Measurements of Cell Size and Expression of Proliferation Markers in Mammary Epithelial Cells of Juvenile Macromastic Patients

1.1 研究背景與目的 過去的調查研究發現，理想的成年女性乳房的體積為250至300立方公分，若超過400立方公分則為乳房肥大，特稱為巨乳症。巨乳症病患經常遭受身體與心理的不便，因而需要尋求手術改善。巨乳症依其乳房異常增大的時期，可分為青春期巨乳症與懷孕期巨乳症。巨乳症的成因可能是由於青春期或懷孕期乳房發育基因的表現異常而造成，但目前對於形成巨乳症的分子機制並沒有很詳細的研究。目前與巨乳症相關的研究大多集中於病患的心理生理評估及手術方法的比較。乳房發育的相關研究目前都集中於乳癌與泌乳退乳的機制。器官的尺寸與細胞的尺寸及生長有關，PI3K/Akt及Jak2/STAT5訊息傳遞路徑均曾被提及與乳腺上皮細胞的增生與凋亡有關，因此本研究目的在量測青春期巨乳症病患乳房組織中乳腺上皮細胞的尺寸及細胞增生分子標記，包括Ki-67、Akt-1/2/3、phospho-Akt-1/2/3、及STAT5a，以釐清巨乳症成因與乳腺上皮細胞增生的關係。 1.2 研究材料與方法 我們蒐集西元2003年至2005年因青春期巨乳症而至台大醫院接受縮乳手術且乳房尺寸大於等於E罩杯的病患為巨乳組，對照組則選取同一時期因乳癌於臺大醫院接受乳房切除手術且乳房尺寸小於等於C罩杯的病患，選取其未受乳癌侵犯的乳房組織。排除的因素包括年紀大於29歲或小於18歲、過去曾有懷孕的記錄、月經不規則、BMI大於27、術前三個月內曾有荷爾蒙的使用、或不正常的術前荷爾蒙檢查。切下的乳房組織以福馬林固定製作成為石臘切片，並以蘇木紫及伊紅染色，和免疫組織化學染色，抗體包括Ki-67、Akt-1/2/3、phospho-Akt-1/2/3、及STAT5a。蘇木紫及伊紅切片用來量測乳腺上皮細胞的尺寸，Ki-67免疫組織化學染色切片計算500個乳腺上皮細胞中陽性反應細胞核的百分比，而其他的免疫組織化學染色以Image-pro Plus 5.0影像分析軟體進行陽性反應面積百分比的計算。兩組的比較則使用Wilcoxan rank sum test，以Stata 8.0統計軟體來進行。 1.3 研究結果 三年內共蒐集到八位巨乳症病患及五位對照組，兩組以Wilcoxan rank sum test比較，乳腺上皮細胞的尺寸及Akt-1/2/3免疫組織化學染色方面在統計學上的沒有顯著的差異，而乳腺上皮細胞之Ki-67、phospho-Akt-1/2/3、及STAT5a免疫組織化學染色方面，則均為青春期巨乳症患者組較高，統計學上均有顯著性的差異。 1.4 結論 本研究發現青春期巨乳症的乳房組織與乳癌病患未受癌症感染之乳房組織，在乳腺上皮細胞之尺寸方面並沒有差異，而在乳腺上皮細胞之增生方面，以Ki-67蛋白表現細胞之比例來看，青春期巨乳症患者的乳腺上皮細胞增生較多；在訊息傳遞路徑方面，青春期巨乳症患者的乳腺上皮細胞表現較多量的活化型phospho-Akt-1/2/3及總量之STAT5a。本研究顯示乳腺上皮細胞增生及抑制乳腺上皮細胞計畫性凋亡之分子機制與乳房增大有關。Introduction The ideal breast size of adult women is 250 to 300 cm3. Patients with macromastia not only suffer from physical discomforts but also psychosocial adjustment disorders and seek for surgical intervention. Macromastia is divided into two groups, juvenile and gravid hypertrophy, according to the onset of excessive breast development. The cause of macromastia is not well understood and may be related to the mistakes in gene expression during puberty or pregnancy. The literatures about macromastia are mostly about operation techniques and physical and/or psychological evaluations. There are a lot of researches focusing on the molecular mechanism of breast cancer and lactation, but seldom on macromastia. The size of an organ is determined by the size of the cells and the number of the cells, which depends on cell division and cell death. PI3K/Akt and Jak2/STAT5 signal transduction pathways has been though to be related to the growth of mammary epithelial cell. This study intended to investigate the difference between breast tissues from juvenile macromastia patients and non-tumor invaded breast tissue taken from breast cancer patients, by analyzing mammary epithelial cell size and proliferation marker, Ki-67. Akt-1/2/3, phospho-Akt-1/2/3, and STAT5a levels in the mammary epithelial cells were also evaluated by immunohistochemistry stains. Materials and Methods Patients who received reduction mammaplasty for juvenile macromastia in NTUH from January 2003 to December 2005 are enrolled as macromastia group. The control group includes the patients who received breast