金黄地鼠舌癌发生过程中增殖性细胞核抗原的表达及淋巴管密度和面积与淋巴道转移的相关性

目的 通过观察金黄生鼠舌癌发生过程中增殖细胞核抗原(PCNA)的表达情况,分析淋巴管密度和面积的变化与舌癌恶性程度及淋巴道转移的相关性,探讨淋巴管生成与淋巴道转移的关系。 方法 用1.%丙烯酰胺-N,N-二甲基-N-丁基-N-甲基丙烯酸乙酯溴化铵(DMBA)丙酮溶液合并创伤方法,诱导金黄地鼠舌癌模型42只,每2周取材7只。采用HE染色确定舌癌的病理变化,PCNA和桥粒斑蛋白(Desmoplakin)免疫组织化学双标记法显示淋巴管增生情况,并测量肿瘤和淋巴管内皮细胞增殖指数(PI)、淋巴管密度(LVD)和淋巴管面积(LVA)作统计学分析。 结果 金黄地鼠舌癌的形成经历了非典型增生原位癌早期浸润癌的过程;在非典型增生组PI(91.55),LVA(7570.23),LVD(2.50);原位癌组PI(113.36), LVA(12105.45),LVD(3.73);早期浸润癌组PI(124.67),LVA(14524.33),LVD(5.33),各组间LVA,LVD,PI差异显著（P＜0.05）,且PI与LVA及LVD呈正相关(P＜0.05);淋巴结转移组PI(130.50),LVA(15430.67),LVD(6.17),非转移组PI(113.00),LVA(12711.67), LVD(3.67),两组差异显著（P＜0.05）。 结论 舌癌发生过程中存在新生淋巴管,舌癌形成过程中微淋巴管的LVA和LVD值增大,与舌癌的恶性程度呈正相关,与淋巴道转移呈正相关。     

敲低核仁小RNA宿主基因6(SNHG6)抑制舌癌细胞的增殖及上皮间质转化

目的探究长链非编码RNA核仁小RNA宿主基因6 (SNHG6)在舌鳞状细胞癌组织、Tca1183舌癌细胞中的表达及对舌癌细胞生物学特性的影响。方法使用实时定量PCR检测舌鳞状细胞癌组织与正常组织、Tca1183舌癌细胞与正常舌上皮细胞中SNHG6的mRNA水平,分析舌癌临床病理指标与SNHG6表达的相关性。构建SNHG6干扰质粒并转染至Tca1183舌癌细胞,噻唑蓝(MTT)法检测细胞活力,集落形成实验检测细胞增殖能力,流式细胞术检测舌癌细胞凋亡变化; Western blot法检测细胞上皮间质转化(EMT)相关蛋白β联蛋白(β-catenin)、上皮钙黏素(E-cadherin)、神经钙黏素(N-cadherin)和波形蛋白(vimentin)的蛋白水平。结果与正常组织和正常舌上皮细胞相比,舌癌组织以及舌癌细胞中SNHG6的mRNA水平显著升高。舌癌组织SNHG6 mRNA水平与患者年龄和性别等不相关,与组织分化差、临床分期高和淋巴结转移呈正相关。干扰SNHG6在Tca1183舌癌细胞中的表达能够抑制癌细胞增殖以及促进细胞凋亡,且可以通过促进β-catenin和E-cadherin蛋白表达以及抑制N-cadherin和vimentin蛋白表达抑制Tca1183细胞EMT。结论 SNHG6在舌癌组织中高表达,敲低舌癌细胞中SNHG6能够抑制其增殖和EMT。     

间隙连接蛋白43在舌黏膜癌变过程中的表达

目的 研究间隙连接蛋白43(connexin 43,Cx43)在口腔黏膜癌变过程中的表达变化,探寻其与口腔黏膜癌变的关系及意义.方法 用4亚基硝氧喹啉(4-nitroquinoline-1-oxide,4NQO)诱导SD大鼠口腔黏膜癌变,应用免疫组化方法检测Cx43在口腔黏膜癌变过程中各阶段的动态变化,分析Cx43与口腔黏膜癌变的关系.结果 Cx43蛋白主要表达于大鼠舌黏膜上皮细胞的胞膜、上皮基底层、棘层和颗粒层呈阳性染色,角质层未见表达.随着舌黏膜上皮异常增生程度的增加,Cx43的表达明显下降.在口腔鳞状细胞癌组织中,Cx43染色分布于癌细胞胞质以及鳞癌组织的角化珠内.Cx43在正常舌黏膜、轻度上皮异常增生、中度上皮异常增生、重度上皮异常增生、口腔鳞癌组织中阳性表达率分别为100.00％(10/10)、85.71％(12/14)、66.67％(8/12)、40.00％(4/10)、33.33％(4/12),差异有统计学意义(P＜0.05).结论 4NQO诱发舌黏膜癌变的过程中,Cx43蛋白表达水平随病变程度加重而显著下降,提示Cx43表达异常参与了口腔黏膜的癌变过程.Cx43表达下降是口腔黏膜癌变的早期事件.     

