兔VX-2舌鳞癌移植瘤模型的建立及生物学特性观察

目的：建立可供实验研究的稳定的兔VX2舌鳞癌移植瘤模型,观察其主要生物学特性。方法：制备VX2肿瘤荷瘤种兔后,将荷瘤种兔新鲜鳞癌组织块移植于24只新西兰大白兔的舌侧缘中1/3黏膜下,制成兔VX2舌鳞癌移植瘤模型。荷瘤动物随机分为2组,第1组（n=6）不同时期分批处死,进行大体观察、组织病理及电镜检查;第2组（n=14）作为对照组,观察荷瘤动物的自然生存期。结果：成瘤率为83.3%（20/24）;移植瘤的组织学特征与移植于兔其他部位的VX2肿瘤基本一致,在舌体内浸润性生长并向周围侵袭、扩散,符合中分化鳞状细胞癌的特征。结论：成功建立了兔VX2舌鳞癌移植瘤模型。该模型成瘤率高,潜伏期短,生长迅速,生物学特性稳定,易于复制,接近人舌鳞癌的临床特征,较客观地反映了舌癌的生物学行为,为舌鳞癌的研究提供了较理想的大型动物模型。     

兔舌不同部位VX-2鳞癌与颈淋巴结转移模型的生物学特性

目的:制作兔舌不同部位VX-2鳞癌和颈淋巴结转移模型,并对其生物学特性进行研究,为舌癌多学科治疗提供研究平台。方法:将新西兰白兔20只,随机分为4组,每组5只,分别植入VX-2鳞癌瘤块。观察瘤块植入后肿瘤生长、体积变化、成瘤率、颈淋巴结转移率、转移的部位、数目以及兔生存期等指标。数据采用SPSS11.5软件包进行游程检验、独立样本t检验和χ2检验。结果:总成瘤率75%,其中舌缘80%,舌中线区70%,颈淋巴结转移率均70%,舌缘转移至同侧颈淋巴结,舌中线区转移至双侧颈淋巴结。平均生存期为舌缘(33.25±6.20)d,舌中线区(39.00±3.74)d。各区间无统计学意义(P>0.05),舌癌和颈部转移淋巴结均为低分化鳞癌。结论:瘤块移植法能成功制作不同部位兔舌VX-2鳞癌模型,成瘤率和转移率均较高,生存期为4～6周。其生物学特性与人舌癌基本一致,生长迅速,容易转移,是研究舌癌较理想的动物模型。     

Cal－27舌鳞癌动物模型的建立及其意义

目的：建立Cal－27舌鳞癌动物移植瘤模型,研究肿瘤细胞的生物学行为及转移瘤的特性。方法：将107／0．2 mL Cal－27细胞悬液通过在裸鼠左腋背部皮下注射接种,建立移植瘤模型。达到成瘤标准后,定期测量肿瘤大小并记录体质量变化,4周后处死裸鼠,通过组织切片HE染色法观察组织结构异型性和细胞异型性,并观察肺、肝有无转移。结果：实验裸鼠在接种后平均14天达到成瘤标准,24只裸鼠移植瘤中,成瘤率100%。未见有肺、肝脏的转移。结论：在细胞接种的数量、肿瘤接种的部位、实验动物的周龄及状态等内外部条件都适宜的情况下,成瘤率可以达到100%。该模型较客观地反应了人舌鳞癌的生物学行为,为以后的动物模型研究提供了较为适宜的注射剂量,为实验研究提供了理想的动物模型。     

Tca8113细胞系下颌下淋巴结转移动物模型的建立

目的:建立人口腔鳞癌Tca8113裸鼠舌体及颊黏膜移植瘤淋巴结转移模型,研究分析两种转移模型的特性。方法:通过在裸鼠舌体及颊黏膜内注射接种Tca8113细胞,建立移植瘤动物模型。舌体及颊部原发灶分别在2周和4周时被切除,观察6周时下颌下淋巴结转移情况。通过组织切片HE染色法计算淋巴结转移率,并采用CK蛋白免疫组织化学染色方法计算淋巴结微灶转移率。结果:13只舌体接种移植瘤裸鼠中,有3只出现下颌下淋巴结转移,5只出现淋巴结微灶转移,转移率达到61.54%。15只颊黏膜接种移植瘤裸鼠中,3只出现下颌下淋巴结转移,3只出现淋巴结微灶转移,转移率为40.00%。结论:裸鼠舌体及颊黏膜接种Tca8113建立的转移模型具有一定的转移率,并能较好地模拟临床口腔鳞癌的转移,为实验研究提供了良好的动物模型。     

