【作者】 杜秀敏；

【导师】 白增亮；

【作者基本信息】 山东大学 ， 发育生物学， 2009， 博士

【摘要】 背景和目的原发性肝细胞癌(HCC)是世界三大癌症相关死亡之一,是最常见的预后较差的肝脏恶性肿瘤。目前研究者建立了各种肿瘤异种移植模型用于人肝癌生物学和化疗研究,但由于其采用的模型动物主要是免疫缺陷鼠或诱导免疫耐受鼠,致使以此模型进行肿瘤免疫治疗的研究受到限制。因而构建免疫力正常的异种肿瘤移植模型,研究肿瘤与宿主的相互作用将有助于我们更好地理解肿瘤的免疫治疗。然而早先的研究证实肿瘤异种移植物仅可以生长于一些免疫豁免位点,如大鼠的颊囊、眼前房,或环孢霉素A处理的小鼠或大鼠的肾囊下生长。异种肿瘤移植物在免疫力正常宿主体内成功存活的主要障碍被认为是T细胞介导的免疫排斥及组织微环境。T细胞介导的异种免疫排斥的重要性已在裸鼠模型中得到证实,而异种肿瘤移植物与组织微环境的关系研究较少。肝脏作为人肝癌细胞移植的常见位点,近年被公认为一个复杂的免疫与耐受器官。它富含天然免疫细胞,如NK、NKT、枯否细胞和树突状细胞,尤其是NKT细胞,近年被称为免疫系统的瑞士军刀,其活化后可激活各类免疫细胞参与多种免疫反应。此外,经典CD4~+T和CD8~+T细胞在肝脏中的比例也与外周血相反。所有这些天然免疫和获得性免疫细胞决定着肝脏微环境的肿瘤监视,但它们在肿瘤异种移植物存活中所扮演的角色却知之甚少。本研究尝试在免疫力正常的乳鼠肝脏内移植入人的肝癌细胞来建立人肝癌—小鼠动物研究模型。其理论依据为:①乳鼠(约出生后0～15d)肝内高表达的甲胎蛋白(alpha-fetaprotein,AFP)具有抑制机体抗肿瘤免疫的生物学特性;②乳鼠胸腺尚未发育成熟,外周免疫系统尚未完善,免疫排斥较弱;③选择“免疫特赦器官”—肝脏作为荷瘤器官,易于诱导免疫耐受。在前期的实验中,我们首次将人肝癌细胞HepG2细胞原位移植到具正常免疫力的昆明种乳鼠肝内,构建了人肝癌HepG2移植性小鼠模型。虽然我们证实在免疫力正常的乳鼠肝内移植人HepG2细胞较成鼠成功率高,移植瘤细胞存活时间较长,但是随着乳鼠发育免疫系统的逐渐完善,肝内异种免疫排斥反应增强,肿瘤细胞在移植后7～9天开始坏死。肿瘤细胞在乳鼠肝内遭遇免疫排斥,何种免疫细胞参与了这一过程?肿瘤细胞在肝内被清除,肝内的免疫微环境发生了怎样的变化?尤其是数量丰富的NKT细胞的活化和功能有着怎样的改变?分化程度不同的异种肿瘤细胞,在乳鼠肝内的命运是否一样?CsA免疫抑制的荷瘤乳鼠肿瘤组织病理切片中并未发现淋巴细胞的浸润和肿瘤细胞的坏死,其对细胞介导的异种免疫排斥有着怎样的影响?围绕这些问题,本研究将生长特性不同的人肝癌细胞株(HepG2和HCCLM3)移植到免疫力正常的乳鼠体内,探讨人肝癌细胞在乳鼠肝内的存活情况、肝内细胞介导的免疫排斥反应、NKT细胞活化及功能的改变。为更好地阐明这一点,我们同时采用成鼠及低剂量CsA处理的乳鼠和成鼠做移植受体,进行对比研究。本研究为HCC异种模型的建立提供了一种新的尝试,为NKT细胞介导的肿瘤异种排斥呈现了一个新的视角。而且它有助于更好地了解CsA的免疫抑制机制,无论是在临床还是动物模型的研究中。研究方法1、DMAHAS体外标记细胞与细胞毒性分析1)DMAHAS标记人肝癌HepG2细胞并荧光成像;2)DMAHAS标记细胞荧光释放分析;3)MTT和中性红法检测DMAHAS处理HepG2细胞的活力;4)考马斯亮蓝法测定细胞总蛋白;5)PI流式细胞术检测细胞周期与凋亡;2、乳鼠和成鼠人肝癌原位移植1)肿瘤细胞培养与体外DMAHAS标记;2)低剂量CsA(10 mg/kg)处理小鼠及其血清浓度检测;3)人肝癌细胞小鼠肝内原位移植;4)异种移植物组织冰冻切片分析及荧光成像;5)异种移植物组织常规HE染色及病理分析;3、荷瘤乳鼠及成鼠不同细胞免疫排斥机理的研究1)人肝癌细胞HCCLM3小鼠肝内原位移植;2)低剂量CsA(10 mg/kg)小鼠皮下注射;3)小鼠肝脾淋巴细胞分离和计数;4)DMAHAS荧光释放法检测CTL杀伤活性;5)荷瘤乳鼠血清肿瘤细胞特异性抗体检测;6)荷瘤小鼠肝脾淋巴细胞流式细胞术免疫表型分析;4、荷瘤小鼠NKT细胞活化及功能的研究1)人肝癌细胞HCCLM3小鼠肝内原位移植;2)低剂量CsA(10 mg/kg)小鼠皮下注射;3)分离小鼠肝脾淋巴细胞并计数;4)乳酸脱氢酶释放法检测NKT细胞杀伤活性;5)肝脾淋巴细胞NKT活化与CD1d表达分析;6)血清及肝脏组织IL-4和IFN-γELISA定量检测;7)体外免疫细胞特异性清除;8)LDH释放法测定CTL活性。研究结果第一部分DMAHAS体外标记细胞成像DMAHAS可在30分钟内快速标记活细胞或多聚甲醛固定的细胞,最适工作浓度为5μM。DMAHAS定位于细胞质,当受到紫外或长波长光(800nm)激发时,可自发地发射蓝色荧光。DMAHAS体外标记细胞与细胞毒性分析采用工作浓度5μM的DMAHAS标记HepG2细胞培养24小时进行MTT、NR、CB和PI流式细胞术检测表明DMAHAS对标记细胞无显著影响(P＞0.05)。DMAHAS标记肿瘤细胞体内示踪和荧光成像DMAHAS标记的人肝癌HepG2和HCCLM3细胞可在受体鼠肝内存活4周以上,移植肿瘤细胞随时间延长逐渐减少,在移植后六周受体鼠肝内未检测到肿瘤细胞或荧光信号。而且检测过程中未见DMAHAS荧光信号的衰减。乳鼠肝脏移植瘤组织病理分析人肝癌HCCLM3细胞原位移植于乳鼠肝内后,第8天左右肝内正常组织出现淋巴细胞的募集,移植瘤周围出现淋巴细胞浸润,无明显的炎性细胞如中性粒细胞或巨噬细胞介入。部分肿瘤移植物仍可存活4周以上,未见明显的肝内转移。低剂量CsA处理可有效减少淋巴细胞浸润,延长肿瘤细胞存活期至5周以上。第二部分荷瘤小鼠肝脾淋巴细胞变化荷瘤乳鼠肝脏淋巴细胞数量于移植后的10天和16天增加(P＜0.05),同时脾脏淋巴细胞数量减少,同样的结果可见于移植后4天、8天和10天的成鼠。并且荷瘤小鼠脾脏或肝脏淋巴细胞未见明显凋亡。荷瘤小鼠肝脾淋巴细胞免疫分析移植后第16天,乳鼠肝脏CD4~+T细胞绝对计数和百分比显著增加(P＜0.01),NK和CD8~+T细胞数量绝对计数在肝脏内增加,而在脾脏中减少(P＜0.05)。成鼠于移植后2天即见CD8~+T细胞在肝内的增加与脾内的减少。荷瘤小鼠脾脏淋巴细胞杀伤功能检测荷瘤16天的乳鼠脾脏淋巴细胞在10:1,20:1和50:1效靶比时对HCCLM3细胞的杀伤显著增加(P＜0.05)。低剂量CsA对移植瘤存活及其引发的免疫反应的影响低剂量CsA处理可显著地减少乳鼠肝内淋巴细胞侵润,延长肿瘤移植物存活期至5周以上。然而其对移植物在成鼠肝内的存活无明显影响。除对CD4~+和CD8~+细胞有抑制效果外,低剂量CsA可有效地抑制NKT细胞的活化。