臺灣博碩士論文加值系統

鈣離子調控許多訊息傳導路徑，像是釋放cytochrome C 、形成緊密的結合以及增加細胞移動的能力，扮演一個二級訊息傳導物質。胚胎和子宮內膜有許多複雜的作用導致胚胎的附著，發生在胚胎著床的過程中。鈣離子主要在調節滋養層細胞和子宮內膜上皮細胞的相互作用在胚胎著床位置，這個機制目前還不是相當的清楚。本論文主要為了釐清鈣離子在滋養層細胞和內膜上皮細胞之間的交互作用中所調控的功能。將RL95-2細胞株種在96孔的培養皿中形成滿的單層細胞扮演子宮內膜的上皮細胞，當作實驗的基質，BeWo形成滋養層細胞的球狀體再植入與內膜上皮細胞共同存在的96孔培養皿中，共同培養24小時。為了阻斷外界鈣離子的內流，利用EDTA、EGTA、verapamil和nifedipine這些藥劑，還有利用BAPTA-AM來阻斷內部的鈣離子變化，這些處理都可明顯觀察到滋養層細胞在內膜上皮細胞擴張的區域大小有受到抑制的效果，並且有濃度梯度抑制的反應。在子宮中會分泌的降血鈣素，是一個32個胺基酸組合而成的蛋白賀爾蒙，會調節體內的鈣離子恆定，在大鼠懷孕的第四天，分泌的量會達到最高而造成胚胎著床的成功。在我們利用上述共同培養兩種細胞的模式中，利用EDTA、EGTA、verapamil和nifedipine這些藥劑來阻斷外界鈣離子的內流，利用BAPTA-AM來阻斷內部的鈣離子減少，上述的這些處理，都可明顯觀察到滋養層細胞在內膜上皮細胞上擴張的區域會降低。另一方面我們利用降血鈣素，在共同培養的模式中會增加滋養層細胞在內膜上皮細胞上擴張的區域。在我們實驗中已經證實降血鈣素會增加RL95-2和BeWo細胞的內部鈣離子濃度，利用鈣離子影像系統的方式會呈現一個濃度梯度反應的現象。降血鈣素所對RL95-2和BeWo細胞增加的內鈣離子濃度，可以被EDTA，EGTA，BAPTA-AM以及完全無鈣離子的環境中來抑制。可以利用verapamil和nifedipine這兩種專一阻斷L形式的鈣離子通道的藥劑，來抑制降血鈣素所造成在RL95-2細胞中增加的內鈣離子濃度，但是在BeWo細胞的反應並不相同。綜合上述的發現，我們推測滋養層細胞在內膜上皮細胞上擴張的區域，是會被細胞內部鈣離子濃度訊息傳遞來調控的。在內部鈣離子濃度的增加是會被降血鈣素所調節的，主要在RL95-2細胞是由L形式的鈣離子通道來媒介，但是在BeWo細胞中並沒有這些現象。鈣離子扮演一個重要的角色在滋養層細胞和子宮內膜上皮細胞的相互作用中，在調節下游的訊息傳導中包含protein kinase C (PKC)的活化。在前人的研究，細胞骨骼的重組和移動能力的增加，都會被PKC的活化所調控。在我們以staurosporine和calphostin C這兩種PKC的抑制劑在共同培養的模式中，會降低滋養層細胞在內膜上皮細胞上擴張的區域。在前處理staurosporine和calphostin C這兩種PKC的抑制劑之後，再加入降血鈣素的共同培養模式中，觀察到降血鈣素所造成的增加滋養層細胞在內膜上皮細胞擴張的區域，會被PKC的抑制劑來降低。因此我們推測降血鈣素所造成的鈣離子內流會造成PKC的活化。

The Ca2+ ion as a secondary messenger, mediated many signaling pathway, including cytochrome c release, tight junction formation and cell migration ability. During implantation, complex embryo-endometrium interaction result in blastocyst adhesion. At the implantation site, the Ca2+ ion play an important role to mediate the interaction between trophoblast and endometrial epithelial cells, the function is still unclear. To role out the function of Ca2+ mediated the trophoblast and endometrial epithelial cells at the implantation stage. RL95-2 cells grow to confluent monolayers of a EEC line, using as the matrices, trophoblast spheroid were dispensed into 96-wells plates containing the monolayers and incubated for 24 hours. Prevention of Ca2+ influx by EDTA, EGTA, verapamil or nifedipine significantly inhibited the trophoblast spheroids outgrowth, whereas prevention of Ca2+ mobilization with BAPTA-AM also had the inhibition of trophoblast spheroid outgrowth area in a dose-dependent manner. The uterine secretion of calcitonin, a 32-amino acid peptide hormone involved in calcium homeostasis, peaks on day 4 of pregnancy in rat to mediated implantation successful. In our co-culture system, removed Ca2+ ions from the medium by EDTA, EGTA, verapamil or nifedipine respectily, prevention of Ca2+ mobilization by BAPTA-AM was due to make the decrease of [Ca2+]i, all of above experiments showed the reduced the area of trophoblast spheroid outgrowth. On the other hand, we treated calcitonin which well to know increase [Ca2+]i, shown increase of the area. In BeWo and RL95-2 cells, increase [Ca2+]i was induced by calcitonin using calcium image system in a dose-dependent manner. The increase [Ca2+]i was induced by calcitonin could be depressed by pretreated EDTA, EGTA and BAPTA-AM, or at the calcium free environment. The calcitonin induced calcium influx could be depressed in RL95-2 cells by verapamil and nifedipine, the specific L-type calcium channel blocker, partly effect in BeWo cells. Concluding above data, we suggested that the area of trophoblast outgrowth was mediated by intracellular calcium signaling. The calcium influx was induced by calcitonin, mainly across by L-type calcium channel in RL95-2 cells, but in BeWo cells were not. Interaction between trophoblast and EEC, Ca2+ play an important role to stimulate the downstream signaling pathway, involved PKC activation. In previous studies, the cytoskeleton rearrangement and migration ability was induced by PKC activation. We treated PKC inhibitor, staurosporine and verapamil, shown the decrease of the area of trophoblast spheroid outgrowth. We suggested that PKC pathway involved in trophoblast outgrowth on EEC monolayers. Pretreated with staurosporine and calphostin C could depress the expansion area of trophoblast spheroid outgrowth. We speculated that calcitonin elicited calcium influx to activate PKC pathway.

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