树突状细胞体外诱导淋巴细胞抑制舌癌细胞生长的实验研究

目的 :探讨树突状细胞 (dendriticcell,DC)通过淋巴细胞介导的免疫反应对舌癌细胞生长的影响。方法 :外周血单核细胞 ,在GM CSF IL 4的诱导下体外培养。其中一组加入TNF α ,另一组不加TNF α ;以不加任何细胞因子作为对照。用Tca8113细胞冻融抗原冲击后 ,加入自体淋巴细胞一起培养。再按 10 0∶1效靶比与Tca8113共培养 ,设未致敏淋巴细胞 Tca8113组 ,单独Tca8113组作对照。行形态学观察 ,免疫细胞化学检测DC的CD1a、CD83、HLA DR表达状况 ,MTT法检测Tca8113的存活率。结果进行统计学分析。结果 :( 1)加入TNF α组更高表达CD83 ,不加入TNF α组经肿瘤抗原刺激后 ,CD83表达急剧增加。 ( 2 )DC激活的淋巴细胞可显著抑制舌癌细胞的生长。结论 :GM CSF IL 4诱导的单核细胞来源的DC ,能体外摄取肿瘤抗原而进一步成熟 ,通过激活淋巴细胞抑制舌癌细胞生长 ;TNF α能促DC成熟 ,诱发更强的抑制作用。     

口腔癌患者外周血树突状细胞的体外培养及表型分析

目的：研究口腔癌患者外周血单核细胞来源的树突状细胞（dendritic cell，DC）形态、表型，与正常人进行比较并分析其临床意义。方法：15例口腔癌患者（实验组）及正常人（对照组）外周血单核细胞经GMCSF，IL4及TNF-α诱导培养，获取成熟树突状细胞，光镜和电镜观察细胞形态，流式细胞仪检测细胞表型CD1a、CD83、CD80、CD86和HLA-DR的表达变化。结果：培养7d后，2组均诱导出典型形态和表型的树突状细胞，实验组CD83、CD1a的表达率较对照组低，差异有统计学意义（均P〈0．001），但CD86、CD80、HLADR表达率2组差异无统计学意义（均P〉0．05）。结论：口腔鳞癌患者外周血单核细胞来源的DC经细胞因子诱导培养，能扩增出较大量高纯度并保持功能活性的成熟DC，为进一步开展以DC为基础的生物治疗和研究打下了基础。     

低剂量环磷酰胺联合树突状细胞瘤苗对荷舌鳞癌裸鼠的治疗观察

目的：观察低剂量环磷酰胺与树突状细胞瘤苗联合应用对裸鼠体内人舌癌细胞的抑瘤作用。方法：培养人外周血单核细胞来源的树突状细胞（DC），冻融法制备Tca8113舌癌细胞可溶性抗原并体外致敏DC，将同源人T淋巴细胞与之共同培养诱导出抗原特异性的CTL，联合低剂量环磷酰胺（Cy）共同接种至荷人舌癌的裸鼠。结果：抗原致敏的DC瘤苗与小剂量环磷酰胺联合后能比单用DC瘤苗更有效的治疗荷舌癌的裸鼠，与对照组相比，肿瘤生长明显抑制，生存率有显著提高（P〈0．05）。结论：以冻融抗原致敏的DC激活产生肿瘤特异性的CTL联合小剂量Cy能更有效的促进荷瘤宿主的免疫反应，具有显著的体内抑制舌癌的效果。     

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目的    研究不同年龄阶段口腔癌患者外周血单核细胞来源的树突状细胞（dendritic cell,DC）的形态、表型差异并分析其临床意义。方法    将2007—2010年兰州大学第一医院及第二医院口腔科住院的口腔鳞癌初诊患者30例,分为40～50岁及≥60岁2组,每组各15例。取患者外周血单核细胞经重组人粒细胞集落刺激因子（rhGM-CSF）、重组人白细胞介素4（rhIL-4）及肿瘤坏死因子-α（TNF-α）诱导培养,获取成熟树突状细胞,光镜观察细胞形态,流式细胞仪检测细胞表面协同刺激分子CD1a、CD83、CD80、CD86的表达。结果    培养7d后,2组均诱导出典型形态和表型的树突状细胞,其中≥60岁组DC培养过程中细胞逐渐死亡,培养至第7天大部分细胞死亡,获得的DC数量较少,有3例未能培养出DC;而40～50岁组培养细胞的活力保持较好,至第7天均可获得数量较多的DC。结论    2组口腔鳞癌患者外周血单核细胞来源的DC经细胞因子诱导培养,均能扩增出成熟DC,但较40～50岁组,≥60岁组外周血培养的DC体外生长活力明显降低,可能与老年患者免疫功能明显降低有关。     

