【作者】 杨开颜；

【导师】 周韧；

【作者基本信息】 浙江大学 ， 病理学与病理生理学， 2004， 硕士

【摘要】 目的： 作为肿瘤抑制基因p53家族的新成员p73和p51基因在肿瘤的发病过程中所起的作用尚未完全明了，研究p73、p51基因在乳腺癌组织中的表达情况，以期了解p73、p51基因与乳腺癌发病的关系。 方法： 应用RT-PCR、TUNEL等技术检测乳腺癌组织p51、p73基因的表达及肿瘤细胞的凋亡。蜡块按常规切片，TUNEL检测操作按细胞凋亡原位检测试剂盒说明书规范进行。设阳性和阴性对照。TUNEL染色阳性细胞棕黄色颗粒位于细胞核。每张切片选择多个高倍视野，共观察1000个肿瘤细胞，计算平均阳性细胞比例，以百分率代表肿瘤细胞凋亡率。RNA的提取采用Tripure分离试剂，按试剂盒说明书规范操作提取RNA。然后进行定量、检测纯度等。先将RNA反转录成cDNA，用于PCR扩增。我们用上游引物：5′-AACCATGGCCACGCAGTG-3′和下游引物：5′-GTTGCTAGAGCGGAGCAGCT-3来分析p73的表达。用上游引物：5′-CAGCCTGCTTCAGGAATGAG-3′和下游引物：5′-CCAATGGCCGGTCTAAGGAT-3′来分析p51的表达。β-actin作为浙江大学硕士学位论文参照。PCR扩增后取1 oulPCR产物经1 .5%琼脂糖凝胶电泳、澳化乙锭染色后，紫外灯下观察结果。在66 lbP位置出现条带为p一actin表达阳性，在23 lbp位置出现条带为p73表达阳性;在348bp位置出现条带为psl表达阳性。 结果:1.45例原发性乳腺癌中P73基因表达阳性率71.1%(32/45)，其中 浸润性癌35例阳性率52.9%(29/35)(单纯癌13/16、髓样癌9/11 和硬癌7/8)，导管内癌30%(3/10)，两者差异有显著性(P<0.01)。 35例纤维腺瘤p73基因表达阳性率14.3%(5/35)，与乳腺癌相比 差异有显著性(P<0.001)。2.45例原发性乳腺癌psl基因表达阳性率46.7%(21245)，其中浸 润性癌35例阳性率54.3%(19/35)(单纯癌9八6、髓样癌5八l和 硬癌5/8)，导管内癌20%(2/10)，两者差异有显著性(P<0.05)。 35例纤维腺瘤psl基因表达阳性率1 1 .4%(4/35)，与乳腺癌相比 差异有显著性(P<0.001)。3.淋巴结转移者21例p73基因表达阳性者21例(100%)，无转移 者24例中p73基因有n例表达阳性(45.8%)，两者差异有显著 性意义(P<0.001)。淋巴结转移者21例中P51基因表达阳性者19 例(90.5%)，而无淋巴结转移的24例中P51基因仅2例阳性表达 (8.3%)，两者差异有显著性意义(P<0.001)。4.P73基因表达阳性的肿瘤细胞凋亡率10.05士1.40，阴性的为 9.24士1 .42，两者无显著性差异(P>0.05);P51基因表达阳性的浙江大学硕士学位论文 21例肿瘤细胞凋亡率9.73士1 .54，24例阴性的为9.90士1 .36，两 者无显著性差异(P>0.05);肿瘤细胞的凋亡率与P51、p73基因 表达无相关性。5.27例雌激素受体阳性者有18例p73基因表达阳性(66.7%)，18例 雌激素受体阴性者有14例p73基因表达阳性(77.8%)，两者没有 显著性差异(P>0.05);23例孕激素受体阳性者p73基因表达为 16例(69.6%)，22例阴性者表达p73基因为16例( 72.7%)，两 者也没有显著性差异(P>0.05)。6.27例雌激素受体阳性者有13例psl基因表达阳性(48.1%)，18例 雌激素受体阴性者有8例P51基因表达阳性(44.4%)，两者没有显 著性差异(P>0.05);23例孕激素受体阳性者P51基因表达为n 例(47.5%)，22例阴性者表达psl基因为10例(45.5%)，两者 也没有显著性差异(P>0.05)。 结论:1.在乳腺癌的发病过程中，p73基因、psl基因起了一定的作用(乳 腺癌发病可能与P51基因、p73基因表达增多有关)。2.与早期乳腺癌相比，p73基因、psl基因在中晚期乳腺癌中作用 可能更明显;3.p73基因、P51基因并不一定通过增强细胞的凋亡在乳腺癌发病 中起作用;4.p73基因、psl基因表达与雌、孕激素受体无相关性。更多还原

