【作者】 马小洁；

【导师】 栾信庸；

【作者基本信息】 山东大学 ， 耳鼻咽喉科学， 2008， 博士

【摘要】 【研究背景与目的】喉癌是耳鼻喉科常见的恶性肿瘤之一,发病率处于耳鼻咽喉各部分恶性肿瘤的第三位。在喉癌的病理类型中,以喉鳞癌最为常见,一般占全部喉恶性肿瘤的95～99%。目前喉癌的治疗以手术、放疗及化疗为主。近年来,虽然手术、放化疗技术发展迅速,但喉鳞癌患者特别是晚期患者的的长期生存率在过去10年中未见明显提高。因此,进一步改善喉癌患者预后的研究势在必行。以往的研究证实,抗肿瘤免疫在肿瘤发生发展的过程中有重要作用。机体的抗肿瘤免疫应答是以T淋巴细胞、NK细胞和巨噬细胞介导的细胞免疫应答为主。在此过程中,首先由抗原递呈细胞（antigen-presenting cell,APC）捕获、加工处理抗原并将抗原信息传递给T淋巴细胞,才能进而引发特异性免疫应答。树突状细胞（dendritic cell,DC）是已知体内功能最强的APC,能在体内外直接激活初始T细胞（na（?）ve T cell）,促进辅助性T细胞和细胞毒性T细胞的生成,同时也能够促进B淋巴细胞生成抗体,是启动、调控并维持免疫应答的中心环节。由于DC在体内免疫应答中重要而独特的作用,近年来,以DC为基础的肿瘤免疫治疗研究越来越受到关注。已有研究证实,通过体外细胞因子联合培养扩增DC,模拟DC体内成熟过程,以不同形式的抗原物质,如人工合成肿瘤多肽、肿瘤细胞裂解产物、肿瘤蛋白抗原、凋亡肿瘤细胞、DNA或RNA负载DC,再将致敏DC回输体内作为疫苗可诱导机体产生有效的抗肿瘤免疫应答。树突状细胞应用于抗喉鳞癌的研究尚处于初级阶段。由于目前尚未发现喉癌肿瘤特异性抗原,因此肿瘤全抗原成为制备疫苗的主要手段。已有报道应用喉癌细胞裂解产物负载树突状细胞或用电融合的方法制备疫苗诱导产生有效的抗肿瘤免疫反应。最近研究发现,以RNA负载DC能够引发更为强大的抗肿瘤效应,而且已有实验证实了其优越的安全性及广阔的应用前景。但以RNA负载DC的抗喉鳞癌研究国内外尚未见报道。另外,树突状细胞免疫学功能的强弱取决于DC的免疫状态。众所周知,肿瘤患者的DC存在缺陷,但由于T细胞活化的MHC限制性,临床上应用DC诱导抗肿瘤免疫应答必须使用肿瘤患者自身的DC,因而研究恶性肿瘤患者DC的数目及功能显得尤为重要。另一方面,虽然DC为基础的免疫治疗有其独特的优势,但近期内仍无法作为主要的临床治疗手段。对于喉鳞癌患者,手术及放疗仍然不失为目前最有效的治疗方法。因此研究喉鳞癌患者DC免疫状态,除了考虑疾病本身对DC的影响,尚需考虑目前常规治疗方法对DC的影响。本研究检测手术及术后辅助放疗对喉癌患者外周血循环DC亚型及外周血单核细胞来源DC（monocyte-derived DCs,MoDes）功能的影响,以探讨喉癌免疫治疗的时机;同时,以EGFP标记的喉癌Hep-2株总RNA转染MoDCs,研究其诱导Hep-2细胞特异性的细胞毒性T淋巴细胞（cytotoxic T lymphocyte,CTL）的能力及对喉鳞癌的特异性杀伤效应,为临床以DC为基础的抗喉癌治疗提供实验基础和理论依据。第一部分健康人外周血树突状细胞亚型检测及培养、鉴定【目的】建立简便可靠的外周血树突状细胞亚型检测方法,培养鉴定外周血单核细胞来源树突状细胞。【方法】采集15例健康人外周血,采用三色流式细胞计量术检测外周血HLA-DR⁺lineage^-树突状细胞及其亚型myeloid DC（mDC）、plasmacytoid DC（pDC）在外周血WBC所占百分比并计算其绝对数。采用淋巴细胞分离液自外周血分离获得单核细胞,体外经人重组细胞因子GM-CSF、IL-4和TNF-α诱导培养,获得成熟外周血单核细胞来源DC。倒置显微镜下观察DC形态,透射电镜观察DC超微结构,流式细胞仪检测其表型,混合淋巴细胞反应（MLR）检测刺激自体T淋巴细胞增殖的能力。【结果】健康人LIN^-DR⁺细胞在WBC中所占百分比为0.41±0.13%,绝对数为22.75±5.22/μl,mDC在WBC中所占百分比为0.28±0.13%,绝对数为15.12±4.66/μl,pDC在WBC中所占百分比为0.068±0.032%,绝对数为3.78±1.40/μl;培养获得具有典型形态特征的外周血单核细胞来源DC;健康人LIN阴性细胞中,共刺激分子CD80（B7-1）、CD86（B7-2）阳性细胞所占百分比分别为46.5±7.1%和51.1±9.6%,同时多数DC表达DC成熟标志CD83和HLA-DR,阳性细胞百分比分别为62.6±10.3%和73.4±8.1%,表明单核细胞经细胞因子诱导后大部分分化为成熟DC;证实少量DC即可刺激自体淋巴细胞产生较强的增殖效应。【结论】流式细胞仪检测外周血DC亚群简便、可靠;联合应用细胞因子GM-CSF、IL-4和TNF-α能够自外周血中成功分离、诱导培养具有典型形态特征及功能活性的单核细胞来源DC。第二部分手术及术后放疗对喉鳞癌患者外周血树突状细胞亚型及功能的影响【目的】研究手术及术后辅助放疗对喉鳞癌患者外周血树突状细胞亚型及外周血单核细胞来源树突状细胞功能的影响。【方法】采集46例经病理证实的喉鳞癌患者及15例健康志愿者外周血。46名喉鳞癌患者中男性44例,女性2例,年龄分布为32～76岁,平均年龄为57.57±10.09岁。根据2002年国际抗癌联盟（UICC）制定的TNM分期标准,Ⅰ期14例,Ⅱ期8例,Ⅲ期11例,Ⅳ期13例。46例患者入院后均接受手术治疗,28例患者手术后接受颈部直线加速器放射治疗。接受单纯手术治疗的患者（n=18）及接受手术+放疗的患者（n=28）均于治疗前、治疗中及治疗后分三次采集外周血标本。采用三色流式细胞计量术检测外周血HLA-DR⁺lineage^-树突状细胞及其亚型mDC、pDC在外周血WBC所占百分比并计算其绝对数。采用淋巴细胞分离液自外周血分离获得单核细胞,体外经人重组细胞因子GM-CSF、IL-4和TNF-α诱导培养,获得外周血单核细胞来源DC,流式细胞仪检测其表型,混合淋巴细胞反应（MLR）检测刺激自体T淋巴细胞增殖的能力。【结果】治疗前喉鳞癌患者外周血mDC计数、MoDC表面分子表达及刺激自体T淋巴细胞增殖能力均低于健康人;经手术治疗后,接受辅助放疗和未接受辅助放疗的患者其mDC计数、MoDC表面分子CD80,CD83,HLA-DR的表达均明显高于治疗前,但仅在术后未接受辅助放疗的患者中观察到CD86表达及刺激自体T淋巴细胞增殖能力的恢复。【结论】喉鳞癌患者DC存在缺陷;手术治疗可提高喉鳞癌患者外周血mDC计数及MoDC的功能;术后辅助放疗不影响外周血mDC计数的正常化,但对MoDC功能具有抑制作用。