此外,低剂量CsA处理可有效抑制荷瘤乳鼠脾脏效应细胞的功能。第三部分HCCLM3异种移植物诱导的肝脏NKT细胞活化HCCLM3移植于3日龄乳鼠肝内后,其肝脏NKT细胞NK1.1分子在移植后5天和10天表达丢失。16天时表达增加并伴随NKT细胞活性增强。成鼠在移植后2天和4天可见NK1.1分子的大量丢失,并于10天时恢复至正常水平。脾脏NK/NKT细胞表面NK1.1分子的表达在移植前后无显著变化。荷瘤小鼠肝脏和脾脏淋巴细胞CD1d表达乳鼠在移植后10天时肝脏NK1.1~+细胞表面CD1d的表达开始增加,随后CD1d的表达上调可见于移植后16天和28天肝脏CD3~+T和NK1.1~+细胞。类似的结果可见于移植后4天和10天时的成鼠肝脾淋巴细胞。荷瘤小鼠血清IL-4和IFN-γ检测血清取自荷瘤10天和16天的乳鼠或4天和8天的成鼠。IFN-γ的水平在移植后的乳鼠和成鼠血清中均有增加(P＜0.05),而IL-4水平的升高仅见于移植后10天的乳鼠和4天的成鼠血清(P＜0.05)。低剂量CsA对NKT细胞活化和功能的影响低剂量CsA处理可显著抑制荷瘤乳鼠肝脏NKT细胞的功能(P＜0.05)及移植2天时的成鼠NKT细胞活化。此外低剂量CsA可显著抑制受体鼠血清IL-4和IFN-γ的分泌,而并未显著下调肝脾T细胞或NK1.1~+T细胞表面CD1d的表达。荷瘤乳鼠淋巴细胞体外免疫清除CTL活性分析体外特异性清除荷瘤乳鼠淋巴细胞中的CD3或CD8细胞,可显著降低CTL细胞的杀伤活性(P＜0.05);而特异性清除CD4或NK1.1~+细胞却无明显影响。研究结论1.首次应用新型双光子荧光染料DMAHAS标记人肝癌细胞及荧光成像研究,并初次证实其对标记细胞的活力、总蛋白合成和细胞周期与凋亡无显著影响。体内研究表明DMAHAS可作为一种安全可靠的探针用于肿瘤细胞示踪和荧光成像研究。2.初步证实乳鼠肝脏较成鼠肝脏更适宜于异种肿瘤细胞的存活与生长。3.淋巴细胞在荷瘤小鼠脾脏中的减少及肝脏中的增加暗示脾脏淋巴细胞以某种方式参与了肝脏异种肿瘤移植物的免疫排斥反应。4.乳鼠和成鼠肝内发生了对肿瘤移植物不同的细胞免疫排斥反应。参与乳鼠肿瘤延迟排斥反应的免疫细胞主要是CD4~+T、NK1.1~+细胞及CD8~+T细胞,而参与成鼠肿瘤急性排斥反应的免疫细胞主要是CD8~+T细胞、NK1.1~+细胞及大量的炎性细胞,如中性粒细胞或巨噬细胞。5.肝脏NKT细胞在异种肿瘤的排斥反应早期活化、功能及活性增强,表明其在排斥反应的早期起着重要作用。6.CD1d分子在荷瘤小鼠肝脏淋巴细胞不同时期表达的增加,表明其参与了异种肿瘤抗原的免疫呈递过程及异种移植物的免疫清除。7.低剂量CsA处理可显著地抑制乳鼠的免疫排斥反应,延长肿瘤移植物存活期,然而其对成鼠肝内肿瘤移植物存活无明显影响。8.除对CD4~+和CD8~+T细胞功能有抑制作用外,低剂量CsA可有效抑制肝脏NKT细胞的活化及功能,而且这种抑制方式可能不依赖于CD1d。更多还原

【Abstract】 Background and objectiveHepatocellular carcinoma(HCC),the third most common cause of cancer-related death worldwide,is the most frequent hepatic tumor with poor prognosis.Tumor xenograft models are widely used for human tumor biology and chemotherapy analysis, but they may be insufficient for studying tumor immunotherapy because most of them are established in immunodeficient hosts.Studies to elucidate the immune mechanisms of tumor-host interactions in immunocompetent tumor xenograft models will benefit our understanding of tumor immunotherapy.Previous studies have demonstrated that tumor xenografts can grow in some immunologically privileged sites,such as the anterior chamber of the eye,the hamster cheek pouch,or under the kidney capsule of mice or rats with limited cyclosporine A treatment.The potential barriers to successful xenograft survival are believed to be T cell mediated xenorejection and tissue microenvironments.The importance of T cells in xenorejection can be manifested by the growth of xenografts in mutant nude mice.However,the interaction between tumor xenografts and local microenvironments where the xenografts grow have received little extent attentions.As a common site for human HCC transplants,the liver is accepted as a complex immune or tolerance organ in recent years.It contains abundant innate immune cells, such as natural killer(NK),natural killer T(NKT) cells,kupffer cells and dendritic cells(DCs).Besides,classic CD4~+ T and CD8~+ T cells in the liver are present at a reversed ratio other than they are in the