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研究生:連韋雄
論文名稱:應用小鼠萊吉氏細胞體外培養系統篩選具激性腺功效之中藥
論文名稱(外文):Screening Chinese medicinal herbs on gonadotropic activity by in vitro culture system of mouse laydig cells
指導教授:林仁壽林仁壽引用關係吳兩新
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:畜產學研究所
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:89
中文關鍵詞:萊吉氏細胞中藥小鼠激性腺功效
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本試驗之目的,旨在建立小鼠萊吉氏細胞(Leydig cell)體外(in vitro)培養系統及睪固酮酵素免疫分析法,進而利用此二項技術,篩選具激性腺素作用之中草藥。
本研究所建立的睪固酮酵素免疫分析法主要係利用雙抗體(double antibodies)之原理(10Ab:rabbit anti-testosterone antiserum;20Ab goat anti-rabbit IgG)。其靈敏度可達15.6 pg/ml,分析內及分析間的變異係數分別為11.4%與12.2%。
以6-8週的成熟小鼠(ICR)睪丸組織為材料,以酵素解離法(collagenase及DNase I)分離取得萊吉氏細胞後,將細胞數調整為105/ml M199,以48孔的細胞培養盤於370C、5% CO2及95% 空氣條件下進行培養。經收集4、24、48及96小時的細胞培養液後,以酵素分疫析法分析其睪固酮的含量。結果發現,小鼠萊吉氏細胞分泌睪固酮具有時程(time course)反應,於培養24小時後即有顯著增加(P<0.05)。另外,經額外添加不同劑量的oLH(0.1、1、5及10 ng/ml)及cAMP(0.1、1及10 mM),亦可使細胞分泌睪固酮的能力明顯升高(P<0.05),且具有劑量反應(dose response)。由以上結果顯示,萊吉氏細胞體外培養系統已建立成功。
將中草藥粗萃液以1、10及100 mg/ml的劑量加入萊吉氏細胞體外培養液中,並測定4、24及48小時細胞培養液之睪固酮含量。結果發現,屬於補氣類的絞股藍(Gynostemma pentaphyllum),當使用10及100 mg/ml等較高劑量時,於48小時具有明顯的刺激睪固酮分泌之作用(P<0.01);而屬於補陽類的淫羊藿(Epimedium macranthum),其所純化的淫羊藿(icariin),以10 mg/ml之劑量,於培養24小時後,即可見到明顯(P<0.01)的刺激作用。而在其他種中草藥植物的粗萃液中,如百合、仙茅、桑椹子、括樓實、人參花及人參單體(Rb)等,也同樣具有刺激萊吉氏細胞分泌睪固酮之能力。本論文結合萊吉氏體外培養系統與睪固酮酵素免疫分析法,已能有效率地篩選出具激性腺活性之中草藥。
The purpose of this study was firstly to established the mouse Leydig cells in vitro culture system and enzyme immunoassay (EIA) of testosterone, and than secondly to screen Chinese medicinal herbs with can stimulate testosterone secretion from Leydig cells.
The testosterone EIA was use (10Ab: rabbit anti-testosterone antiserum;20Ab: goat anti-rabbit IgG) double antibody system, and the sensitivity of the EIA was 15.6 pg/ml. The coefficients of variation of the inter- and intra-assay were 11.4 and 12.2% respectively.
Testis were obtained from mature ICR mouse with cycle between 6-8 weeks and dissociated with collagenase and DNaseⅠ. Aliquots of approximately 105 viable cells /ml M199, were incubated in vitro at the condition of 37℃、5% CO2 and 95% O2 in a 48 wells culture plate. After 4、24、48 and 96 hours incubation, the culture media were collected for testosterone assay. In this culture system, the testosterone basal secretion showed time course. Furthermore, ovine luteinizing hormone (oLH, 0.1、1、5 and 10 ng/ml) and cAMP (0.1、1 and 10 mM), stimulated Leydig cells screened testosterone in a dose response pattern. The results indicated that the mouse Leydig cells in vitro culture system was success fully established.
The Chinese medicinal herbs medicine extracts with 1、10 and 100 ml/ml were added to Leydig cells culture medium. After 4、24 and 48 hours, the culture media were collected for testosterone assay. The results showed that the testosterone secretion from the Leydig cells was significantly highest than the basal by Gynostemma pentaphyllum extract, with 10 and 100 mg/ml after 48 hours incubation and the icariin with 10 mg/ml for 24 hours incubation. In other Chinese herbals medicine: Lilium brownii、Cucrculigo orchioides、Morus、Panax ginseng and Trichosnthes kirilowii as well to have stimulated testosterone secretion of mouse in Leydig cells. This study combining with mouse Leydig cells in vitro culture system and testosterone EIA, already has established on efficiency screen for the gonadotropic activity of Chinese medicinal herbs.
目 錄
頁 次
摘 要 ……………………………………………….. 1
緒 言 ………………………………………………. 3
文獻檢討 ………………………………………………. 5
材料與方法 ………………………………………………. 32
結 果 ………………………………………………. 43
討 論 ………………………………………………. 63
結 論 ………………………………………………. 70
參考文獻 ………………………………………………. 71
英文摘要 ………………………………………………. 81
附 錄 ………………………………………………. 83
簡 歷 ………………………………………………. 89
圖 次
頁次
圖1. 性類固醇內泌素合成途徑…………………………... 7
圖2. 小鼠萊吉氏細胞於培養4-96小時後之睪固酮基礎分泌量………………………………………………... 44
圖3. oLH處理對於小鼠萊吉氏細胞體外分泌睪固酮之影響…………………………………………………... 45
圖4. 8-bromo-cAMP處理對於小鼠萊吉氏細胞分泌睪固酮能力之影響………………………………………... 46
圖5. 酵素-睪固酮複合體之蛋白質濃度………….……… 49
圖6. 酵素-睪固酮複合體之免疫及酵素活性…….……… 50
圖7. 酵素免疫分析法之雙抗體原理……………………... 51
圖8. 睪固酮酵素免疫分析法之標準曲線………………... 52
圖9. 睪固酮酵素免疫分析法之靈敏度測試……………... 53
圖10. 睪固酮標準液及細胞培養液之平行試驗…………... 55
圖11. 以EIA及RIA測定睪固酮標準液的相關…………… 56
圖12. 以不同劑量之絞股藍 (butanol層)萃取物處理,對於萊吉氏細胞分泌睪固酮能力之影響……………... 61
圖13. 以不同劑量之淫羊藿 (icariin)對於萊吉氏細胞分泌睪固酮能力之影響…………….……………….. 62
表 次
頁次
表1. 由睪丸產生且作用於萊吉氏細胞之主要影響因子... 12
表2. 補虛壯陽中草藥分類表………………………….….. 29
表3. 抗睪固酮抗血清與類固醇之交叉反應性…………... 37
表4. 睪固酮酵素免疫分析法之分析內及分析間變異.….. 54
表5. 以EIA測定M199中睪固酮濃度之回收率………… 54
表6. 應用小鼠萊吉氏細胞體外培養系統篩選具激睪固酮分泌之中草藥……………………………………... 59
表7. 具激睪固酮分泌活性之中草藥……….…………….. 60
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