【Abstract】 Aim:The roles of p73gene and p51 gene, as two novel genes of p53 family of tumor suppressor, are not clear yet in carcinogenesis. To determine the relationship between p73 gene,p51 gene and breast cancer, we investigated the expression of p73 gene p51 gene in breast cancer.Methods:The expression of p73 gene p51 gene in breast cancer tissues were detected by RT-PCR, and tumor apoptotic cells were detected by TUNEL. The paraffin-embedded tissues were performed under the direction of TUNEL kit instructions. Positive and negative controls were done. Brown-yellow pellets were observed in nuclei when the specimen was positive. Apoptosis index (AI) per case were calculated respectively by counting TUNEL positive cell of 1000 tumor cells. RNAs of all cases were extracted using the Tripure extraction kit according to the manufacture’s protocol. Samples of cDNA were then generated from RNA by RT. The forward primer was5’-AACCATGGCCACGCAGTG-3’ and the reverse primer was 5’-GTTGCTAGAGCGGAGCAGCT-3’ for p73 gene; the forward primer was 5" -CAGCCTGCTTCAGGAATGAG-3’ and the reverse primer was 5’-CCAATGGCCGGTCTAAGGAT-3’ for p51 gene. -actin was also performed as control. The PCR products were analyzed on 1.5% agarose gel by electrophoresis. actin positive band was observed in 661bp; p73 gene in 231 bp and p51 gene in 348 bp. Results:1. There were 32 cases detected the expression of p73 gene among 45 cases breast cancer(71.1%),29/35 positive in invasive carcinoma (simple carcinoma(13/16),medullary carcinoma(9/11),scirrhous carcinoma(7/8)) and 3/10 positive in intraductal carcinoma,the expression of p73 gene is significantly higher in breast invasive carcinoma than in breast intraductal carcinoma (P<0.01).There were 5 cases detected the expression of p73 among 35 cases fibroadenoma ,the expression of p73 gene is significantly higher in breast cancer than in breast fibroadenoma (P<0.001) .2. There were 21 cases detected the expression of p51 gene among 45 cases breast cancer(46.7%),19/35 positive in invasive carcinoma(simple carcinoma(9/16),medullary carcinoma(5/ll),scirrhous carcinoma(5/8)) and 2/10 positive in intraductal carcinoma,the expression of p51 gene is significantlyhigher in breast invasive carcinoma than in breast intraductal carcinoma (P<0.05).There were 4 cases detected the expression of p51 among 35 cases fibroadenoma ,the expression of p51 gene is significantly higher in breast cancer than in breast fibroadenoma (P<0.001) .3. There were 21 cases the expression of p73 gene among 21 cases of lymph nodes metastasis ,there were 11 cases the expression of p73 gene among 24 cases no lymph nodes metastasis ,the difference is significant in positive metastasis of lymph nodes and in negative ones(P<0.001); There were 19 cases the expression of p51 gene among 21 cases of lymph nodes metastasis ,there were only 2 cases the expression of p51 gene among 24 cases no lymph nodes metastasis ,the difference is significant in positive metastasis of lymph nodes and in negative ones (P<0.001);4. The apoptosis index of p73 gene positive cells(32) was 10.05 1.40, p73 gene negative cells(13) was 9.241.42 , there was not significantly different between the positive and negative ones (P>0.05) . The apoptosis index of p51 gene positive cells(21) was 9.73 1.54, p51 gene negative cells(24) was 9.90 1.36, there was not significantly different between the positive and negative ones (P>0.05) .5. There were 18 cases detected the expression of p73 gene among 27cases of ER positive breast cancer. There were 14 cases detected the expression of p73 gene among 18 cases of ER negative breast cancer , the expression of p73 gene was not significantly different between ER positive breast cancer than ER negative breast cancer (P>0.05) .There were 16 cases detected the expression of p73 among 23 cases of PR positive breast cancer, there were 16 cases detected the ex更多还原