第三部分以EGFP标记的喉癌Hep-2细胞株总RNA转染树突状细胞的特异性抗喉癌实验研究【目的】探讨以EGFP为标记观察喉鳞癌Hep-2细胞株总RNA转染树突状细胞的可行性;探讨转染后DC诱导特异性识别Hep-2细胞的细胞毒性T淋巴细胞的可能性及特异性杀伤效应。【方法】采用淋巴细胞分离液自健康人外周血分离获得单核细胞,体外经人重组细胞因子GM-CSF、IL-4诱导培养,获得未成熟DC,观察细胞形态,流式细胞技术鉴定其表型;培养喉癌Hep-2细胞株,采用脂质体转染技术将携带绿色荧光蛋白质粒pEGFP-N1转染至Hep-2细胞株,通过G418筛选稳定表达EGFP的细胞株;Trizol一步法提取稳定转染的Hep-2细胞株总RNA,检测其完整性及纯度;以共孵育及电穿孔法将RNA导入未成熟DC,并以TNF-α促成熟;应用荧光显微镜观察并计算转染效率;通过流式细胞技术、混合淋巴细胞反应检测RNA转染后对DC表型、刺激T细胞增殖能力的影响;将转染后DC与同种异体T淋巴细胞共培养7天,收集效应细胞作为特异性CTL,通过⁵¹Cr释放法检测其对Hep-2细胞株的特异性杀伤效应。【结果】pEGFP-N1质粒转染喉癌Hep-2细胞株,经G418筛选可获得稳定表达EGFP的细胞株,荧光显微镜下可见绿色荧光,经20次传代后荧光仍无消失,Hep-2细胞形态及生长特性未见明显改变;经Trizol一步法提取稳定转染的Hep-2细胞株总RNA 80～100μg/10⁷细胞,分光光度法测定OD260/OD280=1.83,RNA电泳显示18S、28S条带,证实RNA的完整性;共孵育及电穿孔转染法转染效率分别为7～9%和21～23%;RNA导入未成熟DC后,细胞表达绿色荧光持续20～24小时;Hep-2-EGFP-RNA转染组DC表面分子CD83、HLA-DR表达较转染前及对照转染组显著升高,阳性细胞百分比达89.5%、95.4%,刺激同种异体T淋巴细胞增殖的能力显著提高;Hep-2-EGFP-RNA转染组DC体外诱导的CTLs可特异性杀伤喉癌Hep-2细胞,但对结肠癌HT-29细胞及卵巢癌OVCAR3细胞无杀伤活性。【结论】绿色荧光蛋白可作为喉癌Hep-2细胞RNA转染DC的标记;转染喉癌Hep-2细胞总RNA促进DC成熟,提高DC刺激同种异体T淋巴细胞增殖的能力,并能够产生喉癌Hep-2细胞特异性杀伤效应。更多还原

【Abstract】 Laryngeal squamous cell carcinoma（LSCC）is one of the most common malignancies, with an increasing occurrence of new cases and deaths annually.Despite all the progresses made in surgical,radiotherapy and chemotherapy techniques,long-term survival of LSCC patients has not been improved significantly over the past three decades,which poses a formidable challenge for physicians in ENT department.Thus, studies of certain key issues pertinent to LSCC,such as tumor immunology,are of great importance in getting insight into the mechanisms underlying the pathogenesis of LSCC and,in turn,resulting in effective therapeutics eventually.It has been suggested that anti-tumor immunity plays a pivotal role in the initiation and progression of malignancy.Induction of an effective anti-tumor immune response requires the activation of effector cells by antigen-presenting cells（APCs） that are responsible for the presentation of tumor-specific antigens.Of particular interest is the role of dendritic cells（DCs）,the key APCs which are specialized in taking up,processing,and presenting tumor antigen to stimulate na（?）ve T cells and to initiate strong tumor antigen-specific immune responses.Because of the important role in initiating,modulating and keeping immune response,DC-based immune therapy becomes the focus of present antitumor researches.It has been demonstrated that DCs could induce in present of combination cytokines.DCs pulsed with tumor antigens administered in different forms,such as peptides,cell lysates,proteins,apoptotic cells,DNA or RNA,could induce specific antitumor immune response.DC-based anti laryngeal carcinoma immune therapy is at its very beginning.Because of the fail in recognizing tumor specific antigens of laryngeal squamous cell carcinoma,DC pulsed with whole tumor-derived material present as a main