致敏树突状细胞抗舌癌作用的实验研究

目的 Tca83人舌黏膜鳞状细胞癌细胞冻融抗原致敏DC.观察其对荷瘤裸鼠的押瘤作用,探讨致敏树突状细胞疫苗用于口腔癌患者免疫治疗的可行性.方法 用重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)、重组人白细胞介素4(rhIL-4)、重组人肿瘤坏死因子α(rhTNF-α)协同诱导人外周血单核细胞成为成熟DC.Tca83舌癌细胞可溶性抗原体外致敏DC,将同源人T淋巴细胞与之共同培养诱导出抗原特异性的细胞毒性T淋巴细胞(CTLs),共同接种至荷瘤裸鼠体内行免疫治疗.结果 人外周血单核细胞在细胞因子协同诱导下可获得功能正常的DC,用致敏Dc CTLs免疫治疗的荷瘤裸鼠与对照组相比,实验组肿瘤生长速度减慢,实验结束时(第21天)肿瘤体积明显小于对照组,明显抑制肿瘤生长(P<0.05).结论 采用舌癌细胞冻融抗原体外致教DC,诱导产生肿瘤抗原特异性CTLs具有显著的抑瘤效应,实验证明致敏Dc治疗舌鳞状细胞癌是一种有效的方法.有望成为口腔癌免疫治疗的新疗法.     

舌癌exosomes诱导CTL细胞体外抗瘤效应的观察

目的：研究舌癌exosomes诱导CTL体外抗肿瘤效应。方法：通过连续离心及超滤法从Tca8113细胞培养上清中分离、纯化出exosomes,然后与树突状细胞（DC）共培养,诱导T淋巴细胞活化为CTL。通过乳酸脱氢酶法测定特异性CTL对Tca8113的杀伤效应。采用SPSS11.0软件包对数据进行t检验。结果：exosomes可促使DC成熟。杀伤实验表明：负载exosomes的DC诱导CTL对Tca8113细胞的杀伤率显著高于未用exosomes负载的DC诱导的CTL及加IL-2培养的T细胞,其差异有显著性（P〈0.01）。结论：Tca8113细胞来源的exosomes负载DC后,可以诱导T细胞成为抗原特异性CTL,具有特异性杀伤肿瘤细胞的功能。     

腺样囊性癌细胞凋亡抗原负载的树突状细胞诱导抗肿瘤效应的体外研究

目的 探讨负载腮腺腺样囊性癌细胞凋亡肿瘤抗原的树突状细胞,诱导淋巴细胞介导的免疫反应,观察其体外杀伤腺样囊性癌细胞的细胞毒性效应.方法 外周血单核细胞,在粒细胞/巨噬细胞集落刺激因子(Granulocyte-macrophage colony-stimulating factor GM-CSF) 白细胞介素-4(Interleukin-4 IL-4)的诱导下体外培养,用凋亡肿瘤细胞抗原冲击后,流式细胞仪检测树突状细胞抗原负载前后CD1a、CD83表达量的变化.采用MTT比色法检测同种混合淋巴细胞反应和诱导细胞毒性T细胞(Cytotoxic T lymphocyte CTL)反应.结果 凋亡肿瘤抗原刺激后,CD83 表达急剧增加(P＜0. 01),而CD1a表达量下调(P＜0.01).负荷凋亡肿瘤抗原树突状细胞体外诱导出明显的细胞毒性反应,并刺激同种T淋巴细胞增殖.结论 GM-CSF IL-4 诱导的单核细胞来源的树突状细胞 ,能在体外摄取凋亡肿瘤抗原而进一步成熟,通过激活淋巴细胞杀伤癌细胞.     

树状细胞疫苗抑制肿瘤的实验观察

目的观察经肿瘤抗原致敏的树状细胞（DC）疫苗对荷瘤裸鼠的抑瘤作用。方法重组人粒细胞-巨噬细胞集落刺激因子（rhGM-CSF）、重组人白细胞介素4（rhIL-4）、重组人肿瘤坏死因子α（rhTNF-α）协同诱导人外周血单核细胞成为DC。Tea8113舌癌细胞可溶性抗原体外致敏DC，将同源人T淋巴细胞与之共同培养诱导出抗原特异性的细胞毒性T淋巴细胞（CTLs），共同接种至荷瘤裸鼠体内行免疫治疗。结果人外周血单核细胞在细胞因子协同诱导下可获得功能正常的DC，用致敏DC＋CTLs免疫治疗的荷瘤裸鼠与其他实验组相比，明显抑制肿瘤生长，肿瘤倍增时间显著提高（P〈0．05）。结论从人外周血来源的单核细胞在多种细胞因子作用下可诱导为功能正常的DC、冻融抗原体外致敏的DC，经其激活的肿瘤特异性CTLs对荷舌鳞癌裸鼠有明显的免疫治疗作用，可望成为口腔颌面部舌癌免疫治疗的新途径。     

