大鼠舌癌癌变过程中琥珀酸脱氢酶的表达变化

目的：探讨大鼠口腔癌变过程中琥珀酸脱氢酶（Succinate dehydrogenase,SDH）的表达及其生物学意义。方法：0.002%4-硝基喹啉-1-氧化物（4NQO）饮水喂养Wistar大鼠9-32周建立大鼠舌癌变模型,用实时荧光定量PCR法检测30例大鼠舌癌变过程不同病理阶段组织中SDH的表达水平。结果：轻度异常增生、中度异常增生、重度异常增生。鳞癌组织病理分级中,中度异常增生、重度异常增生、鳞癌组织中SDH表达显著低于正常黏膜（P〈0.05或P〈0.01）。SDH表达与组织的病理学分级相关（P〈0.01）。结论：SDH基因表达与口腔癌发生发展相关,三羧酸循环在口腔黏膜癌变过程中可能起到重要作用。     

大鼠舌白斑癌变过程中线粒体琥珀酸脱氢酶活性变化的研究

目的:探讨大鼠口腔癌变过程中线粒体琥珀酸脱氢酶(Succinate dehydrogenase,SDH)的活性变化及其生物学意义.方法:2×10-5 4-硝基喹啉-1-氧化物(4NQO)饮水喂养Wistar大鼠9~32周建立大鼠舌癌变模型,提取30例大鼠舌癌变过程不同病理阶段的舌背组织线粒体,酶标仪检测SDH酶活性的变化.结果:在大鼠舌白斑癌变过程中,线粒体SDH活性呈逐渐下降趋势,其中,重度异常增生、鳞癌组织中SDH酶活性显著低于正常黏膜(P<0.05). SDH的基因表达及酶活性变化趋势较为一致. 结论:SDH酶活性和mRNA表达水平的下降与口腔癌发生发展相关,三羧酸循环在口腔黏膜癌变过程可能起到重要作用.     

4-硝基喹啉-1-氧化物诱导C57BL/6小鼠舌黏膜癌变的研究

目的 用4-硝基喹啉-1-氧化物（4NQO）诱导C57BL/6小鼠舌黏膜癌变。方法 将85只小鼠随机分为蒸馏水对照组（n=5）、1,2丙二醇对照组（n=5）、实验组（n=75）,分别给予蒸馏水、1,2丙二醇及4NQO溶液饮用。实验组小鼠再分为15笼,每2周处死一笼,对照组小鼠于28周处死。对小鼠进行称质量和大体观察及组织病理学检查。结果 实验组死亡1只。实验组小鼠体质量在24周后体质量较对照组明显降低（P<0.05）。实验动物的舌、口底、上颚、颊部共出现79处肉眼可见的病变,其中舌部病变70处（88.6%）。实验组动物在不同时间点黏膜病变不同,28周时均发生高分化鳞状细胞癌。对照组未见病变。结论 舌癌动物模型被成功建立。实验12~16周及20~28周可作为研究舌黏膜癌变早期和中晚期阶段的时间点。     

4NQO诱导大鼠舌黏膜癌变过程中HSF1的表达及意义

目的：探讨热休克因子1（ HSF1）在4?硝基喹啉?1?氧化物（4NQO）饮水法诱导大鼠舌黏膜癌变过程中的表达及意义。方法：80只Wistar大鼠随机分两组,实验组用0．001％～0．004％4NQO递增性喂养大鼠8～28周,对照组则用普通自来水喂养,分别于8、16、20、24、28周处死大鼠,通过大体标本、HE染色观察大鼠舌部组织学改变,同时采用免疫组化染色方法观察HSF1在大鼠舌癌变全过程的表达情况。结果：随4NQO作用时间延长和浓度递增,大鼠舌背后部黏膜相继出现颗粒状、白色斑块、疣状突起、菜花状新生物和溃疡状改变。诱导后8、16、20、24、28周,舌癌的发生率分别为0、20．0％、50．0％、66．7％、100％。HSF1在对照组大鼠舌背黏膜上皮中不表达或者微弱表达,而在实验组大鼠随着舌黏膜异常增生程度的增加,HSF1表达明显增强;在原位癌中,HSF1弥漫分布于整个癌巢中;在舌黏膜浸润癌中HSF1在癌上皮中表达有所降低,而在癌基质成纤维细胞中HSF1表达增强。结论：4NQO饮水法可诱导大鼠舌黏膜发生癌变,成功建立大鼠舌癌模型,同时HSF1在大鼠舌癌发生、发展过程中发挥重要作用。     