strategy.It has been reported that DCs loaded with tumor lysates and fusions of DCs with tumor cells could induce effective anti-tumor effects in LSCC.Recently,it has been demonstrated that DCs transfected with specific or whole cell ribonucleic acid（RNA）is more effective and safe than DNA vector constructs. However,little is known about the antitumor effects of RNA pulsed DCs in LSCC.However,patients with advanced cancer including squamouse cell carcinoma of the head and neck（SCCHN）are known to be immunologically compromised,which allows malignant cells to evade the host’s anti-tumor defenses.The MHC restriction made it necessary to use autologous DCs in immune therapy.On the other hand,it would be ideal to use DC-based immunotherapy as a first choice,but in fact,it is more likely to be of use as an adjuvant treatment together with the conventional therapies.In concerning of laryngeal SCC,surgery and radiotherapy is still regarded as the most efficient treatment strategy world wide.Therefore,it is important to understanding changes of DCs in malignant tumor patients not only caused by the disease per se,but also by other aspects,such as different therapeutic regimens or other clinical features.In this study,we firstly investigate the effects of surgery and adjunctive radiotherapy on both circulating dendritic cells（DCs）and MoDCs of LSCC patients. Then we investigate the influence of Hep-2 cells total RNA transfection on monocyte derived dendritic cells（DCs）and the possibility of induction of antigen specific anti-tumor effects,as well as the feasibility of EGFP as a marker during RNA transfection.The aims of this current research are providing insight into the immune system status of LSCC patients,meanwhile assisting us in developing the proper DC-based anti-cancer immunotherapeutic strategies. PARTⅠCirculating Dendritic Cell Subsets detection by FACS and Induction of DC from Human Peripheral Blood MonoeyteObjective To investigate the convenient and reliable assay for circulating dendritic cell subsets detection,induct DC from peripheral blood monocyte of healthy donors.Methods Peripheral blood samples were collected from 15 healthy donors.Using multicolor flow cytometry,the percentages of lineage negative（LIN^-）and HLA-DR⁺ DC precursors,as well as their HLA⁺LIN^-CD11c+（myeloid）and HLA⁺LIN^-CD123⁺ （lymphoid）subsets,were determined in whole blood.Moreover,monocyte-derived DCs were generated utilizing granulocyte macrophage colony-stimulating factor （GM-CSF）,interleukin-4（IL-4）and tumor necrosis factor-α（TNF-α）.The morphous of cultured cells were observed using inverted microscope and electron microscope, then the phonotype of MoDCs was measured by flow cytometry and the ability to stimulate autologous T cells were tested in mixed leukocyte reaction（MLR）.Results The percentages of HLA-DR⁺ DC