林仁壽2006-07-262018-06-292006-07-262018-06-292002http://ntur.lib.ntu.edu.tw//handle/246246/16446本實驗之目的旨在建立一個穩定的小鼠睪丸萊吉氏細胞(Leydig cell)分離及培養系統，並探討刺蒺藜與桂枝對萊吉氏細胞的睪固酮分泌機制。 利用取自性成熟公ICR 小鼠的睪丸，經由酵素(collagenase)與機械性解離後取得的精間細胞，使用Percoll 溶液產生的不連續相濃度梯度(discontinuous gradients)離心法分離細胞，將取得的萊吉氏細胞培養於M199 中，同時添加不同劑量的中草藥粗萃取液，於培養後收集不同時程的培養液，並以酵素免疫分析法測定培養液中睪固酮(testosterone)含量。實驗結果如下： 1. 使用10、20、30、40 及50%的不連續相Percoll 濃度梯度分離細胞，將回收的每層細胞利用排卵素(LH)刺激與3β-HSD 染色法，結果證實萊 吉氏細胞主要是位於50% Percoll 層中，而回收的萊吉氏細胞對不同的外源刺激物(LH、cAMP 等)具有良好的睪固酮分泌反應性。由此結果顯示小鼠萊吉氏細胞分離及培養系統已建立。 2. 利用上述的培養方式，發現刺蒺藜萃取物(H13)在培養24 小時後具有刺激小鼠萊吉氏細胞分泌睪固酮的效果，同時具有劑量反應。經由不同訊息傳遞之相關試劑處理的結果，顯示H13 透過胞內PKA 與PKC 之激活而傳遞其刺激類固醇生合成的路徑，並同時涉及到胞內蛋白質的轉錄與轉譯。 3. 利用上述的培養方式，發現桂枝萃取物在培養4 小時後具有抑制基礎與oLH 的刺激睪固酮的分泌量，經由添加訊息傳遞的活化物質包括 CTX、FSK、cAMP、PMA 與A23187 與類固醇生合成的中間受質包括:膽固醇、22R-OHC、P5、P4、17a-OH-P4與andostenedione 均無抑制效果表示桂枝的抑制效果是在類固醇生合成以外的路徑。The purpose of this study was firstly to isolate purified mice Leydig cells and use enzyme immunoassay (EIA) for testosterone production. Selected Chinese medicinal herbs extracted by hot water extraction procedure were co-cultured with the isolated cells and their effect on testosterone secretion vis a vis basal and stimulants were investigated. Testis were obtained from mature ICR mouse of 6-8 weeks age, decapsulated and finely minced before dissociating with collagenase. The digested tissue is then sieved and Percoll discontinuous gradients method applied to separate the Leydig cells. The harvested Leydig cells were than incubated in M199 in a 48 well culture plate, different doses of Chinese medicinal herbs extracts, various stimulants and signal transductor inhibitors were added to the Leydig cells culture system. The medium was than collected at different time periods and assayed using EIA technique. The results and findings of our present work can be summarized as follows: 1. Investigating for 10, 20, 30, 40 and 50 % Percoll discontinuous gradients and using LH stimulation and 3-HSD staining it was revealed that 50% Percoll gradients has better results. The Leydig cells when stimulated (LH, cAMP) had good testosterone production both at dose and time response indicating the successful establishment of mouse Leydig cells in vitro cell culture system. 2. Tribulus terrestris L. hot water extracts (H13) stimulated testosterone secretion of mouse Leydig cells, in 100 µg/ml dose almost double to that of basal. It also showed time course (24 and 48 hour) and dose response (1, 10 and 100µg/ml). It also revealed that the H13 mediated stimulation via PKA and PKC activation leading to protein transcription and translation. 3. Cinnamomum cassia Presl extracted by hot water extraction (10 and 100 µg/ml) showed decrease in testosterone production over the basal and LH stimulated secretion of mouse Leydig cells in 4 and 24 hour time course. From our data by addition of signal transduction agents, it was suggested that Cinnamomum cassia Presl decrease LH stimulated testosterone secretion function without acting on the steroidogensis pathway.application/pdf34454 bytesapplication/pdfzh-TW國立臺灣大學動物科學技術學系暨研究所萊吉氏細胞睪固酮中藥刺蒺藜桂枝Leydig cellstestosteroneChinese herbsTribulus terrestris LCinnamomum cassia Preslreporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/16446/1/902313B002301.pdf