人舌鳞状细胞癌细胞系WU-TSC-1的建立及鉴定

目的:建立1株人舌鳞状细胞癌细胞系WU-TSC-1并鉴定其基本生物学特性.方法:将所获得的舌鳞状细胞癌患者手术切除的组织块标本进行原代培养,传代后建立舌鳞状细胞癌细胞系WU-TSC-1,显微镜下观察并记录其细胞形态和生长特性,行基因组DNA短串联重复序列分析,并对其倍增时间、凝集反应、细胞表面标记物、染色体核型和裸鼠成...     

野生型p53基因对口腔鳞状细胞癌细胞系生长的影响

目的 :研究 p5 3基因对口腔鳞状细胞癌细胞系生长的影响。 方法 :以人鳞状细胞癌细胞系Tca83和Tca8113为实验对象 ,将载有人野生型p5 3cDNA的重组腺病毒 (Ad5CMV -p5 3 )感染鳞状细胞癌细胞系 ,观察Ad5CMV -p5 3对鳞状细胞癌细胞生长的影响。 结果 :鳞状细胞癌细胞系Tca83和Tca8113被转染Ad5CMV -p5 3病毒后 ,均可以诱导其发生调亡 ,使细胞系生长受到明显的抑制。结论 :Ad5CMV -p5 3介导的野生型p5 3基因治疗口腔鳞状细胞癌可能是一种有效的辅助手段     

严重联合免疫缺陷小鼠异种移植人舌鳞状细胞癌的初步研究

目的 建立稳定的人舌鳞状细胞癌(TSCC)严重联合免疫缺陷(SCID)小鼠异种移植瘤模型,初步研究其组织形态学以及生物学行为.方法 将未经治疗的新鲜人TSCC标本移植于SCID小鼠腹部皮下,当肿瘤生长至10 ～ 15 mm时,取出移植瘤再次移植传代,对比研究各代移植瘤的组织学形态,原位杂交以及免疫组织化学法检测肿瘤组织的来源.结果 75%(8/12)标本成功建立起稳定移植瘤模型,最长传至第5代.组织形态学对比显示移植瘤保持了原代瘤的组织学特征和异质性;免疫组织化学证实肿瘤细胞的人源性和基质细胞的宿主源性.结论 成功建立人TSCC SCID小鼠异种移植瘤模型,较好的保持了原代肿瘤的特性.     

直流电对鳞状细胞癌Tca-83细胞系抑杀作用的体外研究

目的:探讨直流电对人舌鳞状细胞癌Tca-83细胞系的抑杀作用。方法:对体外培养的人舌鳞状细胞癌细胞系Tca-83采用不同的电处理参数进行了直流处理。观察处理后癌细胞的形态、结构、生长情况、生物学特性;检测电处理后培养液的酸碱度、电解质的改变情况。结果:直流电处理对舌鳞状细胞癌有明显的杀伤及抑制作用。在电压一定的情况下增加电量可提高抑杀效率。电处理后的培养液pH,电解质,有机成分发生较大改变,其对癌细胞的生长有一定的抑制作用。电处理后未被杀死的癌细胞增殖能力也明显减低。结论:直流电处理可通过电解、电泳、电渗作用直接杀伤舌癌细胞,亦可通过改变细胞生长的外环境抑制舌癌细胞修复、生长、增殖     

共培养体系下血管内皮细胞中破骨细胞相关因子的表达

目的 检测血管内皮细胞和口腔鳞状细胞癌经体外共培养后内皮细胞细胞核因子κB受体活化因子配体( receptor activator of nuclear factor-κB ligand,RANKL)表达,了解肿瘤性骨破坏中肿瘤细胞、内皮细胞和破骨细胞之间的关系.方法 选取3对细胞系,即:小鼠鳞状细胞癌和血管内皮瘤细胞系、人口腔鳞状细胞癌和人脐静脉血管内皮细胞系、人鳞状细胞癌手术标本细胞与人脐带所得内皮细胞,3对细胞共培养.分别对培养前、后的内皮细胞进行RANKL的免疫组化染色观察.结果 共培养前,3种血管内皮细胞RANKL免疫组化染色阴性;经共培养后,内皮细胞出现RANKL的阳性表达.结论 血管内皮细胞和鳞状细胞癌细胞可成功共培养;共培养后血管内皮细胞在鳞状细胞癌细胞诱导下可以有RANKL的表达,为其分化活化提供条件.     

