臺灣博碩士論文加值系統

β-defensins (BD) 家族為小分子 (3~4 kD) 之陽離子胜肽，具有廣範圍之抗微生物作用。β-defensins主要來自於上皮細胞，參與先天與後天免疫反應。子宮內膜屬於先天粘膜免疫之一環。目前已有研究指出cBD1、cBD102及cBD103具有抗微生物的功能。於人類子宮的研究中，β-defensins會因為動情週期而有不同的表現高峰。然而，目前犬子宮內膜於正常動情週期間以及發生子宮蓄膿 (pyometra) 時，其β-defensins表現仍未明瞭。本研究目的是想了解β-defensins在犬子宮內膜中之表現與作用情形。本研究樣本來自經手術取下之犬子宮卵巢及胎盤組織，包括正常動情週期以及子宮蓄膿之子宮，經半定量RT-PCR之結果顯示cBD1 mRNA於子宮懷孕期、hydrometra以及mucometra時，表現有顯著增加之情形。當子宮受到感染時，可見到cBD1表現向上調節，與子宮內膜厚度、腺體量多寡以及分泌物多寡有關。另一方面，本研究結果顯示犬子宮中β-defensins表現並非受到TLR2及TLR4所調控。總之，β-defensins可能於犬子宮免疫反應中扮演著重要的保護角色，且可能與腺體量以及分泌物多寡有關。

β-defensins (BD) are a family of small cationic proteins that have been shown to be associated in defense against pathogenic organisms. The endometrial epithelium is the first defensive line against pathogenic microbes. To date, the expression of β- defensins in epithelial surfaces have been reported in respiratory tract, gastrointestinal tract, skin, and reproductive tract. However, little is known about β-defensins ex- pression in bitches during estrus cycle and with pyometra. The aim of this study is to extend our knowledge on expression and action of β-defensins in the canine endometrium. We studied canine β-defensins (cBDs) expression between healthy uteri and pyometra samples obtained from bitches presented for ovariohysterectomy. The results of this study demonstrated that the expression of cBD1 were upregulated during pregnancy in uteri under semi-quantitative RT-PCR analysis. And cBD1 expression was markedly increased in hydrometra, mucometra, and pyometra compared with healthy uteri group. The expression of cBD1 upregulated may correlate with abundant glands, thickness in the endometrium. On the other hand, the β-defensins were not regulated by TLR2 and TLR4. In conclusion, the CBDs expression may play an essential role in the innate immune defense of the canine endometrium.

目 次
中文摘要 i
英文摘要 ii
目 次 iii
圖 次 v
表 次 vii
第一章 前言 1
第二章 文獻探討 3
第一節 犬子宮動情週期 (estrus cycle) 與子宮內膜形態學 3
第二節 子宮內膜囊腫性增生與子宮蓄膿 6
第三節 雌性動物生殖道之先天免疫 10
第四節 Defensins 11
第五節 β-defensins 16
第三章 材料與方法 20
第一節 組織樣本採集與保存 20
第二節 組織病理切片製作 20
第三節 組織學檢查 21
第四節 細菌分離與純化 21
第五節 細菌培養核酸萃取與鑑定 22
1. 革蘭氏陽性菌核酸萃取 22
2. 革蘭氏陰性菌核酸萃取 23
3. 細菌16S ribosome DNA (rDNA) 之PCR鑑定 23
第六節 樣本分組 24
第七節 β-defensins及TLRs之mRNA表現量檢測 25
1. 組織Total RNA萃取 25
2. cDNA (complement DNA) 製備 26
3. 測定之標的基因專一性引子合成 26
4. 反轉錄聚合酶鏈鎖反應 27
5. 洋菜膠體 (Agarose gel) 電泳分析 28
6. 反轉錄聚合酶鏈鎖反應之特異性產物純化與定序 29
7. 標的基因mRNA表現量之半定量測定 29
第四章 結果 31
第一節 犬正常卵巢、子宮角與胎盤之組織構造 31
第二節 犬異常子宮之組織學形態 32
第三節 細菌分離鑑定結果 33
第四節 β-defensins及TLRs之mRNA表現量之測定 33
第五節 β-defensins及TLRs之mRNA表現量之統計分析 36
第五章 討論 55
參考文獻 61

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