臺灣博碩士論文加值系統

已知高泌乳素血症是引發哺乳類動物性功能障礙或不孕症的原因之一，主因性腺睪酮分泌功能不佳所致。過去對其致病機轉的解釋一直圍繞在下視丘－腦下腺軸系引起的荷爾蒙分泌失調。但是這種單一的解釋並不完美，常見矛盾例証。對於泌乳素如何直接干擾睪酮之分泌，論述不多。本研究論文乃利用異體腦下腺移植於腎包膜下的方式建立高泌乳素血症的大鼠動物模式，觀察高泌乳素血症大鼠於活體時接受人絨毛膜性腺促素(hCG)刺激睪酮分泌異常的情形。分離離體之睪丸間質細胞(TIC)，觀察人絨毛膜性腺促素，cAMP生成相關酵素，或睪酮生合成所需各階段前驅物刺激後睪酮釋放的情形。更進一步，再從睪丸間質細胞中純化分離萊氏細胞進行與睪丸間質細胞培養類似的刺激，比較其睪酮釋放的變化。另一方面更將睪丸間質巨噬細胞(TIM)分離出，觀察其分泌出的腫瘤壞死因子a(TNF-a)在高泌乳素血症誘發睪酮分泌障礙中所扮演的角色。
結果顯示，由異體腦下腺前葉移植誘發之高泌乳素血症雄鼠，於活體時經人絨毛膜性腺促素刺激出現睪酮反應不足的現象。睪丸間質細胞經人絨毛膜性腺促素、泌乳素、8-Br-cAMP或睪酮生合成前驅物的刺激，睪酮的釋放，以高泌乳素血症組遠低於對照組者。然而，大鼠萊氏細胞培養接受類似刺激後，睪酮的釋放卻出現與間質細胞培養相反的結果，即高泌乳素血症組睪酮之分泌遠高於對照組者。暗示萊氏細胞本身睪酮生合成機制會因高泌乳素血症而強化。由於睪丸間質細胞與萊氏細胞培養間最大的差別在間質細胞中存在為數不少的巨噬細胞。巨噬細胞可以分泌以腫瘤壞死因子a為主的細胞激素以影響周遭細胞的功能。高泌乳素血症組的間質細胞培養液中可以測得較多的腫瘤壞死因子a，以腫瘤壞死因子a外加於萊氏細胞培養後，即出現隨劑量增加的睪酮分泌抑制效應。
以上的結果，得到以下的結論與推論：(1)高泌乳素血症誘發的睪酮分泌不足可以在動物模式上重現。(2)高泌乳素血症誘發的睪酮分泌不足可以在離體睪丸間質細胞模式再現。(3)高泌乳素血症動物其離體萊氏細胞睪酮生合成機制其實並未受到抑制，相反的，其表現是增強的。(4)睪丸內除了萊氏細胞外的某種間質細胞可能掌握了解開此機制秘密的鎖鑰。(5)導致高泌乳素血症誘發睪酮分泌不足的原因，必須探究睪丸間質巨噬細胞的角色。巨噬細胞的主要細胞激素（腫瘤壞死因子a）可以在培養液中測得，且在高泌乳素血症組有較高的分泌量，而巨噬細胞很可能就是扮演這個角色的細胞。
以上的結論及推論除了為泌乳素影響睪丸、睪酮分泌提供重要且有意義的資訊外，並且也開啟了睪丸間質巨噬細胞對生殖內分泌功能上研究的大門。

Hyperprolactinemia (hyperPRL) might induce sexual impotence or infertility in male mammals. The phenomenon is because of the appearance of hypogonadism. The mechanism of hyperPRL-induced hypogonadism has been explained by a dysfunction of hypothalamus-pituitary axis. However, this explanation is not satisfied in all circumstances. A direct effect of prolactin (PRL) to testicular steroidogenesis has been proposed. How PRL influence, in a direct way, the testicular testosterone (T) release is still elusive. The present study was to explore the detailed mechanisms regarding hyperPRL effects on the testicular T release.
The hyperPRL rat model was conducted by allografting anterior pituitary (AP) glands to the renal sub-capsular space. The in vivo study was designed to confirm a decreased T response after human chorionic gonadotropin (hCG) stimulation in the hyperPRL animals. The in vitro T responses after the stimulation of hCG, PRL, cyclic adenosine mono-phosphate (cAMP) related enzymes and steroidogenic precursors were observed in both testicular interstitial cells (TIC) and Leydig cells stimulations. The testicular interstitial macrophages (TIM) were also isolated to characterize the role of TIM-released tumor necrosis factor-a (TNF-a) in hyperPRL-induced hypogonadism. The concentrations of T, PRL and cAMP were measured by radioimmunoassay (RIA). The measurement of TNF-a was by enzyme-linked immuno-sorbent assay (ELISA). The data was analyzed by the analysis of variance (ANOVA) and Duncan''s multiple range tests.
