犬乳腺肿瘤中MUC1的表达及临床意义

为了检测MUC1在乳腺肿瘤的侵袭、转移及病理分级中的作用,本研究通过real-time PCR检测了47例犬乳腺肿瘤组织(20例良性,27例恶性)和3例健康犬乳腺组织中MUC1基因的转录情况,通过IHC检测MUC1在犬乳腺组织中的表达情况,并分析二者与肿瘤恶性程度分级的关系。结果发现:MUC1基因和MUC1在犬乳腺肿瘤组织中高表达,且在不同的恶性分级中差异显著(P0.05)。研究显示MUC1基因和MUC1可作为犬乳腺肿瘤诊断和恶性程度评估的重要指标之一。     

NLRP3炎症小体及其下游炎症因子在犬乳腺肿瘤组织中的表达

本研究旨在明确NLRP3炎症小体及下游炎症因子在犬乳腺肿瘤中的表达及临床意义.采用RT-qPCR方法检测45例犬乳腺肿瘤组织(15例良性肿瘤和30例恶性肿瘤)以及16例肿瘤旁正常乳腺组织中的NLRP3、Caspase-1、IL-1β和IL-18 mRNA的表达水平,并分析NLRP3与肿瘤临床病理学特征的关系(肿瘤大小、...     

实时荧光定量RT-PCR检测犬乳腺肿瘤血管内皮生长因子(VEGF)基因方法的建立及初步应用

为了准确地分析血管内皮生长因子（Vascular endothelial growth factor,VEGF）在犬乳腺肿瘤形成过程中的作用,试验设计了针对犬VEGF基因的特异性引物,提取乳腺肿瘤组织RNA并扩增出目的片段,将PCR产物克隆到pGEM-T Easy载体,经质粒PCR扩增、酶切和测序鉴定重组质粒,构建标准曲线等,成功建立了乳腺肿瘤VEGF基因实时荧光定量PCR检测方法。运用实时荧光定量PCR检测了40例不同的犬乳腺肿瘤病例、2例正常乳腺组织。结果发现：在犬恶性乳腺肿瘤组织中,VEGF基因表达量明显高于良性乳腺肿瘤和正常乳腺组织（P〈0.001）;在伴有淋巴结转移的犬乳腺癌组织中,VEGF基因表达量明显高于没有发现转移的乳腺癌组织（P〈0.01）,但VEGF基因的表达量与肿瘤的大小和发病动物年龄无关（P〉0.05）。VEGF基因在犬乳腺癌组织中的表达量远高于其在正常乳腺和良性乳腺肿瘤组织中的表达,且与淋巴结是否发生转移相关。因此,VEGF可作为犬乳腺癌转移、复发的预测指标之一。     

犬p27蛋白在犬乳腺肿瘤中的表达分析

为分析犬p27蛋白在犬乳腺肿瘤组织中的表达情况,本研究构建了原核表达重组质粒pET-p27,进行重组蛋白的表达,并将纯化的重组蛋白免疫新西兰白兔,制备了抗犬p27蛋白多克隆抗体。将制备的犬p27抗体对临床84例犬乳腺肿瘤组织中的犬p27蛋白表达情况进行了检测。结果显示,制备的抗体效价达1∶12 800以上;western blot检测显示,该多克隆抗体能够与重组蛋白发生特异性反应;免疫组织化学试验表明,犬p27蛋白在犬恶性乳腺肿瘤组织中表达率低于犬良性乳腺肿瘤组织,提示犬p27的表达水平可能与犬乳腺肿瘤的恶性程度相关,本实验为进一步研究犬p27蛋白的生物学功能奠定了基础。     

Ki-67在犬乳腺肿瘤的表达与分析

本试验通过免疫组化的方法,检测了从中国农业大学教学动物医院收集到的6个正常乳腺组织和135例乳腺肿瘤组织(良性30例,恶性105例)Ki-67的表达率。结果犬乳腺肿瘤组织中的Ki-67蛋白表达率明显高于正常乳腺组织(P0.05);犬恶性肿瘤组织中的Ki-67蛋白表达率明显高于良性肿瘤(P0.05)。表明Ki-67在恶性肿瘤里高表达,可以作为犬乳腺肿瘤疾病诊断的指标。     