抗感染对伴炎症兔颊VX2鳞状细胞癌组织中树突状细胞表面分子HLA-DR和CD86表达的影响

目的 探讨抗感染对伴炎症兔颊VX2鳞状细胞癌（鳞癌）组织中树突状细胞（DCs）表面分子HLA-DR和CD86表达的影响。方法 通过接种VX2瘤粒、形成机械创伤、饮用高糖黏性牛奶的方式,建立兔颊VX2鳞癌炎症模型。实验分为4组。A组（12只）：颊癌伴炎症模型兔,连续3 d普鲁卡因青霉素肌注和替硝唑片灌胃。B组（12只）：颊癌伴炎症模型兔,连续3 d生理盐水肌注和阿司匹林灌胃。C组（12只）：颊癌伴炎症模型兔,伴有局部炎症的兔颊癌,连续3 d生理盐水肌注和灌胃。D组（10只）：单纯颊癌模型兔,连续3 d生理盐水灌胃和肌注。实验结束后处死所有兔,收集肿瘤标本,流式细胞仪检测肿瘤组织中DCs表面分子HLA-DR和CD86的表达。结果 HLADR单独表达率、HLA-DR和CD86同时表达率均为A组>D组>B组>C组,CD86单独表达率为A组>D组>B组及C组（P<0.05）。结论 抗感染治疗能明显提高伴炎症的兔颊癌组织中DCs表面分子HLA-DR和CD86的表达。     

人口腔鳞癌手术标本组织块裸鼠移植瘤模型的建立

目的：口腔鳞癌是口腔颌面部常见的恶性肿瘤之一，建立与人类口腔癌相似的动物模型，对进一步探讨口腔癌的发生、发展、治疗和预后具有重要意义。方法：选取新鲜的口腔鳞癌手术标本，制备成1mm^3组织块，植入BALB／cnu—nu裸鼠皮下．观察其成瘤情况．并对移植瘤的生长特性、组织形态和对化疗药物的反应进行检测。结果：收集了27例临床标本，成功建立了15个裸鼠皮下移植瘤模型，成瘤率为56％。所建立的口腔鳞癌移植瘤模型能够在裸鼠体内连续成瘤。与细胞系建立的移植瘤模型相比，其组织学特征与人口腔鳞癌标本极为相似。其中，20％（2／10）的移植瘤模型对传统化疗药物顺铂敏感。结论：成功建立了人口腔鳞癌手术标本组织块移植瘤模型。该模型为进一步研究口腔鳞癌提供了基础。     

兔舌癌哨位淋巴结微转移模型的建立

目的:建立兔舌癌哨位淋巴结(sentinel lymph node,SLN)微转移模型,为进一步研究舌癌区域淋巴结转移的发生、发展机制及防治提供实验平台。方法:先正位、多循环移植Vx-2癌瘤于兔舌,每次8只兔,筛选出高淋巴转移潜力移植瘤;再将其移植到40只兔的舌部,分别于移植后第8、10、12、14和16天各随机处死8只,取出兔舌Vx-2移植瘤SLN,经连续切片和免疫组织化学检测其微转移形成情况。应用SPSS 17.0软件包对结果进行统计学分析。结果:经3次正位循环移植后,所得Vx-2移植瘤舌移植后SLN转移率达100%(8/8);该高淋巴转移潜力移植瘤移植兔舌后10~12 d,所获移植瘤SLN微转移发生率为100%(8/8)。结论:应用本研究筛选的高淋巴转移潜力Vx-2瘤移植兔舌后10~12 d,可获得较稳定的兔舌癌SLN微转移模型。     

人内皮抑素基因导入对裸鼠舌鳞癌移植瘤的抑制影响

目的:探讨经体内注入人内皮抑素(humanendostatin,hES)基因治疗口腔鳞癌的可行性。方法:荷瘤人舌鳞癌Tca8113细胞裸鼠瘤体内和肌内分别注射含hES的基因质粒,观察转入hES基因对舌鳞癌移植瘤生长的影响,免疫组织化学检测肿瘤组织中hES及血管内皮细胞生长因子(vascularvesselendotheliacellgrowthfactor,VEGF)的表达,Weidern法行肿瘤微血管密度(microvesseldensity,MVD)计数,流式细胞仪检测肿瘤细胞凋亡率,采用精确概率法和单因素方差分析进行统计学处理。结果:荷瘤注射hES后第16天,瘤体注射组抑瘤率为75.0%,肌内注射组抑瘤率为66.1%(P<0.01)。免疫组织化学检测结果显示,hES在瘤体注射组和肌内注射组肿瘤中的表达率分别为90.0%和85.0%,均高于对照组的20.0%(P<0.01)。2实验组肿瘤MVD值分别为18.60±3.44和16.70±2.63,均低于对照组的22.40±3.41(P<0.01)。流式细胞仪检测发现,瘤体注射和肌内注射hES基因质粒后,肿瘤细胞凋亡率分别为11.36%±3.20%和8.08%±2.00%,均高于对照组的1.80%±0.50%(P<0.01)。结论:注射hES基因具有抑制舌鳞癌Tca8113细胞移植瘤生长的效应。     