The results indicated that the AP-allografting-induced hyperPRL decreased T response to the in vivo challenge of hCG. In vitro TIC cultures showed that the hyperPRL groups had a significantly lower T release after challenge of hCG, PRL, 8-Br-cAMP or steroidogenic precursors. While in the in vitro Leydig cells incubations, the hyperPRL exhibited an opposite results after similar challenge as in the TIC, a tremendously higher T release was observed in the hyperPRL group than in the control. These findings indicated that the effects of high PRL levels boosted the testosterone biosynthesis in Leydig cells. On account of the major difference between TIC and Leydig cell cultures is the presence of TIM. TIM is the major source of TNF-a, which regulates the functions of neighboring cells. TNF-a could be detected in the TIC medium, in particularly to higher levels in the medium of hyperPRL groups. Incubation of Leydig cells with TNF-a created a dose-dependent inhibition of T release.
These results suggest that (1) hyperPRL-induced hypogonadism has been demonstrated in the AP-grafted rats and the in vitro TIC culture; and (2) the T biosynthesis machinery of the Leydig cells in hyperPRL rats was not weakened, to the contrary, the expression of T biosynthesis related protein or enzyme was enhanced in the Leydig cells in the hyperPRL animals. This picture echoes the theory that PRL being a trophic hormone to testicular development. Certain TIC (e.g. TIM) other than Leydig cells must play a role in suppressing the T release by the Leydig cells.
This study not only provided useful information on how PRL affecting testicular T release, but also opened a new door to the research of TIM in hypogonadism.

名詞中英文對照………………………………………………………… 7
中文摘要………………………………………………………………… 8
英文摘要………………………………………………………………… 10
第一章 緒論……………………………………………………………… 12
一 概述 …………………………………………………………………… 13
二 泌乳素………………………………………………………………… 14
（一） 泌乳素簡介……………………………………………………… 14
（二） 泌乳素的分泌與調節…………………………………………… 14
（三） 泌乳素的功能…………………………………………………… 16
（四） 高泌乳素血症…………………………………………………… 17
（五） 高泌乳素血症對下視丘－腦下腺－睪丸軸系的影響………… 20
三睪酮…………………………………………………………………… 22
（一） 雄性激素與睪酮………………………………………………… 22
（二） 睪酮的分泌細胞………………………………………………… 22
（三） 萊氏細胞的調節………………………………………………… 22
（四） 睪酮的合成……………………………………………………… 23
（五） 黃體促素與人絨毛膜性腺促素………………………………… 24
四目的與假說…………………………………………………………… 26
第二章 動物模式的建立………………………………………………… 33
一高泌乳素血症的雄性大鼠動物模式………………………………… 34
二以異體腦下腺前葉移植腎包膜下的方式誘發高泌乳素
血症的方法………………………………………………………… 35
(一) 摘取捐贈鼠腦下腺前葉………………………………………… 35
(二) 接受植入的步驟………………………………………………… 35
(三) 植入成果的檢驗………………………………………………… 35
三 腦下腺前葉植入誘發之高泌乳素血症大鼠其血中激素基礎值的測定 37
(一) 血漿基礎泌乳素濃度測定……………………………………… 37
(二) 血漿基礎性促素(gonadotropins)濃度測定…………………… 37
第三章 激素及生化物質之檢測與分析……………………………… 43
一 泌乳素之放射免疫測定…………………………………………… 44
二 睪酮之放射免疫測定……………………………………………… 45
三 黃體促素之放射免疫測定………………………………………… 47
四 濾泡促素之放射免疫測定………………………………………… 48
五 環單磷酸腺甘(cAMP) 之放射免疫測定…………………………… 49
第四章 高泌乳素血症對活體雄鼠睪酮分泌的效應………………… 51
一 緒言………………………………………………………………… 52