PTEN 基因在犬乳腺肿瘤组织中的表达及临床意义

有关酪氨酸磷酸酶基因（Phosphatase and tensin homolog deleted on chromosometen,PTEN）在乳腺肿瘤中的检测在人医早有报道。为了研究PTEN基因在犬乳腺肿瘤组织中的表达情况,笔者运用实时荧光PCR定量检测了38例不同的犬乳腺肿瘤组织（包括15例良性乳腺肿瘤和23例恶性乳腺肿瘤）、4例正常犬乳腺组织。结果发现：PTEN基因在犬恶性乳腺肿瘤组织中表达量明显低于其在良性乳腺肿瘤和正常乳腺组织中的表达量,两者差异极显著（P〈0.001）;PTEN在良性乳腺肿瘤组织中的表达与正常犬乳腺组织相比,差异不显著（P〉0.05）;发生了淋巴结转移的乳腺癌PTEN基因的表达量与未发生转移的乳腺癌组织的表达量间差异亦不显著（P〉0.05）,且PTEN的表达量与肿瘤组织的大小和发病动物年龄无关。结论：PTEN蛋白表达异常可能与乳腺肿瘤发生、发展相关,可考虑作为判断犬乳腺肿瘤生物学行为和预测的指标。     

CA15-3在犬乳腺肿瘤中的表达及其临床意义

通过ELISA和IHC分别检测了乳腺肿瘤患犬血清和组织中CA15-3的浓度和表达情况,发现CA15-3在乳腺肿瘤患犬血清浓度(15. 70 U/m L±12. 93 U/m L)明显高于健康犬血液中CA15-3浓度(5. 72 U/m L±4. 82 U/m L)。IHC结果发现,CA15-3 MOD在乳腺肿瘤组织中(0. 21±0. 08)明显高于健康乳腺组织(0. 10±0. 04),且在不同的组织分级中差异显著(P 0. 05)。结论:血清中CA15-3浓度可作为犬乳腺肿瘤早期诊断和预后评估的生物标志物之一,组织中CA15-3蛋白可作为评估犬乳腺肿瘤恶性程度的生物标志物之一。     

犬乳腺肿瘤组织病理学观察及腱糖蛋白C表达的检测

为了探讨腱糖蛋白C(TNC)表达与犬乳腺肿瘤生物学关系,对23例犬乳腺肿瘤临床病例进行病理学观察及分类,然后采用免疫组化SABC方法检测TNC蛋白在23例犬乳腺肿瘤和2例犬正常乳腺组织中的表达情况。结果显示:23例病例中,良性肿瘤有7例,占30.43%;恶性肿瘤有13例,占56.52%;增生病变有3例,占13.05%。在良性肿瘤中良性混合瘤最多,为3例,其余2例为导管内乳头状腺瘤,1例复杂腺瘤及1例纤维腺瘤。恶性肿瘤中,5例未分化癌,3例癌肉瘤,2例筛状癌,2例固体癌,1例纤维肉瘤,1例混合瘤引起的癌。TNC蛋白主要表达于细胞外基质。犬恶性乳腺肿瘤中,TNC蛋白阳性表达率为46.15%(6/13);良性乳腺肿瘤中,其阳性表达率为42.85%(3/7);乳腺增生病变和正常乳腺组织中,未见其表达。研究结果表明,TNC蛋白可以为犬乳腺肿瘤的鉴别诊断提供参考。     