人口底鳞癌裸鼠原位移植模型的建立及其生物学特性

目的：建立一种与临床更为相关的人口底鳞癌动物模型。方法：30只6周龄裸鼠,雌雄各半。对人口底鳞癌细胞HSC-2传代培养、消化、离心后调整细胞浓度达4×108。口底黏膜下注射细胞悬液0.1mL。4周后切除移植瘤并剪成1mm3瘤块,植入裸鼠口底一瘤块,共传5代。观察记录移植成功率,生长潜伏期,每周2次测量瘤体体积。对每代移植瘤和裸鼠口底,颌下组织,肉眼可见的淋巴组织以及下颌骨进行病理学检查,透射电镜观察第5代瘤体,制备染色体标本进行染色体分析。结果：移植成功率为93%。移植瘤病理检查结果符合鳞状细胞癌典型特征,染色体检查表明其属于人类染色体。7只裸鼠发生口底和颌下浸润,其中一只转移至颌下淋巴结。癌细胞呈侵袭性生长,浸润肌肉,神经和周围血管。透射电镜观察：肿瘤成巢状生长,细胞分化高,核成圆形,细胞器发达。结论：本模型客观的反应了人口底鳞癌的生物学行为,为口底鳞癌的生物学及实验治疗的研究提供了较为理想的动物模型。     

放射诱导启动子介导p53上调凋亡调控因子基因靶向治疗舌鳞状细胞癌裸鼠移植瘤

目的探讨放射诱导启动子介导p53上调凋亡调控因子(PUMA)基因靶向治疗舌鳞状细胞癌(简称舌鳞癌)裸鼠移植瘤的疗效。方法构建放射诱导启动子介导PUMA基因表达的真核表达质粒pcDNA(+)3.1/E-PUMA,建立人舌鳞癌裸鼠移植瘤模型,以脂质体为载体进行瘤内转染,辅以3Gy放疗,描述肿瘤生长曲线,反转录聚合酶链反应(RT-PCR)检测PUMA的mRNA水平,免疫组化检测细胞核增殖抗原的表达,原位末端标记法检测细胞凋亡。数据分析采用SPSS 11.0统计软件,两样本均数比较采用χ2检验。结果放射诱导启动子介导的PUMA基因转染对舌鳞癌裸鼠移植瘤生长有明显抑制作用,诱导放疗显著增强了PUMA的mRNA表达水平,RT-PCR检测发现其电泳条带灰度值从(0.325±0.075)上调至(0.371±0.096),P<0.01;诱导放疗可显著提高疗效,增殖指数由32.27%下降至22.77%,P<0.01;凋亡指数从14.43%上升为27.98%,P<0.01。结论放射诱导启动子作为基因治疗分子开关可介导PUMA基因在人舌鳞癌裸鼠移植瘤中靶向表达促进细胞凋亡,为舌鳞癌放射基因治疗提供了新思路。     

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.     

Preliminary study of indirect CT lymphography-guided sentinel lymph node biopsy in a tongue VX2 carcinoma model

The feasibility of using indirect CT lymphography (CT-LG) to guide sentinel lymph node (SLN) biopsy in a rabbit model of tongue VX2 carcinoma with cervical lymph node metastasis was studied. Tongue VX2 carcinoma with cervical lymph node metastasis was induced in 19 rabbits by injecting VX2 carcinoma suspension into the tongue submucosa. SLN biopsy was performed under the guidance of indirect CT-LG. SLN identification was performed by indirect CT-LG combined with blue dye injection in 2 rabbits. Tongue SLNs were identified preoperatively by indirect CT-LG and blue-stained SLNs were visualized intraoperatively. Only one SLN was enhanced in each side of the neck, lateral to the larynx-tracheal region. CT attenuation values of the enhanced SLNs were 782.4 ± 46.6, 443.1 ± 68.5, 180.3 ± 20.6 and 80.5 ± 10.7 HU at 1, 5, 15 and 20 min after contrast injection. Overall, ipsilateral SLN identification rate was 97.4% and contralateral SLN identification rate was 100%. Ipsilateral SLN metastasis was verified in all rabbits (100%), bilateral SLN metastasis occurred in 8 rabbits (42%), and micrometastasis was found in 3 rabbits (16%). Indirect CT-LG may be useful for guiding SLN biopsies in tongue cancer. Combining indirect CT-LG with blue dye injection may improve preoperative and intraoperative SLN identification.     