Laminin和survivin的表达与胆囊癌临床病理特征的关系

目的:研究laminin和survivin蛋白在原发性胆囊癌组织中的表达情况,及其与癌组织类型、病理分级和转移状况的关系。 方法:应用免疫组织化学方法检测49例原发性胆囊癌、21例胆囊腺瘤和13例慢性胆囊炎组织中laminin和survivin蛋白的表达。 结果:胆囊黏膜内癌或原位癌细胞基底膜laminin线性染色完整；NevinⅡ期胆囊癌组织表现为基底膜不完整,变薄,断裂,或缺损；临床NevinⅢ、Ⅳ、Ⅴ期胆囊癌，则无基底膜形成，在肿瘤组织周围，laminin表达类型呈碎片状或断线状和连续线状，部分癌组织内laminin染色消失和癌细胞浆内有laminin弱染色。胆囊癌组织中survivin阳性表达率明显高于胆囊腺瘤和慢性胆囊炎组织，但survivin的阳性表达与胆囊癌细胞分化程度、病理分级和转移无关(P>0.05)。且胆囊癌组织中laminin的连续线断裂或缺失,与胆囊癌组织中survivin的表达情况无相关性。 结论:胆囊癌基质中laminin的表达类型与胆囊癌的侵袭和转移有关，而survivin在胆囊癌中表达增加，但两者之间似乎无关联。      

miR-132抑制舌癌细胞增殖、侵袭及迁移

为研究miR-132在舌癌中的表达情况,收集病理诊断确诊为舌癌的20例患者舌癌组织及癌旁组织,通过RT-PCR检测miR-132在舌癌及癌旁组织中的表达水平。应用mimics转染CAL-27细胞株,采用平板克隆、MTT实验检测miR-132过表达组、对照组舌癌细胞的增殖活性。Transwell实验研究miR-132与舌癌细胞迁移作用的关系。结果显示,miR-132在舌癌组织中的表达水平(0.56±0.05)显著低于癌旁组织(1.43±0.12,P0.001)。舌癌组织中miR-132的表达水平随着病理分级恶性程度的升高而降低(P0.05)。细胞克隆形成实验及MTT实验结果显示,miR-132 mimics组的细胞活性及增殖明显受抑制(P=0.003 2,P0.001)。Transwell实验显示,miR-132 mimics组的细胞过膜数量[(89±15)/视野]明显低于对照组[(380±25)/视野](P=0.002)。提示miR-132在舌癌组织中表达下降,miR-132在舌癌的增殖和转移过程中发挥重要作用,其可作为舌癌的潜在临床预后标志物,为舌癌的预后判断及治疗提供参考。     

宫颈鳞癌基底膜Ⅳ型胶原及层粘连蛋白的免疫组化观察

采用免疫组织化学法染色,标记基底膜的特异性成分Ⅳ型胶原、层粘连蛋白对宫颈鳞状上皮不典型增生、原位癌,早浸癌及浸润癌的基底膜变化进行观察研究。结果显示正常及不典型增生情况下基底膜是完整,连续的。原位癌基底膜通常是连续的,偶有中断。早浸癌基底膜通常是断续的,偶尔癌巢周围可出现完整、连续的基底膜。浸润癌基底膜的表现与细胞分化有关,分化好的癌巢周围存在基底膜,分化差的则缺少基底膜。基底膜变化的机理可能在于合成和分解代谢的不平衡。     

