【作者】 路璐；

【导师】 范志民；

【作者基本信息】 吉林大学 ， 外科学， 2012， 博士

【摘要】 在类固醇激素中除了性激素（雌孕激素，雄激素等）外，糖皮质激素也在乳腺组织生长和生理功能中发挥着重要作用。糖皮质激素不仅能够影响原代培养的乳腺上皮细胞的分化，还对泌乳的发生与维持起着重要的作用。大量的体外研究已经证实人类的乳腺癌细胞具有对类固醇激素的反应，此外在不同的乳腺癌细胞系也证实糖皮质激素具有抑制细胞生长的作用。但是截至目前为止，糖皮质激素在乳腺癌发生及发展中的作用机制及其在乳腺癌局部组织中的调节方式尚不清楚。通过配体结合实验研究发现：糖皮质激素受体（GR）在大约33-50%的乳腺癌中表达。最近的体外和体内的异种移植模型研究中发现：地塞米松激活的糖皮质激素受体能够抑制雌激素依赖性乳腺癌的生长。在糖皮质激素靶组织中，11β-羟化类固醇脱氢酶(11β-HSD)能够调控细胞内糖皮质激素水平。11β-羟化类固醇脱氢酶分为1型和2型，具有催化肾上腺皮质酮和皮质醇相互转化的作用。其中，11β-HSD1能够催化皮质酮转化为皮质醇，而11β-HSD2则作用相反。在小鼠的基因敲除实验中发现11β-HSD2具有维持循环中糖皮质激素水平的作用，并使肝脏中糖皮质激素水平保持较高水平。而抑制11β-HSD2的活性则能够增强糖皮质激素的抗增殖作用。最近采用基因连锁的研究发现，11β-HSD1基因可能是绝经后妇女乳腺癌患病风险的候选基因。为了进一步明确11β-HSD1和GR在乳腺癌发生和发展中的作用，首先构建11β-HSD1相对应的蛋白，为氨基酸位置42-225的肽序列，分多点注入4只新西兰兔体内，取得相应抗血清，并纯化，采用免疫印记法检测抗体纯度。然后，分别应用纯化的特异性11β-HSD1抗体和GR以及CDC-47、ERα、PRB等特异性抗体，采用免疫组化方法测定11β-HSD1以及GR、CDC-47、ERα、PRB在84例女性乳腺癌患者的乳腺癌组织及癌旁非恶性乳腺组织中的表达情况，并明确它们在组织及细胞中表达的定位情况，进一步探讨了其表达与相关临床参数及其他相关指标的关系。结果：1、11β-HSD1在乳腺癌细胞浆中弱表达，阳性率为64.3%（54/84）。周围基质细胞无特异性染色。在非恶性癌旁组织腺泡和腺管上皮细胞的细胞质和细胞核中11β-HSD1均有染色，染色主要位于腺泡上皮细胞双层结构中的外层。在乳腺癌和癌旁组织中小血管壁染色明显，偶尔可见到间质细胞染色，阳性率为91.7%（77/84）。阴性对照组未见到阳性表现。研究结果表明：该酶在乳腺癌组织中的表达明显低于癌旁非恶性乳腺组织（p<0.001）。2、类固醇激素受体（ERα，GR和PRB）与CDC47一样，在乳腺癌细胞核和非恶性乳腺组织上皮细胞的细胞核中表达。在84例乳腺癌组织的检测中， ERα阳性表达的患者为71.4%，GR阳性表达为72.6%，PRB阳性表达为78.6%。而在非恶性乳腺组织中，ERα阳性表达为67.9%，GR阳性表达为95.2%，PRB阳性表达为64.3%。乳腺癌组织中GR的阳性表达明显低于癌旁非恶性乳腺组织（p<0.0001），而PRB的阳性表达反而明显高于正常周围组织（p<0.05）。CDC47作为检测肿瘤增殖的标记性抗原，它在阳性肿瘤的百分比明显高于非肿瘤周围组织（77.4%vs.15.5%；p<0.0001）。阴性对照组（应用相应抗原免疫吸收抗血清后）无阳性表达。3、相关性检验结果：在乳腺癌组织中11β-HSD1表达与腋窝淋巴结有明显相关（X2=8.192，P=0.042）。11β-HSD1表达与GR表达呈负相关（r=-0.327，p=0.002），与CDC-47呈负相关（r=-0.232，p=0.033），与PRB表达的呈正相关（r=0.218，p=0.047）；GR表达和ERα表达呈负相关（r=-0.262，p=0.016），与PRB表达呈负相关（r=-0.262，p=0.016）。在癌旁正常乳腺组织中，PRB表达与11β-HSD1表达呈正相关（r=0.269，p=0.013），与ERα表达呈正相关（r=0.351，p=0.001），与CDC-47表达呈正相关（r=0.492,p=0.000）。结论（创新点）：1、11β-HSD1在癌旁正常乳腺组织中表达的阳性率明显高于癌组织，表明11β-HSD1可能对乳腺癌发生和发展有抑制作用。CDC-47与11β-HSD1在癌组织中表达的相关性也证实了11β-HSD1对乳腺癌的抑制作用。2、GR的在癌组织中的表达明显低于在癌旁组织中的表达，证明GR与乳腺癌发生和发展有关。3、在乳腺癌组织中，11β-HSD1表达与GR表达呈负相关，表明了局部组织中11β-HSD1发挥作用可能不仅依赖于糖皮质激素，还存在其他直接作用方式，尚需进一步研究。4、与临床参数检验中发现，11β-HSD1表达与腋窝淋巴结转移数明显相关，表明11β-HSD1可能在乳腺癌转移中发挥作用，提示我们调节局部组织中11β-HSD1可能能够抑制乳腺癌转移，为临床研究提供新思路。5、对于ER阴性或低表达患者，糖皮质激素治疗可能会有效，本研究为ER阴性乳腺癌治疗提供一种新的思路。本实验是在加拿大拉瓦尔大学分子内分泌及肿瘤研究中心进行的，相关文章发表在“Pathology&Oncology Research”SCI(Impact Factor:1.483)收录杂志。更多还原

【Abstract】 Sex steroid hormones as well glucocorticoids exert important roles in the development andfunctions of the mammary gland. Glucocorticoids have been shown to induce differen-tiation ofmammary epithelial cells in primary culture and to be essential for the initiation and maintenanceof lactation. The responsiveness of human breast cancer cells to steroids has been extensivelyinvestigated in vitro. Thus, glucocorticoid-dependent inhibition of cell growth has beendemonstrated in different breast cancer cell lines. But the mechanism of glucocorticoid functionin the development and progression of the breast cancer and the regulation of glucocorticoid in localtissue