peripheral blood.All these innate and adaptive immune cells residing in liver dominate the local tumor surveillance,but their role in tumor xenograft survival has been inadequately explored.In this study we attempt to establish a human-mouse HCC model by transplanting human HCC cells to the liver of immunocompetent newborn mice.We based on three reasons:first,the elevated expression of alpha-fetaprotein(AFP) in the liver of newborn mice(at day 0～15 after birth) can suppress the anti-tumor immunity;second, newborn mice can generate weak immune rejection because of their incomplete thymus and peripheral immune organs;the third,as a immune privilege organ,the liver is apt to induce immune tolerance.In our previous experiments,we transplanted human HepG2 cells into the liver of immunocompetent newborn KM mice for the first time.We demonstrated that human HepG2 cells survived longer than those in adults.However,following the immune systems of newborn mice maturing,the immune xenorejection enhanced,and tumor cells began necrosis or apoptosis.The tumor cells encountered immune rejection in the liver of newborn mice,which immune cell participated in that process? Transplanted tumor cells were cleared from the liver,how the immune microenvironment of liver changed? In particular the aboundent NKT cells,how their activation and function changed? Whether the fate of different cells in the liver of newborn mice is same or not? No lymphocyte infiltration was detected in tumor tissues of CsA treated newborn mice,how the effect of CsA on cell-mediated xenorejection?Trying to solve these problems,we transplanted different human HCC lines (HepG2 and HCCLM3) to immunocompetent newborn mice.We intended to examine the fate of transplanted human tumor cells,the cell-mediated immune rejection,and the changes of activation and function of NKT cells.For better understanding this,we adopted low-dose CsA treated newborn and adult mice as controls.This study provides new information on tumor xenograft models in immature animals and throws new insight into NKT-mediated tumor xenorejection.Moreover,it adds to our understanding of the immunosuppressive effects of CsA.Methods1.In vitro cell imaging and Cytotoxicity analysis by DMAHAS1) HepG2 cells labeled with DMAHAS and cell imaging;2) Fluorescence release analysis of DMAHAS labeled cell;3) Cell viability was determined using MTT and neutral red assays;4) Total protein content of cells was measured by Coomassie brilliant blue assay;5) Cell proliferation and apoptosis were analyzed using the propidium iodide(PI) flow cytometric assay; 2.Orthotopic implantation of human HCC cells in young and adult mice1) In vitro culture of tumor cells and labeled by DMAHAS;2) Mice were treated with lose-CsA(10mg/kg) and sera CsA was measured;3) Orthotopic implantation of human HCC cells in