GTC对DMBA诱发大鼠乳腺导管增生的抑制作用

目的探讨绿茶提取物绿茶儿茶素(green tea catechins,GTC)对二甲基苯蒽(DMBA)诱发SD大鼠乳腺癌前增生性病变的抑制作用。方法42只7周龄雌性SD大鼠,随机分为A(空白对照)、B(DMBA)、C(GTC)、D(DMBA GTC)4组。①A、B组自由饮用自来水;②C、D组自由饮用自来水,并各鼠每天接受1次GTC水溶液灌胃(80mg/d);③B、D组各鼠于实验首日接受一次性DMBA油溶液灌胃(70mg/kg)。实验持续20周。第20周末时,断颈处死各组大鼠,取其全部乳腺组织,常规制备石蜡HE切片,镜下计数4组大鼠乳腺癌前增生性病变;以SP法和TUNEL法分别检测B、D组大鼠乳腺上皮细胞的PCNA表达(增生指数)和凋亡指数。结果①GTC灌胃(C、D)组大鼠的乳腺上皮增生性病变少于非GTC灌胃(A、B)组,其中的D(DMBA GTC)组少于B(DMBA)组(P<0·05);②D组大鼠乳腺上皮细胞的PCNA表达少于B(DMBA)组(P=0·001);③D组与B组大鼠乳腺上皮细胞的凋亡指数差异无显著性(P=0·177)。结论①绿茶提取物GTC对DMBA诱发SD大鼠乳腺癌前增生性病变具有抑制作用,这可能与乳腺上皮细胞的增生受抑有关,未显示与乳腺上皮细胞凋亡指数的相关性;②GTC对未接受致癌剂DMBA作用的大鼠乳腺上皮细胞未显示增生抑制作用。     

Aquaporin 1在中国人肺鳞癌细胞的表达

目的研究Aquaporin 1(AQP1)对中国人肺鳞癌细胞分化过程的影响。方法采用HE染色和AQP1免疫组织化学法对高、中、低分化的人肺癌细胞进行染色,光学显微镜观察并显微摄影。结果AQP1在高、中分化的肺鳞癌细胞呈阳性表达,在低分化的肺鳞癌细胞呈阴性表达;在高分化鳞癌中,角化珠旁的癌细胞呈较强阳性表达,癌巢周围的癌细胞呈阴性表达。结论AQP1参与人肺麟癌细胞的分化,其表达可能同分化程度相关。     

肺癌间质血管与肺癌的转移

我们对32例肺鳞癌及癌周正常肺组织的免疫组化研究表明:肺癌间质血管内皮细胞Ⅷ因子表达明显增强,主要组织相容抗原(HLA-DR)几乎不表达,Ⅳ型胶原在基底膜上的表达与鳞癌的分化程度密切相关,分化越低,其间质血管基底膜越不完整,这些形态和功能的改变都有利于肺癌细胞的浸润生长和转移。但从核仁形成区的研究才明:无论肺癌的分化程度如何,其间质血管内皮细胞核内均为一个Ag-NORs颗粒。推测间质血管内皮细胞的生物学行为并没有改变。     

肾脏原发性鳞状细胞癌5例临床病理学分析

目的：探讨肾脏原发性鳞状细胞癌临床病理学特征、诊断、鉴别诊断、治疗及预后。方法回顾性分析5例肾脏原发性鳞状细胞癌的临床表现、组织学形态和免疫表型特征,并复习相关文献。结果5例均为男性,平均年龄54．3岁,其中2例伴有肾盂结石。镜下见癌细胞呈巢状、片状排列,部分癌巢周边细胞呈栅栏状排列,可见细胞间桥及角化珠,伴有坏死。免疫表型：癌细胞 CK5/6和 p63均(+)、EMA(灶状+),CK20、CK7和vimentin均(-)。结论肾脏原发性鳞状细胞癌较少见,可能来源于化生性癌或起源于肾原基的未分化干细胞。免疫组化染色可辅助确诊,其恶性程度比肾透明细胞癌高,预后差。     

Protective Effect of Withaferin-A on Micronucleus Frequency and Detoxication Agents During Experimental Oral Carcinogenesis

Our aim was to investigate the effect of Withaferin-A on bone marrow micronucleus frequency and buccal mucosa detoxication agents during 7, 12-dimethylbenz[a]anthracene (DMBA) induced hamster buccal pouch carcinogenesis. Oral squamous cell carcinoma was developed in hamsters'' buccal pouches by painting 0.5% DMBA in liquid paraffin, three times per week for 14 weeks. We observed 100% tumor formation in DMBA painted hamsters. Elevated frequency of bone marrow micronucleated polychromatic erythrocytes (MnPCEs) and decrease in buccal mucosa phase II detoxication agents were noticed in tumor bearing hamsters. Oral administration of Withaferin-A significantly reduced the micronucleus frequency and brought back the status of phase II detoxication agents in DMBA painted hamsters. Our study thus demonstrated the protective effect of Withaferin-A on DMBA-induced micronucleus frequency in the bone marrow of golden Syrian hamsters. Also, Withaferin-A maintained the status of buccal mucosa detoxication agents during DMBA-induced hamster buccal pouch carcinogenesis.     

A proposed selective cell carcinogenesis in mammary tumors.