兔舌癌哨位淋巴结C/Gd复合物间质磁共振造影的实验研究

目的:探讨新型显影剂C/Gd纳米复合物间质磁共振造影（IMRLG）诊断兔VX2舌癌模型颈部哨位淋巴结转移的应用价值。方法:将VX2癌组织块植入16只健康新西兰大白兔左侧舌缘。25 d后,于兔双侧舌缘黏膜下注入造影剂C/Gd纳米复合物0.25 mL。分别于注射前及注射后5、10、15、20、30、40 min及24 h进行磁共振淋巴成像。计算特定时间节点的信号强化率(E%),定位哨位淋巴结,观察淋巴系统。注射造影剂24 h后,于双侧舌下黏膜注射0.2 mL亚甲蓝,行活体淋巴染色,解剖颈部淋巴结,行组织病理学检查。采用SPSS19.0软件包对数据进行统计学分析。结果:成功建立兔VX2舌癌模型16只（16/16）,其中发生淋巴结转移16只（16/16）。注射造影剂后10 min,肿瘤侧与对照侧淋巴结磁共振成像的E%具有显著差异（P<0.05）。IMRLG判断转移哨位淋巴结18个,在亚甲蓝活体染色下,解剖出淋巴结32个,其中病理学检测转移淋巴结17个,IMRLG的准确率为94.45%（17/18）,两者诊断SLN转移无统计学差异（P>0.05）。结论:应用C/Gd纳米复合物行IMRLG,可较准确地判断兔舌癌哨位淋巴结状况,为判断舌癌淋巴结转移提供了新的思路,有潜在应用价值。     

Evaluation of the VX2 rabbit auricle carcinoma as a model for head and neck cancer in humans.

INTRODUCTION: This study investigates whether the VX2 carcinoma cell line, transplanted into the rabbit auricle, can be used as a head and neck cancer model. The biologic behaviour of this model is evaluated, comparing tumour transplantation with either tissue pieces or cell suspensions. MATERIAL: Thirty-six adult NZW rabbits received s.c. injections of VX2-suspensions (Group S) and 11 rabbits received solid VX2-pieces (Group P) into both auricles. METHODS: In Group S, 16 rabbits were sacrificed at various days before (S1) and 15 after (S2) the 28th day following transplantation. In the other five rabbits transplantation failed. Animals from Group P were sacrificed every 2 weeks after the 28th day. At autopsy the size of the primary tumours and of lymph node, lung and other metastases were assessed. If transplantation failed, the maximal tumour size and the time at which regression took place were recorded. Exponential trend lines were used to create growth curves of metastases. Differences between groups were evaluated with the chi2 test, correlations between parameters with Kendall's tau. RESULTS: The tumour take-rate in Groups S and P was 78% and 59% respectively. The maximal size and time at which regression occurred was significantly different, amounting to 83 +/- 7 mm2 at 10.4 +/- 1.6 days (Group S) and 243 +/- 30 mm2 at 20.9 +/- 2.0 days (Group P), respectively. Development of lymph node metastases was not different. In Groups P and S2, over 90% of the necks contained lymph node metastases. There was a higher incidence of lung metastases in Group S2 when compared to Group P (47% vs. 14%) but it was not statistically significant. A significant correlation (p<0.05) between weight loss and the size of lung metastases was found. CONCLUSION: Transplantation of the VX2-tumour with cell suspensions produces a useful head and neck cancer model for locoregional disease in which anti-tumour regimens against both the primary and lymph node metastases can be tested. Transplantation with tumour pieces is not advised as the take-rate is low and spontaneous remissions occur at a late stage.     

T2 carcinoma of the tongue: the histopathologist's perspective.

The histological characteristics and extent of tumour spread at the primary site and in the neck are described in a series of 50 patients with a T2 squamous cell carcinoma of the oral tongue. The likely site of origin was lateral tongue in 29 cases and ventral tongue in 21 cases. Simultaneous tumours were evident in four resections and multifocal dysplasia in a further 13. Lymph node metastasis was diagnosed histologically in 27 cases, including one case with bilateral metastases. Comparison of the groups, with and without metastasis, showed significant differences in tumour thickness, tumour diameter, multifactorial histological malignancy grade, vessel invasion and nerve invasion. The number of positive lymph nodes per patient ranged from 1 to 16 (mean 3), and 17 dissections showed spread beyond the nodal capsule. Postoperative follow-up ranged from 1 to 6 years and, at the time of analysis, 18 patients had died of or with disease. Loco-regional failure was related to 'high-risk' features at the primary site and extracapsular spread in the neck.     