are still not clear.By ligand-binding assays, glucocorticoid receptors (GR) have been foundto be present in about33–50%of human breast cancer. Recently, it has been shown thatactivation of glucocorticoid receptor by dexamethasone inhibited estrogen-dependent breastcancer growth in vitro and in xenograft model.In glucocorticoid target tissues, intracellularglucocorticoid levels are modulated by two11β-hydroxysteroid dehydroge-nase (11β-HSD)isoenzymes, type1and type2, which catalyze the interconversion of glucocorticoids from theirinactive (cortisone) to their active form (cortisol).11β-HSD type1is the isoenzyme whichconverts cortisone to cortisol while17β-HSD type2converts cortisol into cortisone in severaltissues. Gene deletion experi ments in mice suggest that the role of17β-HSD type2is both tomaintain circulating glucocorticoid levels and to provide higher local concentration ofglucocorticoids in the liver. On the other hand, inhibition of the activity of11β-HSD type2, theisoform which converts cortisol to the inactive steroid cortisone, has been shown to enhance theantiproliferative effect of glucocorticoids. The report has recently identified11β-HSD type1from a genome-wide linkage analysis as a possible candidate gene for breast cancer risk inpostmenopausal women.To better understand the role of11β-HSD type1and glucocorticoid receptors in breastcancer development and/or evolution, we prepare the protein of11β-HSD type1and thepeptide sequence located at amino acid position42–225of the human11β-HSD type1. Weinjected sc with1ml at multiple sites of four New-Zealand rabbits. Collect the antiserum of the rabbits and purify it. Positive antiserum was analyzed by immunoblot. The expression of11β-HSD1and CDC-47、ERα、PRB、GR in84specimens of female human breast carcinomaand adjacent non-malignant tissues were studied by immunocytochemistry. We located theenzymes and glucocorticoid receptors in breast cancer tissue and adjacent non-malignanttissues. These results were correlated with the clinical parameters.Results:1, Immunostaining for11β-HSD type1could be detected in the cytoplasm oftumor cells in54cases out of the84cases of breast cancer (64.3%). Usually, the cytoplasmicstaining expression appeared weak. The surrounding interstitial cells were generally devoid ofany specific staining. In non-malignant adjacent tissues, strong staining was seen in epithelialcells bordering the lumen (luminal cells) in both acini and ducts in91.7%of cases. Labeledstromal cells were occasionally observed. No staining could be detected when the antiserumimmunoabsorbed with the antigen (11β-HSD type1) or pre-immune serum was used (notshown). The expression of the enzyme was significantly lower (p <0.001) in tumors than innormal adjacent tissues.2, Steroid hormone receptors (ERα, AR, PR and GR) as well asCDC47immunoreactivity was almost exclusively found in nuclei of both cancer cells andepithelial cells from non-malignant