口腔肿瘤患者外周血单核细胞CD69的表达及意义

目的：测定外周血单核细胞CD69分子的表达,探索口腔肿瘤患者的T细胞的活化功能。方法：实验对象10位健康人作为对照组;8位口腔良性肿瘤患者为实验组Ⅰ;10位口腔鳞状细胞癌的患者为实验组Ⅱ。每位实验对象在实验的当天,抽取10mL的静脉血。用Ficoll—Paque剃度离心法分离PBMC。将PBMC悬浮液分别与TCM,ConA和PHA培养4h和20h,分别收获细胞,用双色和单色单克隆荧光抗体CD4-FITC／CD8-PE和CD69-PE标记,CD69的表达用流式细胞仪和相应的分析软件SYSTEMTMII SOFTWARE分析。结果：在PBMC经ConA刺激4h后,口腔鳞状细胞癌患者的CD69表达的增长率（44．5％）明显低于对照组（67．8％）和实验组Ⅰ（70．8％）,P〈0．05。同样,经PHA刺激后4h,口腔鳞状细胞癌患者的CD69表达的增长率也低与其他两组（P〈0．05）,而且,PBMC的CD69表达率也低与其他两组（P〈0．05）。结论：本研究提示这种免疫反应力的下降可能是由于淋巴细胞的活化或增殖功能低下的原因。由PHA诱导的PBMC的CD69表达可能成为口腔癌患者免疫监视系统的指标之一。     

口腔扁平苔藓损害组织中树突状细胞亚群数量和比例

目的：研究口腔扁平苔藓（oral lichen planw,OLP）组织中树突状细胞（dendritic cell,DC）的亚群数量和比例,探索其在OLP疾病中的作用。方法：收集正常对照（HC组）,萎缩糜烂型OLP（E－OLP组）,斑纹型OLP（R－OLP组）的颊黏膜,通过流式细胞术染色和免疫组化染色,检测朗格汉斯细胞（CD1a＋CD11cint／lo CD207＋MHC II＋）、髓样DC （CD11c＋MHC II＋）、浆细胞样DC（CD11cint／lo CD123＋MHC II＋）和细胞毒性T细胞（CD3＋CD8＋）在组织中的浸润情况。结果：收集样本23例（HC组5例,E－OLP组7例,R－OLP组11例）,Bartlett检验各组数据方差不齐,采用Kruskal－Wallis非参数检验。朗格汉斯细胞的中位数比例分别为0．092％（HC）、0．564％（E－OLP）和0．541％（R－OLP）,3组间无统计学差异;髓样DC的中位数比例分别为0．311％（HC）、0．996％（E－OLP）和0．448％（R－OLP）,其中E－OLP组与HC组之间有统计学差异（P＜0．05）;浆细胞样DC的中位数比例分别为0．090％（HC）、3．490％（E－OLP）和2．010％（R－OLP）,2组OLP组与HC组之间均有统计学差异（P＜0．05）;CD8＋T细胞的中位数比例分别为0．126％（HC）、4．210％（E－OLP）和1．850％（R－OLP）,2组OLP组与HC组之间均有统计学差异（P＜0．05）。结论：DC在OLP中数量和比例增加,可能与疾病的自身免疫发病机制有关。     

Odontogenic ghost cell tumour with clear cell components: clear cell odontogenic ghost cell tumour?

A case of odontogenic ghost cell tumour (OGCT) with clear cell components was encountered in the mandible of a 63-year-old man. The tumour revealed ameloblastomatous-type epithelial components accompanied by clusters of ghost cells and dentinoid juxtaposed to the odontogenic epithelium. In addition, some areas of the tumour tissue showed sheets and islands of clear, glycogen containing epithelial cells, which were separated by a thin fibrous connective tissue stroma. Both ameloblastic and clear cells exhibited positive immunoreactivities for cytokeratin 19 and AE1/3. It is not known whether this tumour represents a clear cell change of a pre-existing OGCT or a separate and distinct neoplasm derived de novo from the odontogenic epithelium. This tumour was given the term 'clear cell OGCT' because it captures the clear cell components, which is one of the most prominent distinguishing features of the tumour.     

