臺灣博碩士論文加值系統

黃連素(berberine)又稱小檗鹼，為一生物鹼，可從黃連、黃柏、三顆針等植物中提取，是傳統上應用很久的中藥。黃連素已被證實具有抗腹瀉、抗菌、抗心律不整、抗血管平滑肌增生與降低膽固醇等療效，在心血管系統與神經系統疾病方面有廣泛的應用；目前已有研究報導指出黃連素可誘發癌細胞凋亡而毒殺癌細胞，但黃連素對胚胎發育的影響至目前為止尚無相關的文獻報導。本研究利用小鼠胚胎與黃連素共同培養，探討黃連素對小鼠胚胎發育與細胞增生的影響。將小鼠囊胚與黃連素 5μM 或 10μM 共同培養 24小 時後，發現隨著共培養的黃連素劑量上升而細胞凋亡亦隨之增加，並造成囊胚發育時細胞數目顯著減少。利用區別染色法和 TUNEL 凋亡分析檢測囊胚細胞的內細胞團細胞和滋養層細胞的變化，結果顯示滋養層細胞比起內細胞團受到較嚴重的傷害；結果顯示黃連素會對小鼠胚胎發育造成傷害且誘導胚胎細胞凋亡。黃連素造成小鼠囊胚細胞凋亡可能是藉由降低抗凋亡因子 Bcl - 2 蛋白表達和增加促凋亡因子 Bax 蛋白的表達，造成凋亡指標 Bax/Bcl-2 比值的增加而誘發胚胎細胞進行凋亡，且觀察到細胞內氧化壓力上升、粒線體內外膜間電位喪失的情形，促使細胞經由粒線體依賴的凋亡路徑而產生凋亡。

Berberine is an alkaloid isolated from Coptis Chinensis, Phellodendron and other plant extracts. Berberine is a traditional Chinese medicine in China which has been applied for a long time. Berberine has been evaluated as anti-diarrhea, anti-bacterial, anti-arrhythmic, anti-vascular smooth muscle proliferation and cholesterol-lowering. Berberine also performs a wide range of applications in the cardiovascular system and nervous system diseases. Several studies have demonstrated that berberine is able to induce apoptosis process in cancer cells. However, a study regarding the embryonic toxicity of berberine has not yet been done. In this study, mouse embryos were used to study the effects of berberine on embryonic development and proliferation. The study results showed that blastocysts treated with 5 or 10 μM of berberine that resulted a significant dose-dependent increase in apoptosis and inhibited cell proliferation. To evaluate the alteration of inner cell mass (ICM) and trophoblast cell (TE) of mouse blastocysts, we used differential staining and TUNEL assay. Statistics showed, TE subject to more damage than ICM. Furthermore, the immunofluorescence staining, revealed that berberine induced apoptosis in mouse blastocysts by reducing expression of anti-apoptotic Bcl-2 protein and increasing expression of the pro-apoptotic Bax protein, which increases the ratio of Bax/Bcl-2. Conclusion, berberine induced embryo cell apoptosis and caused hazardous effects on embryonic development.

目錄
摘要.....................................................Ⅰ
Abstract..............................................,,,Ⅱ
目錄.....................................................Ⅲ
圖目錄.................................................Ⅴ
表目錄...................................................Ⅶ

第一章 前言...............................................1
第一節 研究動機...........................................1
第二節 研究背景...........................................3
2-1 黃連素................................................3
2-2 小鼠胚胎發育過程......................................5
2-3 細胞凋亡..............................................9
第二章 文獻回顧..........................................16
第三章 實驗設計與材料方法................................18
第一節 實驗設計..........................................18
第二節 實驗方法..........................................19
第三節 實驗材料..........................................26
第四章 實驗結果..........................................32
第五章 討論..............................................55
第一節 黃連素抑制小鼠囊胚細胞增生........................55
第二節 黃連素藉由改變Bcl-2家族蛋白誘使小鼠囊胚細胞凋亡..56
第三節 黃連素促使小鼠囊胚細胞凋亡藉由提高氧化壓力與粒線體介
導路徑............................................57
第四節 黃連素對於小鼠胚胎著床前與著床後體外發育的影響....58
第五節 黃連素影響滋養層細胞與內細胞團細胞發育對於胚胎發育的
影響..............................................58
第六章 結論..............................................60
第七章 參考文獻..........................................61


圖目錄
圖一、黃連圖...............................................4
圖二、黃連素分子結構式.....................................4
圖三、小鼠胚胎卵裂.........................................5
圖四、小鼠囊胚.............................................8
圖五、小鼠胚胎著床前發育過程...............................8
圖六、凋亡訊息傳遞途徑....................................12
圖七、外生性細胞凋亡路徑..................................14
圖八、內生性細胞凋亡路徑..................................15
圖九、成功交配後雌鼠陰道口的plug..........................20
圖十、黃連素對於小鼠囊胚外觀 (morphology) 的影響..........31
圖十一、黃連素對小鼠囊胚細胞增生的影響....................34
圖十二、黃連素誘導小鼠囊胚細胞凋亡........................37
圖十三、黃連素對於小鼠囊胚細胞內活性氧族群變化之影響......39
圖十四、黃連素對於小鼠囊胚細胞中粒線體膜電位之影響........40
圖十五、黃連素對於小鼠胚胎Bax 蛋白表現的影響.............42
圖十六、黃連素對於小鼠胚胎Bcl-2 蛋白表現的影響............45
圖十七、黃連素對於小鼠囊胚體外發育的影響..................49
圖十八、黃連素對於 2-細胞期胚胎體外發育的影響.............52
圖十九、黃連素對於小鼠胚胎體內發育的影響..................54

表目錄
表一、細胞凋亡與壞死的比較........................10
表二、實驗設計...........................................18

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