adjacent tissues. In the84tumors examined,71.4%werepositive for ERα,78.6%for PRB and72.6%for GR. In non-malignant specimens, ERα waspositive in67.9%, PRB in64.3%and GR in95.2%of cases. The percentage of tumorsconsidered as positive for PRB was significantly higher (p <0.05) than the percentage ofpositive normal adjacent tissues. On the other hand, the percentage of tumors positive for GRwas significantly lowers (p <0.001) than that of positive normal adjacent tissues. Asexpected, the percentage of tumors considered as positive for CDC47was significantly higherthan that of non-tumoral adjacent tissues (77.4%vs.15.5%; p <0.001). No staining wasdetected when the antisera were immunoab-sorbed with the corresponding antigen (data notshown).3, Correlation between the different parameters studied: In malignant tissue, theexpression of11β-HSD1was significantly correlated with the metastasis of the axillary nodes（X2=8.192，P=0.042）; the expression of11β-HSD1was significantly correlated with GR andCDC-47（r=-0.327,p=0.002; r=-0.232,p=0.033, respectively）.The expression of11β-HSD1wassignificantly correlated with PRB(r=0.218,p=0.047). The expression of GR was significantlycorrelated with ERα and PRB(r=-0.262， p=0.016, r=-0.262,p=0.016, respectively). In adjacentnon-malignant tissues, the expression of11β-HSD type1was significantly correlated with PRB （r=0.269,p=0.013）, the expression of PRB was significantly correlated with ERα and CDC-47（r=0.351,p=0.001, r=0.492,p=0.000, respectively）.Conclusion:1, the expression of the11β-HSD type1was significantly lower in tumorsthan in normal adjacent tissues. It might be suggested that the enzyme might inhibit thepathogenesis and development of breast cancer. This is identified by the relationship between11β-HSD type1and CDC-47too.2, the percentage of tumors positive for GR wassignificantly lower than that of positive in normal adjacent tissues, it suggested GR mightplay a role in pathogenesis and development in breast cancer.3, the expression of11β-HSD1was significantly correlated with GR in breast cancer tissue. It suggested11β-HSD1may playa role through the other way independent of GR and further research is required.4, theexpression of11β-HSD type1was significantly correlated with the metastasis of the axillarylymph nodes, it suggested the enzyme may has a role in the metastasis of breast cancer andregulation of the enzyme in local tissue might be inhibit the metastasis of breast cancer. Thisgives us a new way for clinical research.5,treatment with glucocorticoid may be useful inbreast cancer patients whose expression of ER was negative or lower, this study maybeprovide a new way to treat ER-negative breast cancer.This experiment is conducted in Molecular Endocrinology and Oncology ResearchCenter of Laval University Hospital Research Center in Canada,the paper related to the topicwas published on “Pathology&Oncology Research”(SCI, Impact Factor:1.483).更多还原