The present study was done to ascertain whether a specific carcinogenic agent has a causal effect on the initial proliferation of only one cell type or whether it acts indiscriminately on all cells in the breast secretory unit. Enzymes histochemistry and electron microscopy were performed on DMBA-induced mammary tumors in female Sprague-Dawley rats and on virus-associated spontaneous mammary tumors in C3H/HEJ mice. The results showed that the chemical carcinogen DMBA affects initial myoepithelial cell proliferation, while virus-associated mammary carcinoma originated from ductular epithelial cell proliferation. To determine whether a specific tumor is composed of a single cell type, tumors were grown in tissue culture. The monolayer was fixed in the usual manner for electron microscopy while in Falcon tissue culture plates. The plates were dissolved in xylene and the monolayer was cut into small pieces and embedded in the plastic media. Electron microscopy performed on the tissue culture and the original tissue from the virus-induced tumors showed the presence of viruses in large numbers. It also suggested the differentiation of basal membrane to form basal lamina and apical plasma membrane into microvilli. This study strongly suggests the presence of selective cell carcinogenesis in the mammary gland.     

The assessment of cell proliferation during 9,10-dimethyl-1,2-benzanthracene-induced hamster tongue carcinogenesis by means of histone H3 mRNA in situ hybridization

The aim of this study was to investigate cell kinetics and ultrastructural changes during carcinogenesis using a hamster 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced tongue cancer model. Five squamous cell carcinomas, five dysplastic epithelia, seven hyperplastic epithelia, and four normal epithelia were obtained from 21 hamster tongues by applying 1.0% acetone solution of DMBA on the left lingual mucosa after scratching with a root canal broach. Ultrastructural examination revealed that the number of microvilli increased, whereas that of desmosomes decreased during carcinogenesis. Cell proliferation was analyzed by means of 5-bromodeoxyuridine (BrdU) immunohistochemistry and in situ hybridization (ISH) for histone H3 mRNA. The BrdU and histone H3 mRNA labeling indices (LIs) were lowest for normal epithelium, higher for hyperplastic and dysplastic epithelia, and highest for squamous cell carcinoma. Cytoplasmic histone H3 mRNA and nuclear BrdU were localized in virtually identical areas of serial sections. The correlation coefficient for the relationship between these two LIs was 0.97 (P 0.001). These results suggest that the assessment of cell proliferation using H3 mRNA ISH will be a useful technique for investigating biological behavior during carcinogenesis.     

Evaluation of carcinogenic potential of diuron in a rat mammary two-stage carcinogenesis model

This study aimed to evaluate the carcinogenic potential of the herbicide Diuron in a two-stage rat medium-term mammary carcinogenesis model initiated by 7,12-dimethylbenz(a)anthracene (DMBA). Female seven-week-old Sprague-Dawley (SD) rats were allocated to six groups: groups G1 to G4 received intragastrically (i.g.) a single 50 mg/kg dose of DMBA; groups G5 and G6 received single administration of canola oil (vehicle of DMBA). Groups G1 and G5 received a basal diet, and groups G2, G3, G4, and G6 were fed the basal diet with the addition of Diuron at 250, 1250, 2500, and 2500 ppm, respectively. After twenty-five weeks, the animals were euthanized and mammary tumors were histologically confirmed and quantified. Tumor samples were also processed for immunohistochemical evaluation of the expressions of proliferating cell nuclear antigen (PCNA), cleaved caspase-3, estrogen receptor-α (ER-α), p63, bcl-2, and bak. Diuron treatment did not increase the incidence or multiplicity of mammary tumors (groups G2 to G4 versus Group G1). Also, exposure to Diuron did not alter tumor growth (cell proliferation and apoptosis indexes) or immunoreactivity to ER-α, p63 (myoephitelial marker), or bcl-2 and bak (apoptosis regulatory proteins). These findings indicate that Diuron does not have a promoting potential on mammary carcinogenesis in female SD rats initiated with DMBA.     

Tumor associated proteins of rat skin tumor induced by 7,12-dimethylbenz[a]anthracene

The incidence of tumor and the time of expression, cellular localization and the molecular weight of tumor associated proteins of rat skin tumor induced by 7,12-dimethylbenz[a]anthracene (DMBA) with or without 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were studied. The time of the development of skin tumors in 0.1% DMBA-TPA treated rats was significantly shorter than that in rats which were treated with DMBA alone. In the complete carcinogenesis case, papillomas developed more slowly and were less common and also squamous cell carcinomas appeared much later. From the analysis of the proteins of each experimental group by SDS-PAGE and two dimensional gel electrophoresis, at least three tumor associated proteins were identified (54kd, pl = 5.66; 27kd, pl = 5.85; 11kd, pl = 4.90). Also these proteins were found in rat dorsal skin from 14 days gestation to 21 days postpartum, and disappeared after 28 days. In conclusions, two stage skin carcinogenesis could be successfully demonstrated in Sprague-Dawley rats and abnormal proteins were produced in DMBA or DMBA-TPA induced skin tumor. The tumor associated proteins of skin tumor induced by DMBA or DMBA-TPA were appeared at the late initiation stage or early promotion stage, and they were localized in plasma membrane and were glycoproteins that are thought to be related to the epidermal differentiation process.     