Histological and immunohistochemical evaluation of the chemopreventive role of lycopene in tongue carcinogenesis induced by 4-nitroquinoline-1-oxide

Objective

Increasing attention has been given to the potential protective roles of specific antioxidant nutrients found in fruits and vegetables. However, there are relatively few reports on the cancer chemopreventive effects of lycopene or tomato carotenoids in animal models. Therefore, the chemopreventive efficacy of lycopene with regard to oral carcinogenesis was investigated using 4-nitroquinoline-1-oxide (4-NQO) induced tongue squamous cell carcinoma.

Materials and methods

Twenty albino rats were equally divided into 2 groups. 4-NQO was dissolved in the drinking water (20 ppm) for rats of both groups. Rats in group 2 were administered lycopene at a dose of 2.5 mg/kg body weight by intragastric intubation once a day. Incidence of oral neoplasms and histopathological changes were microscopically evaluated after 32 weeks of administration of the specific treatments. Moreover, immunohistochemical expressions of proliferating cell nuclear antigen (PCNA), E-cadherin and β-catenin were analysed in tongue specimens using an image analyser computer system.

Results

Lycopene treatment significantly decreased the incidence of 4-NQO induced tongue carcinogenesis. A decreased percentage of PCNA-positive nuclei was associated with lycopene treatment. Proliferating cells were mainly confined to the basal and parabasal epithelial cell layers. Increased E-cadherin and β-catenin immunoexpression was recorded in the lycopene treated group in comparison to the carcinogen group.

Conclusion

Results of the present study indicate that lycopene can exert protective effects against 4-NQO induced tongue carcinogenesis through reduction in cell proliferation and enhanced cellular adhesion, suggesting a new mechanism for the anti-invasive effect of lycopene. Further studies are needed to provide more definitive answers to the question of the anticancer effects of lycopene.     

4NQO诱导小鼠舌癌前病变模型的研究

目的 使用4NQO诱导并建立C57BL/6J小鼠舌癌前病变模型.方法 46只6～8周龄C57BL/6J小鼠,随机取16只作为阴性对照,不做任何处理,分别于8、12、16周末处死.实验组30只饮用50μg/ml 4NQO水溶液,分别于8、12、16周末处死.处死前2小时腹腔注射BrdU (50mg/kg),肉眼及组织病...     

大鼠舌癌变过程中生存素、Bcl-2和p53蛋白的表达

目的 探讨生存素(survivin)蛋白在大鼠舌癌变过程中的表达及其与Bcl-2、p53蛋白表达的相关性.方法 0.002%4-硝基喹啉-1-氧化物(4一nitro-quinoline 1-oxide,4NQO)饮水喂养SD大鼠9～36周建立大鼠舌癌变模型,用免疫组化二步法检测60例大鼠舌癌变过程中生存素、Bcl-2及p53蛋白的表达.结果 36例止常组织中生存素蛋白表达仅1例,而11例异常增生组织中其表达为6例,11例口腔癌组织中其表达为10例.生存素蛋白在正常舌黏膜、异常增生黏膜及舌癌中的阳性表达差异有统计学意义(P<0.001),异常增生及口腔癌组织中生存素蛋白表达显著高于正常黏膜中的表达(P<0.01).在17例生存素蛋白表达阳性的大鼠舌组织中,Bcl-2蛋白阳性表达12例,p53蛋白阳性表达8例;生存素蛋白表达与Bcl-2、053蛋白表达呈正相关(P<0.01).结论 生存素蛋白的表达增高和口腔鳞状上皮的异常增生、癌变有关,提示生存素可能参与了口腔癌的发生、发展;生存素、Bcl-2及p53蛋白的表达可能在口腔癌的发生、发展中起协调作用.     