Establishment and characterization of a spindle cell squamous carcinoma cell line

BACKGROUND: Spindle cell squamous carcinoma (SCSC) is a rare and peculiar biphasic malignant neoplasm that occurs mainly in the upper aerodigestive tract. It consists of sarcomatoid proliferation of pleomorphic spindle-shaped cells and squamous cell carcinoma. METHODS: Here, we established a SCSC cell line from a tumour arisen in gingiva. We characterized the feature of a SCSC cell line by immunohistochemistry. To know the biological feature, we examined the cell growth, invasiveness and epithelial-mesenchymal transition markers of a SCSC cell line in comparison with oral squamous cell carcinoma (OSCC) cell lines. RESULTS: By immunohistochemical analyses, the primary tumour expressed cytokeratin and vimentin, indicating carcinosarcoma-like characters. This tumour also showed overexpression of p53 protein. Cultured SCSC cells resulted in bypass of crisis and maintenance over passage 100. The established SCSC cell line was spindle-shaped and showed identical immunohistochemical characters to those of primary tumour cells. Similar to the primary tumour, the cell line showed p53 overexpression and had p53 mutation at codon 132: AAG (lys)-->AAT (asp). The SCSC cell line grew slower than two other OSCC cell lines (MSCC-1 and HSC-2), whereas SCSC cells had remarkable invasiveness in comparison with these cell lines. Moreover, SCSC cells expressed wnt-5a and vimentin mRNA at high levels, but did not express E-cadherin mRNA. This expression pattern of the markers was similar to that of mesenchymal cells, not of epithelial cells. CONCLUSION: In the present study, we newly established a SCSC cell line with strong invasiveness. This is the first report on the establishment of SCSC cell line. The SCSC cell line can be a useful cell model for the study to know the cytodifferentiation and nature of SCSC.     

Craniofacial features of patients with sickle cell anemia and sickle cell trait

Objective:To identify the craniofacial characteristics of patients with sickle cell trait (SCT) and sickle cell anemia (SCA) and to compare these measurements with those of nonaffected subjects.Materials and Methods:Clinically normal patients and those with SCT and SCA were evaluated in this study. The patients were divided into three groups: normal (control), SCA, and SCT (n  =  with 15 in each group). Inclusion criteria were SCA or SCT verified by laboratory methods and no treatment with fixed orthodontics or facial orthopedics. Lateral cephalometric radiographs were carried out and were used to obtain angular and linear measurements of anatomic structures displayed. All markings and measurements were performed by a single examiner.Results:The average ANB was increased in groups with SCA (5.47 ± 2.0°) and SCT (3.80 ± 1.4°), indicating a tendency to Class II. The mean SNA angle was 83.0 ± 3.8° and 82.1±3.5° for SCA and SCT, indicating a proper positioning of the jaw from the skull base. There was an interaction between the group and sex factors for the variable SN-GoGn; measures were higher for men in the SCA group.Conclusion:Patients with SCA and SCT exhibited characteristics of Class II skeletal pattern because of mandibular retrusion. Most patients showed no compensatory maxillary expansion, which was determined by the normal jaw length and absence of maxillary protrusion.     

舌癌单细胞培养建系与癌干细胞相关标志的检测

目的 以舌癌Tca8113M1细胞系单细胞培养建系为基础,观察Tca8113M1细胞系中舌癌干细胞存在的现象及其相关标志的变化规律。方法 选取Tca8113M1细胞系,以有限稀释法进行体外单细胞培养并建立细胞亚系,在证实其成瘤性的基础上,进一步采用流式细胞术检测癌干细胞相关标志CD44、CD184、细胞外可溶性抗原（ESA）的表达情况,着重观察单个细胞培养形成细胞克隆的形态与时间。结果 以有限稀释法获取192个Tca8113M1舌癌单个细胞,在96孔板中进行体外培养,获取12个细胞亚系（获取比例为6.25%）,均有高成瘤性。癌干细胞相关标志CD44 与ESA均为高水平表达,而CD184表达则在12个细胞系之间有差异。在单个细胞培养中,形成完全克隆、部分克隆与旁克隆3种形态,12个细胞亚系均源于单个细胞形成的完全克隆,均可进行连续传代与扩增,而部分克隆与旁克隆则在后续培养中逐渐衰老与消亡。结论 Tca8113M1细胞系中可能存在癌干细胞,而单细胞培养可形成完全克隆并建立细胞亚系,是进行舌癌干细胞后续研究重要的细胞培养模式。     