检测p53基因诊断犬乳腺恶性肿瘤的初步研究

研究犬乳腺恶性肿瘤p53基因在体内的表达,探讨其与犬乳腺肿瘤恶性程度的相关性。根据GenBank中犬p53基因序列以及文献报道的高度保守区域,设计1对引物,同时以GAPDH基因序列作为内参,将临床采集的犬乳腺肿瘤组织进行p53基因PCR检测,分析p53基因的表达量变化趋势。结果表明,良性肿瘤组织p53基因的表达量极小;恶性肿瘤组织中的表达量随着其恶化程度明显增高。由此可推断,检测p53基因的表达量可作为诊断犬乳腺肿瘤、判断肿瘤恶化程度及预后的一个重要指标。     

44例犬乳腺肿瘤的病理学分析及p53和HER-2的蛋白表达

为了对犬乳腺肿瘤分类,分析表皮生长因子受体-2(HER-2)与p53的过表达在肿瘤类型上的差异及影响,对所收集的犬乳腺肿瘤标本,进行石蜡切片、H.E.染色,划分肿瘤的病理类型,应用免疫组织化学法检测犬乳腺肿瘤中HER-2基因与p53基因的表达情况。结果 44例犬乳腺肿瘤病理组织切片中,共观察到纤维腺瘤、叶状肿瘤、导管乳头状瘤、浸润性导管癌、浸润性小叶癌(腺泡型)、小叶癌、黏液癌、髓样癌伴随淋巴细胞浸润、混合样癌9种类型;42例犬乳腺肿瘤的免疫组化结果中,HER-2在良性肿瘤中无表达,在恶性肿瘤中的表达率为38.9%,p53在犬乳腺肿瘤中的表达可达78.6%,而在恶性肿瘤中的表达率100%。结论 HER-2与p53的过表达有肿瘤类型的差异性。     

The expression of carbonic anhydrase in canine mammary gland and mammary gland tumor

Carbonic anhydrase (CA), a metallo-enzyme containing zinc, broadly distributes in mammalian tissues and participates in physiological regulation such as respiration, acid-base balance, ion transport, bone resorption, as well as the development of tumor by the reversible hydration of carbon dioxide. However the expression of CA in the tissue of mammary gland tumor was not documented. In this study we examine the histolocalization and gene expression of CA in both normal canine mammary gland tissue and mammary gland tumor by histochemical examination, and RT-PCR. Four mRNA expression of CA isoenzymes, such as CA II, IV, VI and IX were found under RT-PCR analysis and different band patterns were found between normal canine mammary tissue and canine mammary gland tumor tissue. CA II, IV, VI and IX gene mRNA expression were found in the normal mammary gland tissue, indicating CA II, IV, VI and IX are likely to be the essential enzymes to maintain the normal physiological condition of canine mammary gland tissue cells. However the expression of CA IV was not found in the tissue of malignant mammary gland tumor that may become the marker for the prognostic recognition of canine mammary gland tumor.     

Expression patterns of the slit subfamily mRNA in canine malignant mammary tumors

Slit, a secreted protein, functions as a chemorepellent factor in axon guidance and neuronal migration and as an inhibitor in leukocyte chemotaxis. In humans, slit2 protein attracts endothelial cells and promotes tube formation in the tumor angiogenic mechanism. In this study, we cloned a part of the canine slit subfamily and examined the expression of slit subfamily mRNAs in 3 normal canine mammary glands and 11 mammary tumor samples by RT-PCR. The cloned part of the slit gene sequences showed high similarity to those of the human, mouse, and rat. The mRNAs were expressed at low levels in the normal mammary gland. The expression levels of slit1 mRNA were low in both the normal and tumor tissues. In contrast, the expression of slit2 mRNA increased in most of the malignant mammary tumors, and an increase in slit3 mRNA expression was observed in 2 of the malignant mixed tumors. These results suggest that the expression of slit2 plays an important role in tumor angiogenesis in canine mammary gland tumors and that slit2 can be a putative marker for malignancy diagnosis of these tumors.     