波形蛋白在舌黏膜鳞状细胞癌中的表达及其与临床预后的关系

目的：研究舌黏膜鳞状细胞癌（舌癌）中波形蛋白的表达,探讨其在舌癌复发转移中的意义。方法：采用免疫组化方法检测30例舌癌患者组织中波形蛋白的表达,并分析其表达水平与临床病理及预后之间的关系。结果：正常口腔黏膜中不表达波形蛋白。在舌癌病例,约33%（10/30）患者的癌组织中波形蛋白呈高表达;67%（20/30）患者的癌组织中波形蛋白呈阴性或低表达。波形蛋白表达水平与患者的性别、年龄、肿瘤分化程度、临床分期之间无统计学意义;波形蛋白的表达水平与肿瘤大小（P=0.007）、淋巴结转移（P=0.007）及复发（P=0.007）之间在统计学上有显著性差异。结论：舌癌组织中波形蛋白的高表达与肿瘤复发、转移之间密切相关,有可能作为舌癌患者预后的预测指标。     

A cervical lymph node metastatic model of human tongue carcinoma: Serial and orthotopic transplantation of histologically intact patient specimens in nude mice.

PURPOSE: A lymph node metastatic model of human tongue carcinoma using orthotopic and serial transplantation was established in nude mice to study the invasive and metastatic properties of human tongue cancer. Materials and Methods: Lymph node metastatic specimens of human tongue carcinoma were transplanted into nude mice orthotopically. Tumors dissected from the metastatic lymph nodes of the nude mice were serially transplanted into tongues of disease-free nude mice at 4-week intervals. RESULTS: All mice developed aggressive and diffuse well-differentiated squamous cell carcinoma at tongue recipient sites. Tumor cells invaded to lymphatic vessels. In addition, increased cervical lymph node metastasis was noted in the first (3 of 14, or 21%), second (4 of 11, or 36%), third (6 of 10, or 60%), or fourth (11 of 14, or 79%) transplantation. In mice, 2 of 14 lung metastases were found in the fourth round of transplantation. CONCLUSION: After surgical specimens of the lymph node metastasis for human tongue cancer were transplanted into the tongue of nude mice, the clinical characteristics of human tongue carcinoma, especially invasion and metastasis, were observed. This metastatic model involving orthotopic and serial transplantation should be useful for studies on the mechanisms, treatment, and prevention of human carcinoma of tongue.     

量子点荧光探针检测人舌鳞状细胞癌荷瘤裸鼠模型早期下颌下淋巴结转移的研究

目的 利用量子点标记的特异性细胞角蛋白抗体QDs605-CK（AE1/AE3）免疫荧光探针检测人舌鳞状细胞癌荷瘤裸鼠早期下颌下淋巴结转移率及微转移率,并与传统的免疫组织化学（IHC）染色及苏木精-伊红（HE）染色方法进行比较,为舌鳞状细胞癌的早期诊断与治疗提供一种新的检测方法。方法 传代培养人舌鳞状细胞癌Tca8113 细胞,接种于18只裸鼠舌体内（不过中线）,建立人舌癌荷瘤裸鼠下颌下淋巴结转移模型。接种6周后,处死裸鼠,解剖下颌下淋巴结,将同一淋巴结分为两份。一份作石蜡包埋半连续切片,行HE染色和IHC检测;另一份即刻液氮冷冻,制作冰冻切片行QDs605-CK（AE1/AE3）荧光探针检测。分别计算3种方法检测出的淋巴结转移率和微转移率。结果 量子点标记的免疫荧光染色检测出裸鼠下颌下淋巴结转移率为66.7%,其中微转移率为38.9%;IHC染色检测的淋巴结转移率为61.1%,其中微转移率为33.3%;HE染色检测的淋巴结转移率为27.8%。经统计学分析,3种方法的差异有统计学意义（χ2=6.379,P<0.05）,量子点标记的免疫荧光染色和IHC检测都优于HE染色,但是量子点标记的免疫荧光染色和IHC染色间的差异无统计学意义（χ2=0.120,P>0.05）。结论 量子点标记的QDs605-CK（AE1/AE3）免疫荧光探针能准确定位于下颌下淋巴结转移的肿瘤细胞的细胞质内,发出红色荧光,其特异性强,分辨率高,背景清晰,能够用于淋巴结转移灶及微转移灶的检测。