Immunohistochemistry profile of p75 neurotrophin receptor in oral epithelial dysplasia and oral squamous cell carcinoma induced by 4-nitroquinoline 1-oxide in rats

ObjectiveThe 4-nitroquinoline 1-oxide (4-NQO) model for carcinogenesis has been used to investigate cancer stem cells (CSC), but no study has addressed the role of the p75 neurotrophin receptor (p75^NTR) in 4-NQO-induced oral dysplasia and oral squamous cell carcinoma (OSCC). The aim of this study was to evaluate the immunohistochemistry profile of the p75^NTR during 4-NQO-induced oral carcinogenesis in rats and to verify whether this profile has an association with proliferating cell nuclear antigen (PCNA) immunolabeling.DesignFor 28 weeks, rats were exposed to 4-NQO, which was diluted in the drinking water. After 3, 5, 7, 16, and 28 weeks, the animals were euthanized and their tongues were histologically analyzed using p75^NTR and PCNA immunolabeling.ResultsIn animals without 4-NQO exposure, the p75^NTR and PCNA were expressed only in the basal epithelial layer and in a clustered manner. The oral epithelium showed dysplasia and a significant increase in the number of p75^NTR- and PCNA-positive cells, which were localized mainly in the basal and suprabasal epithelial layers during weeks 5–16 of 4-NQO exposure. When the epithelium invaded the lamina propria and well-differentiated OSCC began, the p75^NTR-positive cell frequency drastically decreased in epithelial cords and nests, showing a negative correlation with PCNA expression. p75^NTR immunolabeling during 4-NQO-induced carcinogenesis was similar to that described for human head and neck dysplasia and neoplasia.Conclusionsp75^NTR immunolabeling observed in 4-NQO-induced oral dysplastic and OSCC lesions were related to the early phases of oral carcinogenesis and may help predict cell dysplasia and malignant transformation.     

Homeobox C5 expression is associated with the progression of 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis

J Oral Pathol Med (2012) 41 : 470–476 Background: Aberrant expression of homeobox genes (HOX), normally required for the differentiation of a particular tissue, has been reported in several types of cancer, but poorly addressed in oral squamous cell carcinoma (OSCC). The present study investigated the expression of HOXC5 in OSCC and identified molecular biomarker whose expression is associated with the multistep oral carcinogenesis. Methods: The expression of HOXC5, proliferation cell nuclear antigen (PCNA), and Bcl‐2 was examined by RT‐PCR and Western blot analysis and confirmed by immunohistochemistry and transferase‐mediated dUTP nick end‐labeling (TUNEL) assay in a 4‐nitroquinoline 1‐oxide (4NQO)‐induced rat tongue carcinogenesis model. Results: Homeobox genes C5 was overexpressed in SCC tissues, but not in normal tissues by RT‐PCR and Western blot analysis. Along with the progress of multistep carcinogenesis, the levels of HOXC5 expression of mRNA and protein significantly increased during the dysplasia (moderate to severe dysplasia) when compared with normal and hyperplasia. The levels of PCNA and Bcl‐2 were sequentially increased from hyperplasia to dysplasia and SCC. By immunohistochemistry, HOXC5 expression was significantly increased in dysplasia, whereas PCNA expression was gradually increased during tongue carcinogenesis. TUNEL‐positive cells were increased until dysplasia, but reduced in SCC. Conclusions: These results indicate that overexpression of HOXC5 is correlated with oral carcinogenesis and strongly contributed to the development of OSCC. HOXC5 may be a useful biomarker and has an emerging therapeutic target of OSCC.     

小分子锌指蛋白1在4-硝基喹啉-N-氧化物诱导SD大鼠颊黏膜癌变中的表达研究

目的 研究口腔黏膜癌变中小分子锌指蛋白1(LMO1)在基因转录和蛋白水平的表达变化。方法 取本课题组前期4-硝基喹啉-N-氧化物(4NQO)饮水法构建鳞癌动物模型的49例样本进行苏木精-伊红(HE)染色,依据上皮异常增生程度确定实验组病理分级并确定实验分组;免疫组织化学染色定性确定LMO1的表达部位;实时荧光定量聚合酶链式反应(RT-qPCR)和Western blot检测5组样本中LMO1 mRNA和蛋白的表达。结果 HE染色确定对照组7例,轻度组6例,中度组11例,重度组9例,OSCC组16例。免疫组织化学染色检测可见,LMO1主要表达于细胞质,对照组、轻度组、中度组、重度组和OSCC组的阳性表达率分别为14.3%、33.3%、81.8%、88.9%、93.8%。RT-qPCR检测可见,对照组LMO1 mRNA的表达量最低,OSCC组最高,对照组与轻度组间mRNA表达差异无统计学意义(P>0.05),其余各组组间两两比较,mRNA的表达差异均有统计学意义(P<0.05)。Western blot检测可见,随着上皮异常增生程度的加重,LMO1的蛋白表达量逐渐上...     