Susceptibility of the mammary gland to carcinogenesis. III. The cell of origin of rat mammary carcinoma.

The rat mammary gland epithelium is composed of three cell types: dark cells (DCs), intermediate cells (ICs), and a layer of myoepithelial cells (MCs), which are evenly distributed along the mammary gland tree in rather constant proportions. The present study was carried out for a determination of the effect of the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) on the distribution and proliferative activity of these cell populations. The proportion and the DNA labeling index (DNA-LI) of each cell type were determined in terminal end buds (TEBs), terminal ducts (TDs), alveolar buds (ABs), and alveoli of the normal Sprague-Dawley rat mammary gland and in intraductal proliferations (IDPs) and carcinomas removed at selected intervals after DMBA administration. DMBA-induced changes in cell distribution were limited to TEBs and TDs, whereas ABs and alveoli were unaffected. The alterations consisted in an increment in ICs from 11% in TEBs and TDs to 90% in tumors and a decrease in DCs from 77% in TEBs and TDs to 7% in tumors. MCs were relatively unaffected. The DNA-LI of DCs, which in the normal gland TEB was 14%, was depressed by DMBA to 6%, whereas the DNA-LI of ICs remained unchanged from the basal level of 40% during the process of carcinogenesis. The progressive increment in number of ICs with a steady DNA-LI suggested that the IC is the target cell of the carcinogen and the cell of origin of mammary carcinomas.     

Genistein and daidzein, in combination, protect cellular integrity during 7,12-dimethylbenz[a]anthracene (DMBA) induced mammary carcinogenesis in Sprague-Dawley rats

The status of glycoconjugates (protein bound hexose, hexosamine, sialic acid and fucose) in plasma or serum serve as potential biomarkers for assessing tumor progression and therapeutic interventions. Aim of the present study was to investigate the protective effect of two major soy isoflavones, genistein and daidzein, in combination on the status of glycoconjugates in plasma, erythrocyte membrane and mammary tissues during 7,12-dimethylbenz[a]anthracene (DMBA) induced mammary carcinogenesis in female Sprague-Dawley rats. A single subcutaneous injection of DMBA (25 mg rat(-1)) in the mammary gland developed mammary carcinoma in female Sprague-Dawley rats. Elevated levels of plasma and mammary tissue glycoconjugates accompanied by reduction in erythrocyte membrane glycoconjugates were observed in rats bearing mammary tumors. Oral administration of genistein + daidzein (20 mg + 20 mg kg(-1) bw/day) to DMBA treated rats significantly (p< 0.05) brought back the status of glycoconjugates to near normal range. The present study thus demonstrated that genistein and daidzein in combination protected the structural integrity of the cell surface and membranes during DMBA-induced mammary carcinogenesis.     

The role of the TP53 gene during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

The medium-term tongue carcinogenesis assay is a useful model for studying oral squamous cell carcinomas phase by phase. The aim of the present study was to investigate the expression of p53 by immunohistochemistry and examine the DNA sequence of exons 5, 6, 7, and 8 of Tp53 for mutations during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4NQO). A total of 30 male Wistar rats were treated with 4-nitroquinoline 1-oxide in their drinking water for 4, 12, and 20 weeks at a dose of 50 ppm. Ten animals were used as negative controls. No histopathological changes in the tongue epithelia were observed in the control group or in the treatment group after 4 weeks of 4NQO. Following 12 weeks of treatment, hyperplasia as well as epithelial dysplasia was found in both mild and moderate forms. At 20 weeks, moderate and/or severe oral dysplasia and squamous cell carcinoma of the tongue were found, and the majority of animals had squamous cell carcinoma. The levels of p53 protein were increased (p<0.05) in pre-neoplastic lesions and in squamous cell carcinomas in some of the tumor cells in squamous cell carcinomas. No mutations were found in any of the exons that were evaluated after the 4-, 12-, or 20-week treatments. Taken together, our results suggest that p53 expression may be an important event in the malignant conversion, whereas Tp53 mutations are not involved in the multi-step tongue carcinogenesis of Wistar rats induced by 4NQO.