarian cancer cell lines of different pathological types,4 cells had high expression of uPAR protein;ES-2 cells were uPAR strongly positive cell lines with an expression rate of 95.2%;A2780 was uPAR negative cell line.2.Construction of stably transfected uPAR-overexpressing A2780 cells and its control cells was completed and overexpression of uPAR up-regulates the expression of uPA and PAI-1;construction of uPAR gene knockout ES-2 cells and its control cells was completed.The uPAR protein and mRNA levels of the stably transfected cell lines were significantly different from those of the blank group and the control group.3.Scratch experiments and Transwell chamber experiments show that overexpression of uPAR promotes the migration of ovarian cancer cells;on the contrary,knockdown of uPAR inhibits the migration of ovarian cancer cells.4.uPAR enhances the metastatic capacity of ovarian cancer cells,up-regulates mesenchymal cell phenotype proteins N-cadherin and vimentin,down-regulates epithelial cell phenotype protein E-cadherin,and promotes epithelial-mesenchymal transition process.Part 2:1.In the mouse abdominal cavity tumor model,after the knockdown of uPAR,the ability of the ovarian cancer cell line ES-2 to form abdominal cavity tumors and produce ascites was reduced;2.After the knockdown of uPAR ES-2 cells formed a mouse ovarian cancer model,their uPAR negative expression characteristic was successfully retained.HE staining showed that the tumors formed by uPAR-negative cells had densely arranged cells,deep stained nuclei,and the cell morphology was oval.The tumors formed by uPAR-positive cells had disordered cell arrangement and the cell morphology was spindle-shaped.Part 3:1.The extracellular recognition region of uPAR-CAR-T cells uses ATF as the natural binding fragment of uPAR and named as ATF-CAR-T cells.CD28-CD137-CD3ζ was used as the intracellular region;2.The method of exogenous addition of CD3,CD28 antibodies,and IL-2 factors was used to expand and activate human peripheral blood-derived lymphocytes in vitro.The proportion of CD3 positive was 99.0%,and the proportion of CD8 positive increased significantly.3.The transduction efficiency of CAR lentivirus for T lymphocytes is 61.0%.Part 4:1.1.CAR-T cells have a significant cytotoxicity effect on uPAR-positive ovarian cancer cells,but not on uPAR-negative ovarian cancer cells,indicating that CAR-T cells play a role of target antigen specificity;using different ratios of target ratio 1: 1,5: 1,10: 1,20: 1,it was found that the cytotoxicity effect of CAR-T is effector cell dose-dependent.When the effector target ratio is 10: 1,the lysis rate of ES-2 by ATF-CAR-T cells is 65.2%±2.7%.When the effector target ratio is 20: 1,the lysis rate of ES-2 by ATF-CAR-T cells can reach 70.6% ± 1.7%.Considering the balance between cost and effect,10: 1 is the most suitable effector target ratio.2.When CAR-T specifically kills uPAR-positive cells,the secretion of IL-2,IFN-γ,and TNF cytokines and the release of granzyme B are significantly increased.Conclusions:1.Successfully constructed uPAR stable overexpression and knockdown ovarian cancer cell lines;determined that overexpression of uPAR promoted the upregulation of uPA;overexpression of uPAR gene enhanced the metastatic capacity of ovarian cancer cells;conversely,knockdown of uPAR gene caused ovarian cancer reduced transfer capacity;2.uPAR promotes the metastasis of ovarian cancer cells by affecting the epithelial-mesenchymal transition.uPAR overexpression promotes the generation of mesenchymal cell phenotype and inhibits epithelial cell phenotype.Conversely,knockdown of uPAR has the opposite effect3.In a mouse abdominal ovarian cancer model,knockdown of uPAR of ovarian cancer leads to a reduction of abdominal lesions and a decrease in ascites formation;4.Effector target killing experiments show that CAR-T cells have a cytotoxity effect on ovarian cancer cells,and this effect has uPAR target antigen specificity and CAR-T cell dose dependency,laying a foundation for the development of clinical extrails;5.When CAR-T cells exert a killing effect,the release of IFN-γ,TNF,IL-2 and granzyme B is significantly increased.The feasibility of anti-uPAR-CAR-T cells in the treatment of uPAR-positive ovarian cancer was confirmed.更多还原