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提要：发展一种新的方法用来提高口腔癌的诊断和治疗效果,建立能够模拟人类口腔鳞癌自然发生的动物模型是必需的,应用4-硝基喹啉-1-氧化物（4-nitroquinoline-1-oxide, 4NQO）诱导SD大鼠舌黏膜鳞癌是一种可靠的方法。水溶性喹啉衍生物4NQO可以形成DNA加成物,引起碱基的改变（G→A）或丢失突变。但4NQO要发挥这种诱变剂作用,需要在4NQO还原酶作用下将4NQO转化成4-羟氨基喹啉-1-氧化物及4-乙酰基喹啉-1-氧化物（4-AAQO）,4-AAQO能够以共价键的方式结合到核酸上并破坏染色体的结构,进一步影响抑癌基因和癌基因表达。动物舌根黏膜该酶含量较高,所以可以靶向性在舌根形成癌。饮水中很低剂量的4NQO便可特异性在舌根形成癌,其形成过程和形态学变化都和人的口腔鳞癌发生过程十分相似,该模型是研究口腔癌发生和癌变机制的理想模型。     

4-硝基喹啉-1-氧化物诱导大鼠舌癌过程中Notch1的表达特点

目的：研究 Notch1在大鼠舌癌模型发生发展过程中的表达变化。方法：将40只 SD 大鼠随机分为对照组（A 组10只）和实验组（B、C、D组各10只）,使用质量百分比浓度为0．004％的4-硝基喹啉-1-氧化物（4-nitroquinoline-1-oxide,4NQO）饮用水喂养实验组 SD大鼠,用蒸馏水喂养对照组 SD大鼠。B、C、D组分别于8、16、24周取大鼠舌体组织;A 组在8、16、24周同时各取3只、3只、4只作为正常对照。行常规组织学观察,并使用免疫组织化学法检测 Notch1在病变不同阶段的舌粘膜内的表达。结果：4NQO可以有效诱导大鼠舌黏膜鳞状细胞癌的发生。在不同的给药时间点,可以诱导大鼠舌黏膜产生不同程度的癌前病变。Notch1在正常舌黏膜、癌前病变、舌鳞癌中的表达逐渐增高（P＜0．01）。随着异常增生程度的增加,阳性表达部位逐渐由基底层向表层扩展,且 Notch1的膜型表达逐渐增多。结论：Notch1的高表达及其在细胞膜上的集中表达可能与舌癌的发生、发展过程相关。     

芪蓝颗粒对大鼠舌黏膜癌变中Bcl-2及P53表达的影响

目的观察芪蓝颗粒对4-硝基喹啉-1-氧化物（4NQO）诱导的大鼠舌癌变的抑制及对Bcl-2蛋白、P53蛋白表达的影响。方法SD大鼠150只,随机分为癌变模型组,低、中、高剂量芪蓝颗粒干预组以及正常对照组,以4NQO饮水喂养诱导大鼠舌黏膜癌变,同时给予芪蓝颗粒干预癌变过程,取9、18、27、36周舌标本进行组织病理学观察,采用Polymer法检测大鼠舌组织中Bcl-2蛋白及P53蛋白的表达。结果芪蓝颗粒干预的各组大鼠的癌变率均低于癌变模型组（P〈0.05）。芪蓝颗粒干预各组及正常对照组中Bcl-2蛋白、P53蛋白的阳性表达明显低于癌变模型组（P〈0.01）。结论芪蓝颗粒可抑制口腔粘膜癌变,对细胞凋亡相关的Bcl-2蛋白和P53蛋白的调控可能是其抑癌机制之一。     