口腔黏膜上皮细胞体外癌变模型的建立

目的诱导永生化口腔上皮细胞系（HIOEC）细胞恶性转化，建立口腔黏膜上皮细胞体外癌变模型。方法通过化学致癌剂苯并芘[B（a）P]体外诱导HIOEC细胞，逐步筛选到鳞状细胞癌细胞系HIOEC-B（a）P。通过微分干涉显微镜和HE染色观察细胞形态学改变；用软琼脂集落形成及裸小鼠异体皮下成瘤的实验鉴定HIOEC-B（a）P细胞的恶性表型。结果①HIOEC细胞在B（a）P诱导培养后可以在正常生理钙离子浓度、含胎牛血清培养液中生长；②细胞在诱导过程中形态多变，最后稳定为多角形铺路石样上皮细胞；③第93代HIOEC-B（a）P细胞开始具备软琼脂集落形成能力；④HIOEC—B（a）P第55代细胞在裸小鼠皮下首次形成角化团块，第69代细胞形成分化较好的典型鳞状细胞癌，第74代、第96代细胞均形成与临床病理相似的Ⅰ～Ⅱ级鳞状细胞癌。结论生物因素合并化学致癌因素B（a）P可以诱导永生化口腔黏膜上皮细胞恶性转化，为进一步研究口腔黏膜上皮细胞多因素、多步骤癌变机制提供实验模型。     

人颊粘膜鳞癌细胞株在裸小鼠颊囊粘膜下原位移植及组织病理学和侵袭特性的实验研究

将人颊粘膜鳞状细胞癌细胞株ＢＣａＣＤ８８５细胞移植入裸小鼠颊囊粘膜下，观察原位移植的成瘤、形态学特征（光镜、电镜、免疫组织化学）及其对周围组织的侵袭性。原位移植成瘤所需接种癌细胞量为１．０～２．０×１０６个细胞／０．２ｍｌ，在荷瘤鼠颊部可见肿瘤局部浸润性生长，侵袭颌骨及肌肉组织，未发现淋巴道及血运转移。原位移植瘤的病理学、超微结构及角蛋白表达均与亲代人颊粘膜鳞状细胞癌相似。结果说明：人颊鳞癌细胞株在裸鼠颊囊粘膜下自主生长，其微环境及生物学特征表达较腋下移植更客观地模拟了患者体内的状况。表明移植部位微环境对移植瘤的生长及生物学行为有一定影响。此模型可用于颊癌局部生物学特性及实验治疗的研究。     

耐长春新碱人舌鳞癌细胞系Tca8113/V100的研究

目的建立耐长春新碱（Ｖｉｎｃｒｉｓｔｉｎｅ，ＶＣＲ）人舌鳞癌细胞系。方法采用间歇性加药，逐步提高培养基中ＶＣＲ的浓度，连续体外诱导，培养，建立了一株耐ＶＣＲ人舌鳞癌细胞系Ｔｃａ８１１３／Ｖ１００。对其药物敏感性、倍增时间、裸鼠成瘤性、超微结构及耐多药基因等生物学特性进行了研究。结果Ｔｃａ８１１３／Ｖ１００在含ＶＣＲ１００μｍｏｌ／Ｌ的培养基中稳定生长，其对ＶＣＲ的耐药性为Ｔｃａ８１１３细胞系的１９２倍，对其他几种抗癌药物也有程度不同的耐药性。Ｔｃａ８１１３／Ｖ１００倍增时间３７２小时，具有裸鼠成瘤性，病理诊断为鳞状细胞癌，超微结构观察其微绒毛短粗，ｍｄｒ１ｍＲＮＡ表达水平明显增高。结论Ｔｃａ８１１３／Ｖ１００细胞系已具备多药耐药性的特点，为口腔癌多药耐药性研究提供了细胞模型。     