precursors,HLA⁺LIN^-CD11c+ DCs and HLA⁺LIN^-CD123⁺ DCs in peripheral WBCs were 0.414±0.13%,0.284±0.13%, 0.068±0.032%,respectively.The absolute numbers of these cells were 22.75±5.22/μl, 15.12±4.66/μl and 3.78±1.40/μl,respectively.Mature monocyte derived DCs of healthy donors displayed typical characteristics of morphology,which were non-adherent,displayed many fine spines on surface,rich chondriosome and poor lysosome in endoplasm.They expressed high level of CDS0,CD83,CD86 and HLA-DR,the percentages of positive expression of these surface marks were 46.54±7.1%,62.64±10.3%,51.14±9.6%and 73.44±8.1%.Few MoDCs could induce the proliferation of autologous T cells at the ratio of 1:10.Conclusion Multicolor FACS is convenient and reliable for circulating dendritic cell subsets detection but need proper gating strategy.We could successful isolate and culture mature DCs from peripheral blood of healthy donor in presence of GM-CSF, IL-4 and TNF-α.Those cells maintained the typical morphological characteristics and had immune activities. PARTⅡTherapeutic Influence on Circulating and Monocyte-derived Dendritic Cells in Laryngeal Squamous Cell Carcinoma PatientsObjective To investigate the effects of surgery and adjunctive radiotherapy on both circulating dendritic cells（DCs）and MoDCs of LSCC patients.Methods Forty-six laryngeal SCC patients and 15 age-matched healthy control subjects were enrolled in this study.Both in patients underwent surgery only（n=18） and those accepted adjunctive radiotherapy after tumor removal（n=28）,blood samples were taken before,during and after conventional treatment.Three-color flow cytometry was used for circulating DC subsets determination.Moreover, monocyte-derived DCs were generated utilizing granulocyte macrophage colony-stimulating factor（GM-CSF）and interleukin-4（IL-4）,then the phonotype of MoDCs was measured by flow cytometry and the ability to stimulate autologous T cells were tested in mixed leukocyte reaction（MLR）.Results The pre-operative mDC counts,MoDC surface molecular expression and stimulatory capacity were impaired in patients in comparison with controls.The number of mDC and the expression of CDS0,CD83,HLA-DR on MoDC were significantly increased as compared to those pre-treatment both in patients underwent surgery only and surgery followed with adjunctive radiotherapy.However,the recovery of CD86 expression and allostimulatory activity was only observed in patients accepted surgery only.Conclusion Surgical resection of laryngeal squamous cell carcinoma（LSCC） could be associated with improved circulating myeloid dendritic cell（mDC）number and monocyte-derived dentritic cell（MoDC）function.Although adjunctive radiotherapy after surgery did not effect the normalization of mDC number,it may have an impact on MoDC function. PARTⅢInduction of Laryngeal Squamous Cell