Reduction of syndecan-1 expression during lip carcinogenesis

Background:  Actinic cheilitis (AC) is an oral pre-cancerous lesion that sometimes develops into lip squamous cell carcinoma (SCC). Syndecan-1, a transmembrane heparan sulfate proteoglycan, modulates cell-proliferation, adhesion, migration and angiogenesis. Malignant epithelial cells often down-regulate their own syndecan-1 production, and are capable of inducing aberrant syndecan-1 expression in stromal cells. The aim of this study was to evaluate the variations in syndecan-1 expression during lip carcinogenesis, in normal lip (NL), AC and well-differentiated lip SCC.
Methods:  Biopsies of NL vermillion ( n  = 19), AC ( n  = 23) and lip SCC ( n  = 24) were stained immunohistochemically for syndecan-1.
Results:  Syndecan-1 expression was significantly reduced in AC and lip SCC as compared to NL ( P  < 0.05), with a significant reduction in lip SCC as compared to AC ( P  < 0.0001). In lip SCC lesions, syndecan-1 expression at the epithelium overlying the tumor was increased when compared to the tumor itself ( P  < 0.03), but was significantly reduced as compared to AC and NL ( P  < 0.001).
Conclusion:  The results showed that epithelial syndecan-1 expression is reduced as lip carcinogenesis progresses (NL>AC>lip SCC), suggesting that syndecan-1 could be a useful marker of malignant transformation in the lip.     

Ras gene mutation is not related to tumour invasion during rat tongue carcinogenesis induced by 4‐nitroquinoline 1‐oxide

J Oral Pathol Med (2011) 40 : 325–333 Background: The aim of this study was to investigate whether mutations in the genes H‐ras and K‐ras were related to the mechanism of invasion as a result of the immunoexpression of H‐Ras, Ki‐67, alpha‐smooth muscle actin (SMA) and vascular endothelial growth factor (VEGF) during 4‐nitroquinoline 1‐oxide (4NQO)‐induced rat tongue carcinogenesis. Methods: Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12 and 20 weeks. Ten animals were used as negative control. Results: Although no histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure, Ki‐67 was overexpresssed in the ‘normal’ oral epithelium. In pre‐neoplastic lesions at 12 weeks following carcinogen exposure, the levels of Ki‐67 were increased (P < 0.05) when compared to negative control. Ki‐67, alpha‐SMA and VEGF were also overexpressed in squamous cell carcinomas induced after 20 weeks of treatment with 4NQO. No significant statistical differences (P > 0.05) were found in H‐ras protein expression for all experimental periods evaluated that corresponded to normal oral mucosa, hyperplasia, dysplasia and squamous cell carcinomas. In the same way, no mutations in H‐ras or K‐ras genes were found. Conclusions: Our results support the idea that expression of Ki‐67 plays a crucial role during malignant transformation being closely related to neoplastic conversion of the oral mucosa cells. However, it seems that mutations in the ras genes are not involved to experimental tongue carcinogenesis induced by 4NQO.     

4NQO饮水法诱发小鼠舌癌前病变模型建立的研究

目的：建立与人口腔癌前病变相似的动物模型。方法：采用0．001％4-硝基喹啉1-氧化物(4NQO)饮水法对Balb／c近交系小鼠进行诱发实验。16周和20周时处死小鼠,观察病变情况。结果：舌背黏膜癌前病变的总发病率为100％。16周时以轻度和中度异常增生为主;20周时以中度和重度异常增生为主。结论：4NQO饮水法的致病过程和组织病理学特征与人相似;0．001％4NQO饮用16～20周是舌癌前病变动物模型建立的理想时间。     

Chemopreventive activity of apple extract following medium-term oral carcinogenesis assay induced by 4-nitroquinoline-1-oxide