甘草苷调控PI3K/Akt/m-TOR信号通路对口腔鳞状细胞癌细胞及裸鼠移植瘤小鼠的作用研究

目的:目的探讨甘草苷(Liquiritin,LIQ)对人口腔鳞状细胞癌细胞(OSCCs)的增殖抑制、凋亡诱导作用和分子机制并通过裸鼠模型进行验证。方法:CCK-8检测LIQ对口腔鳞状细胞癌细胞的增殖抑制作用;流式细胞术及TUNEL实验检测凋亡情况;Western blot检测蛋白表达变化;构建裸鼠瘤模型,连续给药20 d,绘制肿瘤生长曲线,并计算抑瘤率。结果:LIQ可以抑制口腔鳞状细胞癌细胞增殖及裸鼠瘤体积增长,并诱导细胞凋亡;LIQ抑制PI3K/Akt/m-TOR信号通路的过度激活。结论:LIQ能够在体内外对口腔鳞状细胞癌产生抗癌效果,其机制可能是通过抑制PI3K/AKT/m-TOR信号通路的过度激活。     

苯丙芘联合佛波酯恶性转化HIOEC细胞的实验研究

目的:诱导永生化口腔上皮细胞系HIOEC细胞恶性转化,建立HIOEC-B(a)P-TPA细胞系。方法:通过递增浓度的化学致癌剂苯丙芘复合促癌剂佛波酯体外诱导HIOEC细胞,筛选得到具有恶性表型的鳞状细胞癌细胞系HIOEC-B(a)P-TPA。通过微分干涉显微镜和HE染色观察细胞形态学改变,免疫组化染色检测细胞角蛋白和波形蛋白表达情况;软琼脂集落形成、裸小鼠异体皮下成瘤性鉴定HIOEC-B(a)P-TPA细胞的恶性表型。结果:(1)HIOEC细胞诱导培养6个月后,可以在正常生理钙离子浓度、含胎牛血清培养液中生长;(2)细胞在诱导过程中形态多变,最后以成纤维样细胞为主,异形性明显;(3)HIOEC-B(a)P-TPA细胞中细胞角蛋白表达减弱,同时表达波形蛋白;(4)有很强的软琼脂集落形成能力,集落形成率为24.5%;(5)HIOEC-B(a)P-TPA细胞尚未观察到成瘤性。结论:生物化学致癌因素B(a)P和TPA可诱导HIOEC细胞恶性转化,反映了口腔黏膜上皮细胞体外恶变发生、发展过程。     

Characteristics of a cancerous cell line, HIOEC-B(a)P-96, induced by benzo(a)pyrene from human immortalized oral epithelial cell line

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the oral and maxillofacial region. The mechanism of carcinogenesis of OSCC is still unclear. In vitro study on OSCC cell lines, especially derived from immortalized oral epithelial cells, is a very useful strategy to understand the mechanism of carcinogenesis. Based on our previous human immortalized oral epithelial cell (HIOEC) line, obtained from normal oral epithelial cells by transfection of HPV16 E6/E7 gene, a new cancerous cell line, HIOEC-B(a)P-96 (HB96), was established from the HIOEC by induction with benzo(a)pyrene. The characteristics of the HB96 cells such as cell morphology, ultrastructure, proliferation ability, invasion ability, and tumorigenesis were studied. The HB96 cells lost contact inhibition with uncontrolled cell division and obvious cell overlap, they were polygonal in shape and ununiform in size with increased ratio between nucleus and plasma. Increased proliferative ability and invasion ability were confirmed by the cell proliferation analysis and cell invasion assay, respectively. The tumorigenicity of well to moderately differentiated squamous cell carcinoma was confirmed in the nude mice experiments pathologically. Increased expression of HPV16 E6/E7 proteins and obvious correlation with decreased expression of p53 and Rb proteins was also confirmed by Western blotting. Thus, this HB96 cell line induced by benzo(a)pyrene from the HIOEC line is a useful tool to study the mechanism of carcinogenesis of OSCC in vitro for future genomic and proteomic analyses. It is also the first in vitro cancerous cell line of OSCC in China derived from immortalized oral epithelial cells.     