二 材料與方法………………………………………………………… 53
(一) 實驗動物………………………………………………………… 53
(二) 高泌乳素血症的誘發…………………………………………… 53
(三) 活體實驗及採血準備…………………………………………… 53
(四) 高泌乳素血症對活體人絨毛膜性腺促素(hCG)
刺激睪酮分泌的效應…………………………………………… 54
(五) 高泌乳素血症對活體性腺釋素(GnRH)刺激黃
體促素(LH)及睪酮分泌的效應………………………………… 54
(六) 睪酮及黃體促素的放射免疫分析測定………………………… 54
(七) 統計分析………………………………………………………… 54
三 結果………………………………………………………………… 56
(一) 高泌乳素血症對活體人絨毛膜性腺促素(hCG)刺激睪酮分泌的效應 56
(二) 高泌乳素血症對活體性腺釋素(GnRH)刺激黃 體促素(LH)
及睪酮分泌的效應…………………………………………………56
四 討論…………………………………………………………………… 57
第五章 高泌乳素血症對離體雄鼠睪丸間質細胞分泌睪酮之效應 61
一 緒言…………………………………………………………………… 62
二 材料與方法…………………………………………………………… 63
(一) 實驗動物…………………………………………………………… 63
(二) 高泌乳素血症的誘發……………………………………………… 63
(三) 睪丸間質細胞(TIC)的備製………………………………………… 63
(四) 高泌乳素血症對離體睪丸間質細胞分泌睪酮之效應 65
(五)高泌乳素血症對離體睪丸間質細胞經泌乳素刺激睪酮分泌的影響 66
(六)高泌乳素血症對離體睪丸間質細胞信息傳遞的影響 66
(七)睪酮及cAMP之放射免疫測定……………………………………… 66
(八)統計分析…………………………………………………………… 67
三 結果…………………………………………………………………… 68
(一) 高泌乳素血症對離體睪丸間質細胞分泌睪酮之效應 68
(二) 高泌乳素血症對離體睪丸間質細胞經泌乳素刺激睪酮分泌的影響 68
(三) 高泌乳素血症對離體睪丸間質細胞信息傳遞的影響 68
四討論…………………………………………………………………… 69
第六章 高泌乳素血症對雄鼠萊氏細胞分泌睪酮的效應……………… 76
一 緒言…………………………………………………………………… 77
二 材料與方法…………………………………………………………… 79
(一) 實驗動物…………………………………………………………… 79
(二) 高泌乳素血症的誘發……………………………………………… 79
(三) 睪丸間質細胞(TIC)的備製………………………………………… 79
(四) 睪丸萊氏細胞的純化與備製……………………………………… 79
(五) 人絨毛膜性腺促素(hCG) 對睪丸間質細胞(TIC)與
萊氏細胞睪酮分泌的刺激效應…………………………………… 80
(六) 25-OH-膽固醇及妊烯醇酮(pregnenolone) 對睪丸間質細胞(TIC) 與
萊氏細胞睪酮分泌的刺激效應…………………………………… 81
(七) Forskolin及8-Br-cAMP對睪丸間質細胞(TIC) 與
萊氏細胞睪酮分泌的刺激效應…………………………………… 81
(八) 睪酮之放射免疫測定…………………………………………… 81
(九) 統計分析………………………………………………………… 82
三 結果…………………………………………………………………… 83
(一) 人絨毛膜性腺促素(hCG) 對睪丸間質細胞(TIC)與
萊氏細胞睪酮分泌的刺激效應…………………………………… 83
(二) 25-OH-膽固醇及妊烯醇酮(pregnenolone) 對睪丸間質細胞(TIC) 與
萊氏細胞睪酮分泌的刺激效應…………………………………… 83
(三) Forskolin及8-Br-cAMP對睪丸間質細胞(TIC) 與
萊氏細胞睪酮分泌的刺激效應…………………………………… 83
四 討論…………………………………………………………………… 84
第七章 睪丸間質巨噬細胞在高泌乳素血症影響雄鼠萊氏細胞分泌睪酮的效應 90
一 緒言………………………………………………………………… 91
二 材料與方法………………………………………………………… 92
(一) 實驗動物………………………………………………………… 92
(二) 高泌乳素血症的誘發…………………………………………… 92
(三) 睪丸間質細胞(TIC)的備製……………………………………… 92
(四) 睪丸萊氏細胞的純化與備製…………………………………… 92
(五) 睪丸間質巨噬細胞(TIM)的分離與計數………………………… 93
(六) 睪丸間質巨噬細胞(TIM)鑑定的方法…………………………… 93
(七) 腫瘤壞死因子a (TNF-a)對人絨毛膜性腺促素(hCG)刺激萊氏細胞睪酮分泌的影響…94
(八) 泌乳素刺激睪丸間質細胞(TIC)腫瘤壞死因子a (TNF-a)分泌的效應………………… 94
(九) 泌乳素與人絨毛膜性腺促素(hCG)對睪丸間質巨噬細胞(TIM)腫瘤壞死因子a (TNF-a)分泌的交互效應…95
(十) 睪酮之放射免疫測定…………………………………………… 95
(十一) 腫瘤壞死因子a (TNF-a)的酵素免疫測定…………………… 95
(十二) 統計分析……………………………………………………… 96
三 結果………………………………………………………………… 96
(一) 腫瘤壞死因子a (TNF-a)對人絨毛膜性腺促素(hCG)刺激萊氏細胞睪酮分泌的影響… 97
(二) 泌乳素刺激睪丸間質細胞(TIC)腫瘤壞死因子a (TNF-a)分泌的效應………………… 97
(三) 泌乳素與人絨毛膜性腺促素(hCG)對睪丸間質巨噬細胞(TIM)腫瘤壞死因子a (TNF-a)分泌的交互效應… 97
四 討論…………………………………………………………………… 99
第八章 結論……………………………………………………………… 107
圖表目錄………………………………………………………………… 109
參考文獻………………………………………………………………… 112
附錄……………………………………………………………………… 122
特殊溶液配製法………………………………………………………… 123
已發表之論文及相關代表作…………………………………………… 126
名詞中英對照表[94]

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