ObjectiveThe aim of this study was to evaluate the chemopreventive activity of an apple extract following medium-term oral carcinogenesis assay induced by 4-nitroquinoline-1-oxide (4NQO).MethodsA total of 30 male Wistar rats were distributed into five groups as follows (n = 6 per group): Group 1, negative control group (non-treated group); Group 2, received 4NQO during 8 weeks in drinking water and treated with apple extract at 1% by gavage between the first and fourth weeks daily (initiation phase); Group 3, received 4NQO for 8 weeks in drinking water and treated with apple extract by gavage at 1% between the fifth and eighth weeks daily (promotion phase); Group 4, received apple extract at 1% by gavage for 8 consecutive weeks only; and Group 5, received 4NQO for 8 weeks in drinking water daily.ResultsHistopathological analysis revealed decreased hyperplasic lesions in Group 2 when compared with Group 5. Likewise, decreased dysplastic lesions in Group 3 were observed when compared with Group 5. In Groups 2 and 3, decreased COX-2 and TNF-alpha gene expressions were observed when compared with Group 5. Cytochrome c and caspase 3 levels increased in Groups 2 and 3 when compared with Group 5.ConclusionIn conclusion, our results demonstrate that apple extract suppresses rat tongue carcinogenesis as a result of anti-inflammatory activity and apoptosis through the intrinsic mitochondrial pathway.     

绞股蓝总甙对鼠舌白斑癌变过程中琥珀酸脱氢酶表达影响的研究

目的：探讨中药绞股蓝总甙（Gypenosides,GP）对实验性口腔白斑癌变过程的阻断作用及其对琥珀酸脱氢酶（Succinate dehydrogenaseS,DH）的影响。方法：Wistar大鼠70只,随机分为癌变模型组（n=25）、中药干预组（n=30）、正常对照组（n=5）和中药对照组（n=10）。以4NQO饮水喂养诱导大鼠舌白斑癌变,中药绞股蓝总甙水溶液灌胃干预癌变过程,取9、13、20、24、32周舌背黏膜标本进行组织病理学观察,用实时荧光定量PCR法检测SDH基因表达水平。结果：中药干预组舌背黏膜异常增生发生率明显低于癌变模型组,SDH表达明显高于癌变模型组（P〈0.01或P〈0.05）,尤其在20、24周时。结论：绞股蓝总甙对实验性口腔白斑癌变有阻断作用,通过影响SDH表达以修复三羧酸循环可能是其抗癌机制之一。     

芪蓝颗粒对大鼠舌黏膜癌变模型淋巴细胞亚群的影响

目的：观察芪蓝颗粒对大鼠舌黏膜癌变模型淋巴细胞亚群的影响,并探讨其阻癌抑癌的免疫学机制。方法：SD大鼠150只,随机分为五组（A、B、C、D、E组）,A、B、C、D组以4NQO饮水喂养诱导大鼠舌黏膜癌变,A组不作干预,作为阳性对照;B、C、D组分别给予不同剂量的芪蓝颗粒干预癌变过程,9、18、27、36周取舌标本进行组织病理学观察,并取新鲜全血,流式细胞术检测各样本全血中CD3^＋细胞、CD4^＋T细胞、CD8＋T细胞的百分比,CD4/CD8比值及NK细胞。E组作为正常对照组。结果：多元方差分析表明,各组及各病理分级的CD3^＋细胞、CD4^＋T细胞百分比存在显著性差异（P〈0.05）。组间比较,芪蓝颗粒干预组的CD3^＋、CD4^＋水平介于正常组与癌变模型组之间,统计表明：E组〉C组〉A组（P〈0.05）。结论：芪蓝颗粒有阻癌抑癌作用,能降低致癌剂对机体免疫系统的影响,稳定CD3细胞水平,主要是通过稳定CD4^＋辅助性T细胞的水平,增强机体的防癌能力。     

Effects of sex hormones on rat tongue carcinoma induced by 4-nitroquinoline 1-oxide (4NQO)

We investigated the regulatory effects of sex hormones on tongue carcinoma initiated by orally administration 4-nitroquinoline 1-oxide (4NQO) to rats. Animals of either sex were classified into three groups. The male rats in each group received an estrogen administration (Me), orchiectomy (Mor), or both treatments (Me/or) while the female rats also received testosterone administration (Ft), ovariectomy (Fov), or both treatments (Ft/ov). The differences in the carcinogenic progress among these groups were examined by macroscopic and microscopic observation of tongue tissues. The incidence of cancer in the tongue tissue was 100% in the group reinforced with testosterone (testosterone+group) (Ft, Ft/ov, Me) but only 56.0% in the group not reinforced with testosterone (testosterone-group) (Fov, Mor). These findings suggest that sex hormones play a role in the onset of 4NQO-induced tongue carcinoma.