- 學位論文
蘆薈大黃素誘發之鈣離子變化對小鼠囊胚發育之影響與調控機制
Impact and regulatory mechanisms of aloe-emodin-induce calcium change on embryonic development in mouse blastocysts
摘要
蘆薈大黃素為富含於蘆薈葉與大黃的根莖部之中的蔥醌類化合物，廣泛運用於中醫作為瀉藥與解毒劑使用。有些研究指出蘆薈大黃素具有抗氧化、抗發炎、抗癌與抗菌活性。在過去的研究中已經證實，蘆薈大黃素會誘導小鼠囊胚內鈣離子上升進而引發凋亡。但對於蘆薈大黃素所引發之鈣離子傳遞路徑尚不清楚。本研究的主要目的為釐清經由蘆薈大黃素處理後的胚胎，其細胞內的鈣離子是否會透過細胞膜上的鈣離子通道傳遞，進而影響與細胞凋亡相關因子的表現。為了進一步探討蘆薈大黃素誘發的鈣離子變化對小鼠囊胚發育的詳細機制，處理過蘆薈大黃素的胚胎將同時處理鈣庫調控通道抑制劑SKF96365並使用fura-2螢光染劑檢測通過鈣庫調控通道引起的鈣離子含量改變。Fura-2螢光染劑可明顯地指出對照組與處理組鈣離子信號的差異。本研究使用TUNEL分析法檢測對照組與處理組的存活率，發現抑制胚胎的鈣庫調控通道將減少胚胎的細胞凋亡。使用凋亡壞死實驗發現抑制鈣庫調控通道可減緩細胞死亡。細胞內活性氧族群(ROS)與一氧化氮(NO)含量分別以2’,7’–dichlorofluorescein diacetate (DCF-DA) 與 3-Amino,4-aminomethyl-2',7'-difluorescein diacetate (DAF-FAM DA)檢測。同時處理SKF96365與蘆薈大黃素的組別其活性氧族群與一氧化氮含量皆比處理蘆薈大黃素的組別低。此外，與蘆薈大黃素共培養12小時不會影響小鼠囊胚的發育能力，但是以SKF96365抑制鈣庫調控通道將會傷害小鼠囊胚發育。綜合以上結果，推測蘆薈大黃素所誘導之細胞質內鈣離子增加，其部分鈣離子來源是透過鈣庫調控通道將膜外鈣離子傳遞進入細胞質之中。
並列摘要
Aloe-emodin (AE), the most abundant anthraquinone compound found in aloe leaves or rhubarb rhizome, has been widely used in traditional Chinese medicines as laxative and detoxifying agent. Several researches showed that AE has anti-oxidant, anti-inflammation, anti- cancer and anti-bacteria activities. Previous study showed that after treatment with aloe-emodin, the calcium levels were elevated in mouse blastocysts resulting in apoptosis. The calcium transport pathway by aloe- emodin-induced is not clear yet. The aim of our study was to clarify the embryos treated by AE, whether its intracellular calcium is transported through the calcium channels in the cell membrane, thereby affecting the processes of apoptosis. To further understand the detailed mechanism of aloe-emodin-induced calcium change on embryonic development in mouse blastocysts, the AE-treated embryos were also incubated with SKF96365, a store-operated channel inhibitor, and then stained with fura-2 to reveal the possible participation of the store-operated channel in the changes of calcium levels. The fura-2 stained calcium signaling was significantly different between the treated and the control embryos. We used the TUNEL assay to determine the survival rate of embryos in untreated and treated groups and found that inhibited store-operated channel of embryos would reduce the apoptosis. Using Annexin V/PI assay, we found that inhibited store-operated channel of embryos would reduce cell death. Intracellular reactive oxygen species (ROS) production and nitric oxide (NO) generation were detected by 2’,7’ –dichlorofluorescein diacetate (DCF-DA) and 3-Amino,4-aminomethyl-2',7'- difluorescein diacetate (DAF-FAM DA), respectively. Both ROS and NO content of co-treated groups were less than the control group. In addition, the in vitro experiment showed that incubation of mouse blastocyst with AE for 12 hrs had no effect on mice blastocyst development, but SKF96365 inhibited the store-operated channel and caused blastocyst to fail from hatching. However, it is not clear the toxic relationship between the AE and calcium signaling and will further investigate the detail regulatory mechanisms in the future. In sum, our study results demonstrated that the store-operated channel were one of the major channel to cause calcium fluctuation in mouse blastocysts.
參考文獻
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