the liver of mice;4) In vitro frozen section analysis and fluorescence imaging of tumor xenografts;5) Conventional HE staining and pathological examination of tumor xenografts:3.Different cellular rejection occurred in young and adult mice was analyzed1) Orthotopic implantation of human HCC cells in the liver of mice;2) Mice treated with lose-CsA(10mg/kg) through subcutaneously injection;3) Isolation of hepatic or splenic lymphocytes and counting;4) Method based on DMAHAS fluorescence release for cytotoxicity analysis;5) Specific antibodies toward minor cells were measured in the serum from mice;6) Phenotypic analysis of hepatic and splenic lymphocytes from mice by FCM;4.Analysis of the activation and function of NKT from tumor-bearing mice1) Orthotopic implantation of human HCC cells in the liver of mice;2) Mice treated with lose-CsA(10mg/kg) through subcutaneously injection;3) Isolation of hepatic or splenic lymphocytes and counting;4) NKT activity analysis by LDH release assay;5) Analysis of NKT activation and CD1d expression of hepatic/splenic lymphocytes;6) Levels of IL-4 and IFN-γwere measured by ELISA using a quantitative kit;7) In vitro immune cells depleted by spcific antibodies;8) Cytotoxicity analysis by LDH release assay.ResultsThe first partCell imaging in vitro using DMAHASWe found that both live and fixed cells took up DMAHAS rapidly within 30min. The final recommended working concentration is 5μM.DMAHAS exhibited strong one photon induced blue fluorescence and stable cytosolic localization. Cytotoxicity of DMAHAS toward HepG2 cellsNo significant cytotoxicity was determined following 48h incubation of HepG2 cells with DMAHAS at a working concentration of 5μM.According to MTT,NR,CB and FCM assay.Tumor tracking in vivo using DMAHAS and fluorescence imagingDMAHAS labeled HepG2 cells could survive in recipient mice for more than 4 weeks and the same results were obtained with mice receiving HCCLM3 xenografts. Smaller tumor xenografts were observed as survival time increased,and tumor cells tended to aggregated.At six weeks after transplantation,no tumor cells or fluorescence were detected in the liver of recipient mice.At any given time,no significant decline in fluorescence intensity was observed.Pathological examination of tumor xenografts in newborn miceLymphocyte infiltration appeared in and around the xenografts at day 8 after implantation of human HCCLM3 cells in the liver of newborn mice,no macrophages or neutrophils was visible,and the recruitment of lymphocytes appeared in the normal liver tissue near the tumor xenografts.Some of them still survived for more than four weeks,and no evidence of distant metastasis was observed.Low-dose CsA treatments significantly reduced the lymphocytes infiltration and prolonged the survival of xenografts in young mice to five weeks.The second