口腔鳞癌中趋化因子受体CXCR4的临床病理及细胞学研究

目的检测CXCR4蛋白在口腔鳞状细胞癌（OSCC）组织和OSCC细胞系中的表达,探讨CXCR4蛋白的表达与OSCC临床病理及SDF-1/CXCR4轴与OSCC细胞增殖的关系.方法用免疫组织化学SP法检测91例OSCC中CXCR4的表达,用细胞培养、免疫组化法检测CXCR4蛋白的表达模式,流式细胞仪直接免疫荧光法测定CXCR4蛋白的表达量,MTT法检测细胞的增殖能力.结果CXCR4在OSCC组织中总阳性表达率为62.6%,与肿瘤的大小、病理分级、淋巴结转移、肿瘤增殖密切相关（P＜0.05）.CXCR4蛋白在OSCC细胞系中亦呈强阳性表达,OSCC细胞在SDF-l作用下增殖反应明显增强,CXCR4抗体可明显抑制肿瘤细胞的增殖.结论CXCR4与肿瘤的大小、病理分级、淋巴结转移及肿瘤增殖密切相关,对评估OSCC的生物学行为有意义.     

Identification and characterization of a highly metastatic epithelial cancer cell line from rat tongue cancer

ObjectivesTongue squamous cell carcinoma (TSCC) is a clinically devastating disease. However, most established TSCC cell lines currently show undesirable malignant behaviours. The purpose of this study is to establish a highly metastatic TSCC cell line to serve as a useful tool for basic research.Materials and methodsTSCCs were induced by 4-nitroquinoline-1-oxide (4NQO) in Sprague-Dawley rats. Tumor cells were obtained from the cancer tissues by primary culture and were then purified by an in vitro invasion assay and a limiting dilution assay. The growth rate, cell cycle distribution, apoptotic rate, tumorigenicity and distant metastatic phenotypes of the rat tongue cancer cells were fully investigated and characterized.ResultsTo date, the rat tongue cancer cell line, named Rca-T, has been continuously cultured in vitro for over 210 passages and exhibit a long spindle-shaped morphology, adherent growth, and a stable epithelial phenotype. The population doubling time of Rca-T cells is 23.35 h. Approximately 39.8% of these cells are in S phase, and the apoptosis rate of Rca-T cells is 7.46%. Furthermore, in immunodeficient nude mice, both the xenograft rate and the incidence of experimental lung metastasis are 100%. The in vitro assays further reveal the highly malignant and epithelial-mesenchymal transition-like properties of Rca-T cells.ConclusionIn this study, the tumorigenic and highly distant metastatic TSCC cell line Rca-T was established. The malignant features of this cell line, especially its metastatic potential, will enable a wealth of functional studies on the molecular mechanisms of TSCC metastasis in the future.     

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.     

Establishment of OC3 oral carcinoma cell line and identification of NF-κB activation responses to areca nut extract

BACKGROUND: Cell lines derived from oral squamous cell carcinoma (OSCC) exposed to variable etiological factors can bestow advantages in understanding the molecular and cellular alterations pertaining to environmental impacts. Most OSCC cell lines have been established from smoker patients or areca chewing/smoker patients, carrying the genomic alterations in p53. METHODS: A new cell line, oral carcinoma 3 (OC3), was established from an OSCC in a long-term areca (betel) chewer who does not smoke. Cellular and molecular features of OC3 were determined by variable assays. RESULTS: The cultured monolayer cells were mainly polygonal and had the expression of cytokeratin 14. The chromosomal analysis using comparative genomic hybridization has revealed the gain in chromosomes 1q, 5q, and 8q, the loss in 4q, 6p, and 8p as well as the gain of entire chromosome 20. Loss of heterozygosity and instability in multiple microsatellite markers in chromosome 4q were also noted. OC3 cells bear wild-type p53 coding sequence and have a high level of p53 expression. Its p21 expression was similar to that in normal human oral keratinocyte (NHOK). Interestingly, activation of nuclear factor kappa B (NF-kappa B) in OC3 cells following the treatment of areca nut extract was observed. CONCLUSION: OC3 cell line could be valuable in understanding the genetic impairments and phenotypic changes associated with areca in oral keratinocyte.     