Carcinoma Specific Anti-tumor Effects Using Dendritic Ceils Transfected with Hep-2 Cells Total RNA Marked with EGFP in VitroObjective To investigate the influence of Hep-2 cells total RNA transfection on monocyte derived dendritic cells（DCs）and the possibility of induction of laryngeal squamous cell carcinoma specific anti-tumor effects,as well as the feasibility of EGFP as a marker during RNA transfection.Methods Immature dendritic cells were generated from peripheral blood monocyte of healthy donor in the present of granulocyte macrophage colony stimulating factor（GM-CSF）and interleukin-4（IL-4）.Hep-2 cells were transfected with plasmid pEGFP-N1 using liposome and stably selected by G418.Total Hep-2-EGFP cellular RNA was generated using Trizol following the manufacturer’s protocol,the integrity and purity of total RNA were detected.Immature DCs were transfected with total RNA of Hep-2-EGFP cells by simple co-incubation and electroporation.Transfection efficiency was detected by fluorescence microscope. After transfection,DCs were incubated with TNF-αfor maturation.The phonotype of matured MoDCs transfected with total RNA was measured by flow cytometry and the ability to stimulate autologous T cells was tested in mixed leukocyte reaction（MLR）. For CTL induction,RNA transfected DCs were incubated with allogeneic T cells for 7 days,and the cytolytic activity of induced CTLs was analyzed by a standard ⁵¹Cr-release assay.Results EGFP was stably expressed in Hep-2 cells,the green fluorescence lasted for at least 20 generations.To attest the intactness of total Hep-2-EGFP cellular RNA, all RNA preparations were subjected to electrophoresis in gel under denaturing conditions with clear visualization of intact 18S and 28S ribosomal bands.Simple co-incubation and electroporation yield transfection efficiency of 7～9%and 21～23% respectively.The expression of maturation marks,such as CD83 and HLA-DR,on DCs transfected with total Hep-2-EGFP cellular RNA was significant higher than on those simply matured.Allogeneic T cell proliferation induced by transfected DCs was significantly higher than untransfected DCs.DCs transfected with total Hep-2 cellular RNA were capable of stimulating CTLs that specific recognized and lysed Hep-2 ceils, but not HT-29 cells（colon cancer）and OVCAR3 cells（ovarian cancer）.Conclusion EGFP could be used as a marker to observe the effect of transfection of DCs with total Hep-2 tumor cellular RNA.Transfection with total Hep-2 cellular RNA could up-regulation the expression of DC maturation marks,promote the stimulatory capacity to allogenic T cells,and induce specific CTL against Hep-2 cells. Thus,toal tumor RNA-transfected DCs may represent a broadly applicable vaccine strategy to induce potentially therapeutic T cell responses in laryngeal squamous cell carcinoma patients.更多还原