Loss of p27 expression in canine mammary tumors and their metastases

The p27 gene is a member of the cyclin-dependent kinase inhibitors, which arrest G1- to S-phase transition of the cell cycle. We have previously shown a significant reduction of p27 mRNA expression level in laser-microdissected mammary carcinomas and their lymph node metastases when compared to non-neoplastic mammary gland of the same dog. Here, p27 expression was analyzed on the protein level in non-neoplastic mammary gland, primary mammary carcinomas, their lymph node metastases and intravascular tumor cells of 49 dogs, adenomas of 49 dogs and non-neoplastic mammary gland of 98 dogs by immunohistochemistry. A significantly (p ? 0.05) decreased percentage of p27 positive tissue samples was found when normal gland was compared with adenomas, carcinomas and lymph node metastases. Specifically, 91% of normal gland epithelium displayed nuclear p27 expression. In contrast, only 22% of the adenomas, 20% of carcinomas, 12% of lymph node metastases and 32% of intravascular tumor cells had p27 reactivity. Cell cycle control by p27 is therefore lost in the majority of canine mammary tumors. The lack of significant differences between benign and malignant mammary tumors indicates that decreased p27 expression is an early step in carcinogenesis of canine mammary tumors and hinders the use of p27 as a marker of malignancy for this tumor type.     

Effectiveness of small interfering RNA (siRNA) against the Mcl-1 gene in a canine mammary gland tumor cell line

The effect of down-regulation of Mcl-1 expression by small interfering RNA (siRNA) against the canine Mcl-1 gene on apoptosis was investigated by transfecting CF33 (canine mammary gland tumor cell line) with siRNA using cationic liposomes. The siRNA against canine Mcl-1 increased the rate of apoptotic cells and decreased the numbers of viable cells. Further, sequence-specific down-regulation of Mcl-1 expression was measured by real time-PCR and Western blot analysis. The siRNA directed against the Mcl-1 gene reduced both the mRNA and protein expression in the CF33. Our study suggests the importance of Mcl-1 in canine mammary tumors for inducing apoptosis and reinforces using Mcl-1 as a putative therapeutic target in canine mammary gland tumor.     

Alteration of somatostatin receptor 2 expression in canine mammary gland tumor

Somatostatin receptor 2 (SSTR2) is a negative regulator of cell proliferation in human breast cancer. Since there is little information about SSTR2 in canine mammary gland tumor (MGT), we clarified its distribution and expression level in normal mammary gland, benign MGT and malignant MGT. SSTR2 expression determined by immunohistochemical staining was observed in the cytoplasm of luminal epithelial cells. The intensity was negatively correlated with malignancy: normal tissues and some of the benign tumors had the highest levels, while the malignant tumors had little or no SSTR2 expression. As for the Western blotting, SSTR2 protein level in benign tumors was significantly lower than the normal mammary gland. On the other hand, SSTR2 protein levels in two of three malignant tumors were higher than the other groups. These results suggest that SSTR2 expression alters according to the malignancy of canine MGT.     

Expression patterns of the BRCA1 splicing variants in canine normal tissues and mammary gland tumors

Human BRCA1 is familial breast cancer susceptibility gene. Recently, decreased BRCA1 mRNA and protein expression has been identified in sporadic breast tumors. In the reported human BRCA1 splicing variants, delta11b lacks the majority of exon11 and is suspected to have a distinct function in normal tissues. The splicing variants display a variety of expression pattern in breast cancer samples. Although mammary gland tumor is important disease in dog, there are few reports for BRCA1 in the canine tumors. In this study, we examined the relative amounts of BRCA1 splicing variants mRNA in canine normal and mammary tumor samples by RT-PCR to investigate whether there is the altered expression of variant mRNAs in the canine tumor as reported in human. The exon11b-defecting RT-PCR products were observed in all the normal tissues examined and the nucleotide sequence was quite similar to that of human BRCA1 delta11b. In some tumor samples, we did not detect the products targeted for exon10-13 and exon14-15, while these products were observed in all the normal samples examined. Especially, the relative amounts of the exon11-defecting products were remarkably decreased in most of the tumors (11/16).