体外成牙环境对牙髓干细胞和外胚间充质干细胞分化的影响

目的利用牙胚细胞（TGC）作为诱导成牙环境,将牙髓干细胞（DPSC）、外胚间充质干细胞（EMSC）分别与其共培养,观察DPSC和EMSC的分化能力。方法利用出生后4 d的大鼠TGC作为诱导成牙环境,将培养的DPSC、EMSC使用BrdU标记及鉴定后与其共培养,免疫荧光双标检测标记细胞表面抗原牙本质涎蛋白（DSP）的表达和碱性磷酸酶（ALP）活性的变化,免疫组化染色鉴定及图像分析DPSC、EMSC在成牙环境中的分化能力。结果共培养7 d后,EMSC共培养组DSP阳性细胞的转化率高于DPSC共培养组（P<0.05）。免疫组化染色图像分析结果表明共培养7 d后,共培养组与TGC单独培养组差异显著（P<0.05）。共培养组3、7 d后ALP活性明显增高,EMSC共培养组较DPSC共培养组ALP活性高。结论混合培养的TGC作为诱导细胞分化的微环境与DPSC、EMSC共培养后,能够诱导细胞向成牙本质细胞分化,EMSC分化能力高于DPSC。     

Basaloid-squamous cell carcinoma of the floor of the mouth: characterization of a cell line

Since it was first described in 1986. basaloid-squamous cell carcinoma (BSC) has been considered a distinct variant of squamous cell carcinoma that occurs in a variety of anatomic sites, including the head and neck region. We report the characterization of the first cell line established from a basaloid-squamous cell carcinoma of the floor of the mouth. The cell line exhibited a highly invasive capacity, indicating that BSC has very aggressive behavior. This cell line may be a useful model for elucidation of the biological characteristics of BSC.     

颌骨巨细胞瘤和巨细胞肉芽肿病变中多核巨细胞性质的探讨

目的 探讨颌骨巨细胞瘤(Giant cell tumor，GCT)和巨细胞肉芽肿(Giant cell granuloma，GCG)病变中多核巨细胞(Multinucleated giant cells，MGCs)的性质及组织来源。方法 对8例颌骨GET和8例GCG(中心性4例、外周性4例)进行酶组织化学和免疫组织化学观察，并与其它含MGICs的口腔病变(如异物反应、淋巴结结核等)进行比较。结果 颌骨GCT和GCG中的MGCs既可表达组织细胞／单核巨噬细胞相关抗原[如CD68、αl-抗胰蛋白酶(Alpha-1-antitrypsin，AAT)、α1-抗糜蛋白酶(Alpha-1-antichymotrypsin，AACT)、溶菌酶等]，又具有破骨细胞特异性酶-抗酒石酸酸性磷酸酶[Tartrate-resistant acid phosphatase(TRAP)]的活性，但不表达增殖细胞标记Ki-67抗原。结论 颌骨GCT和GCG中的MGCs可能是反应性终末分化细胞，由病变中处于不同分化阶段的前体破骨细胞融合而成．同时具有单核巨噬细胞和破骨细胞的某些表型特征。     

Mast cell degranulation and the role of T cell RANTES in oral lichen planus

OBJECTIVES: The present study investigated mast cell degranulation in oral lichen planus (OLP) and the effect of OLP lesional T cell supernatants on mast cell degranulation. MATERIALS AND METHODS: Immunohistochemistry was used to identify mast cell degranulation in both OLP (n = 22) and normal control (n = 14) tissues. OLP lesional T cell lines (n = 5) and HMC-1 (a human leukemia mast cell line) were used to examine the effects of OLP T cell supernatants on mast cell degranulation in vitro. RESULTS: Approximately 60% of mast cells were degranulated in OLP. OLP lesional T cells expressed mRNA for RANTES, and TNF-alpha stimulation upregulated OLP lesional T cell RANTES secretion. OLP lesional T cell supernatants induced degranulation of HMC-1 with release of TNF-alpha and histamine. Human recombinant RANTES similarly induced mast cell degranulation. Anti-RANTES antibody blocked OLP lesional T cell supernatant-induced mast cell degranulation. CONCLUSIONS: This study is the first to show that OLP lesional T cells produce and secrete RANTES which triggers human mast cell degranulation. Degranulating mast cells release TNF-alpha which upregulates OLP lesional T cell RANTES secretion. Such a cyclical mechanism may underlie disease chronicity and future therapies may include blocking RANTES or TNF-alpha activity in OLP.