partNumber changes of hepatic and splenic lymphocytes in mice bearing tumor cellsIn young mice,the absolute number of lymphocytes increased in the liver,but decreased in the spleen at days 10 and 16 postimplantation(P＜0.05).Similar results were found in adult mice at days 4,8 and 10 postimplantation.No significant apoptosis of splenic or hepatic lymphocytes was detected in young mice.Immune analysis of hepatic and splenic lymphocytes in mice bearing tumor cellsIn young mice,the absolute number and proportion of hepatic CD4~+ T cells was significantly increased at day 16 postimplantation(P＜0.01);the absolute number of NK cells and CD8~+ T cells increased in the liver,but decreased in the spleen(P＜0.05). In adult mice,the proportion of CD8~+ cells increased in the liver,but decreased in the spleen at day 2 after transplantation.CTL responses induced by HCCLM3 xenografts in young miceSplenic lymphocytes from tumor-bearing young mice showed higher cytotoxicity against HCCLM3 cells at the ratio of 10:1,20:1 and 50:1(P＜0.05).Effects of low-dose CsA on the survival of xenografts and immune responseLow-dose CsA treatments significantly reduced the lymphocytes infiltration and prolonged the survival of xenografts in young mice to five weeks posttransplantation. However,CsA had no obvious effect on the survival of xenografts in adults.Besides the inhibiting effects on CD4~+ and CD8~+ cells as seen in data from young and adult mice,low-dose CsA treatment efficiently suppressed the activation of hepatic NKT cells at day 2 after implantation in adult mice without the loss of NK1.1 expression on NKT cells.Moreover,low-dose CsA treatment efficiently suppressed the splenic CTL activity in tumor-bearing young mice.The third partHepatic NKT cell activation in mice induced by HCCLM3 xenograftsAfter HCCLM3 cells were transplanted into the livers of 3-day-old mice,the hepatic NKT cells lost expression of NK1.1 at days 5 and 10.NK1.1 molecules reappeared at day 16,and elevated NKT-like activity was detected.In adult mice,a loss of NK1.1 expression was detected in hepatic NK/NKT cells at days 2 and 4 posttransplantation.Then the NK1.1 marker was reexpressed and returned to normal levels at day 10 posttransplantation.There were no significant changes in NK1.1 expression on splenic NK/NKT cells after transplantation.CD1d expression on lymphocytes from the liver and spleen of tumor-bearing mice In young mice,the CD1d expression first increased on hepatic NK1.1~+ cells at day 10 after transplantation.Then up-regulation of CD1d expression was detected on hepatic CD3~+ T cells and NK1.1~+ cells at days 16 and 28 after transplantation.Similar results were detected in hepatic or splenic lymphocytes from adults at day 4 and 10 posttransplantation.Secretions of IL-4 and IFN-γin tumor-bearing young and adult miceSera were collected