operation for breast cancer in NTUH in the same time period, with breast sizes not larger than C cup. The exclusion criteria are age over 29 or less than 18, positive pregnancy history, irregular menstrual cycle, body mass index over 27, hormone exposure within 3 months, and abnormal hormone level. Sections (5 μm) from formalin-fixed, paraffin-embedded blocks were cut for each case, followed by hematoxylin & eosin stain and immunohistochemistry stains (antibodies include Ki-67, Akt-1/2/3, phospho-Akt-1/2/3, and STAT5a). The H&E sections were used for measurement of mammary epithelial cell size. Five-hundred nucleus of mammary epithelial cell were counted on the Ki-67 immunohistochemistry stain sections and the positive rate was used to evaluate cell proliferation. The other immunohistochemistry stain section were photographed and evaluated with computer program Image-pro Plus 5.0 to calculate the positive area of the mammary epithelial cells. Nonparametric Wilcoxan rank sum test was used for comparison of data among the two groups. Results There were 8 patients in the macromastia group and 5 patients in the control group. The result didn’t show significant difference between the two groups. There is no difference between the two groups in mammary epithelial cell size measurement and Akt-1/2/3 immunohistochemistry stain. The levels of Ki-67, phospho-Akt-1/2/3, and STAT5a of juvenile macromastic patients are higher than those of the patients of the control group, and the results are statistically significant. Conclusions The study showed no difference between the juvenile macromastic breast and non-tumor part of cancer breast in mammary epithelial cells. But the proliferation marker, Ki-67, is higher in the macromastia group, which means that the proliferation of the mammary epithelial cells of the macromastia breast is higher. The phospho-Akt-1/2/3 and STAT5a levels were higher in the macromastia group, which implies that the cause of juvenile macromastia may be related to the activity of PI3K/ Akt or Jak2/STAT5 pathways.一、中文摘要 1.1 研究背景與目的…………………………1 1.2 研究材料與方法…………………………1 1.3 研究結果…………………………………1 1.4 結論………………………………………2 二、緒論 2.1乳房的結構………………………………………3 2.2 何謂巨乳症……………………………………4 2.3台灣地區女性的胸罩尺寸………………………6 2.4 乳房的發育……………………………………6 2.5 影響乳房發育及生理功能的內分泌激素及基因………8 2.6 研究目的…………………………………………………10 三、研究方法與材料 3.1 樣本的蒐集……………………………………11 3.2 檢體的處理……………………………………11 3.3 蘇木紫及伊紅染色步驟………………………11 3.4 免疫組織化學染色步驟………………………11 3.5 使用抗體之廠牌………………………………12 3.6 乳房上皮細胞尺寸的測量……………………12 3.7 Ki-67免疫組織化學染色的判讀及計算……13 3.8 免疫組織化學染色的判讀及計算……………13 3.9 統計分析……………………………………13 四、結果 4.1 病患基本資料分析…………………………………14 4.2乳腺上皮細胞尺寸分析……………………………14 4.3 免疫組織化學染色分析……………………………14 五、討論 5.1 巨乳症患者乳房組織中雌性激素受體的表現量……15 5.2 決定器官尺寸的因素……………………………15 5.3 巨乳症對於Ki-67蛋白在乳腺上皮細胞表現的影響……15 5.4 巨乳症對PI3K/Akt訊息傳遞路徑在乳腺上皮細胞表現的影響……16 5.5 巨乳症對於Jak2/STAT5訊息傳遞路徑在乳腺上皮細胞表現的影響…16 5.6 本實驗的限制………………………………………17 六、展望 6.1 本實驗的主要成果及未來方向…………19 6.2 控制乳房尺寸的因子………………………………19 七、論文英文簡述………………………………………20 八、參考文獻……………………………………………22 九、圖表……….…………………………………………2