CXCR4受体在口腔鳞癌细胞系中的表达及对细胞增殖和迁徙的影响

目的:观察口腔鳞癌细胞系(OSCC)中趋化因子受体CXCR4的表达,检测SDF-1/CXCR4反应轴对OSCC增殖的作用,SDF-1对CXCR4阳性肿瘤细胞的趋化作用,探讨CXCR4受体在OSCC中的功能及活性。方法:细胞涂片免疫荧光法检测CXCR4蛋白在OSCC细胞系的表达,流式细胞仪直接免疫荧光法检测OSCC细胞系中CXCR4蛋白的表达量,MTT法检测细胞的增殖能力,体外迁徙实验检测SDF-1/CXCR4反应轴对OSCC细胞的趋化作用。采用SPSS10.0软件包进行ANOVA方差分析和t检验。结果:CXCR4蛋白在OSCC细胞系呈阳性表达,表达率为68.62%。OSCC细胞在SDF-l作用下,其增殖反应显著增强,CXCR4抗体可显著抑制肿瘤细胞的增殖,SDF-1可显著诱导OSCC细胞的移动。结论:CXCR4受体与OSCC细胞增殖、迁徙功能有一定关系。     

化学致癌剂4-NQO饮水法诱导小鼠口腔鳞癌模型的建立

目的 ：使用化学致癌剂4-硝基喹啉-1-氧化物（4-nitro-quinoline 1-oxide,4-NQO）诱导并建立C57BL/6小鼠口腔鳞癌病变模型。方法 将4-NQO加入小鼠饮水,观察小鼠口腔黏膜的变化。于第12、16、20、24周处死,采集病变组织的样本,确定病变性质,并分离、培养肿瘤细胞。结果 实验组小鼠在16周后口腔黏膜有明显的外生性肿物发生,直径约0.2 mm。小鼠口腔黏膜肿物H-E染色结果显示,肿物呈外生性或浸润性生长,可见角化珠和明显的核分裂象。细胞呈多边形,贴壁牢固,CK、Ki-67染色阳性,表明此细胞为鳞癌细胞。结论 该模型诱导过程与人口腔鳞癌发病过程相似,是理想的研究口腔鳞癌发生、发展机制的动物模型。     

Establishment of a highly metastatic tongue squamous cell carcinoma cell line from New Zealand White rabbit

OBJECTIVE: Prior to this study, the widely used tongue squamous cell carcinoma cell lines could only initiate tumours in immunodeficient mice, which greatly delayed studies on immune function during carcinogenesis. This study established a new tongue squamous cell carcinoma cell line named 'RSCC-1', which can initiate tumours in both immunocompetent rabbits and immunodeficient nude mice and has high metastatic ability. DESIGN: Primary tongue cancer was induced by DMBA and local mechanical stimulation in New Zealand White rabbits. The induced cancer was serially transplanted into homogeneous rabbits to establish transplanted models. At the same time, cancer samples were collected, cultured and passaged in vitro. Finally, a cell line named 'RSCC-1' was established. Its growth behaviour, cell cycle distribution and tumourigenicity in rabbits and nude mice were investigated. RESULTS: RSCC-1 cells were cultured continuously in vitro for 19 months (165 passages). They contain between 54 and 196 chromosomes, with a modal number of 75. Tumourigenicity rates were 100% in both homogeneous rabbits and nude mice, with 20% lung metastasis and 50% cervical lymph node metastasis in homogeneous rabbits. CONCLUSION: RSCC-1 is a poorly differentiated, highly malignant rabbit tongue squamous cell carcinoma cell line. Its behaviour in the inoculated animal model closely resembles human tongue cancer, and could metastasise to local lymph nodes and remote organs.     

Increased expression of Cathepsin B in oral squamous cell carcinoma

Previously, an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC) was established with a line of human immortalized oral epithelial cells (HIOECs), a line of cancerous HB96 cells and another type of cell (HB56 cells) at the early stage of carcinogenesis. In this study, comparative proteomic analysis identified a panel of differentially expressed proteins among these cells. Cathepsin B was one of the significantly up-regulated proteins accompanying cellular transformation. Cathepsin B was further validated for its expression in the three cell lines and in clinical samples of tumour tissues and their adjacent normal epithelia from 30 primary OSCC patients. Western blot analysis and real-time PCR detected increased Cathepsin B protein and mRNA levels in the cancerous HB56 and HB96 cells over HIOECs. Immunohistochemistry and real-time PCR showed elevated Cathepsin B protein and mRNA levels in the tumour tissues over the adjacent non-malignant epithelia from OSCC patients. The results presented here suggest that the expression of Cathepsin B increases along with the cancerisation in OSCC both in vitro and in vivo, and it may serve as a candidate biomarker of OSCC.