from the retro-orbital veins at days 10 and 16 in young mice and at days 4 and 10 in adult mice after transplanted with human HCCLM3 cells. Elevated secretion of IL-4 was detected at day 10 in young mice and at day 4 in adult mice(P＜0.05),and elevated secretion of IFN-γat any given time in young and adult mice(P＜0.05),compared with control groups.Effects of low-dose CsA treatment on the activation and function of NKT cellslow-dose CsA treatment efficiently suppressed the activity of NKT cells from tumor-bearing young mice,and the activation of hepatic NKT cells in adult mice at day 2 after implantation.Moreover,low-dose CsA treatment inhibited the secretions of IL-4 and IFN-γin recipient mice(P＜0.05).No down-regulation of CD1d expression on hepatic or splenic T cells and NK1.1~+ cells was detected in mice with low-dose CsA treatment.CTL analysis after in vitro deletion of specific lymphocytesIn vitro deletion of CD3 and CD8 cells efficiently reduced the cytotoxicity against HCCLM3 cells(P＜0.05),however,deletion of CD4 and NK1.1 cells showed no evident effect. Conclusion1.DMAHAS was used to label human HCC cells for fluorescence imaging for the first time.It was demonstrated that DMAHAS had no evident effect on the cell viability,synthesis of total protein or DNA,as well as cell apoptosis.In vivo study indicated that DMAHAS was a reliable probe for tumor cell tracking and imaging.2.It was demonstrated that the liver of newborn mice was more suitable for the survival of tumor xenografts than that of adults.3.Lymphocytes significantly increased in the liver,but decreased in the spleen after tumor implantation without enhanced cell apoptosis,suggesting that splenic lymphocytes may participate in the tumor xenorejection.4.cell-mediated xenorejection in the liver of young mice was different from that in adult liver.In young mice the delayed cellular rejection was mediated mainly by CD4~+T cells,CD8~+T cells and NK1.1~+cells.In contrast,in adult mice the acute cellular rejection was mediated mainly by CD8~+T cells,NK1.1~+cells,macrophages or neutrophils.5.The rapid activation of hepatic NKT cells with enhanced activity and function, suggested that NKT cells acted as an important role in the xenorejection.6.Increased expression of CD1d on the surface of hepatic or splenic lymphocytes, implying that CD1d molecules participate in the tumor xeno-antigens presenting and the subsequent tumor clearance.7.Low-dose CsA treatments significantly prolonged the survival of xenografts in young mice by inhibiting the cell-mediated rejection.However,it had no obvious effect on the survival of tumor xenografts in adults.8.Besides the inhibiting effects on CD4~+ and CD8~+ cells,low-dose CsA treatment efficiently suppressed the activation and function of hepatic NKT cells,maybe through a CD1d-independent manner.更多还原