臨床上已證實肉毒桿菌素A注射於前列腺可改善男性膀胱出口阻塞(bladder outlet obstruction)。本研究實驗一、肉毒桿菌素A於前列腺的作用機轉。實驗二、於大白鼠前列腺注射辣椒素，辣椒素會刺激C感覺神經纖維(C-afferent fiber)而引起神經性發炎，建立大白鼠非細菌性前列腺炎的實驗動物模式。並期將非細菌性前列腺炎的實驗動物模式應用在肉毒桿菌素A於前列腺的止痛及抗發炎研究。實驗一: 於大白鼠前列腺注射不同劑量的肉毒桿菌素A。注射不同劑量肉毒桿菌素A的大白鼠前列腺於一或兩個禮拜後取下，利用蘇木紫-伊紅染色(Hematoxylin-Eosin stain)觀察肉毒桿菌素A對大白鼠前列腺組織學所造成的變化。以免疫組織化學染色觀察增殖細胞核抗原(PCNA; proliferating cell nuclear an-tigen)的表現，以TUNEL染色觀察細胞凋亡等。用西方墨點法評估前列腺a1A 腎上腺素接受器(a1A-adrenergic receptor)和雄性激素接受器(androgen receptor)的蛋白質表現量。實驗二: 於大白鼠前列腺注射不同劑量的辣椒素，以大白鼠行為模式的改變評估注射辣椒素三十分鐘所引起的疼痛反應。取下大白鼠前列腺觀察組織學變化並測量cyclooxygenase(COX) 2 蛋白質表現量。實驗於靜脈內注射Evans blue(50mg/kg)以血漿蛋白質滲漏情況評估發炎反應。另外一組實驗於大白鼠前列腺注射肉毒桿菌素A，七天後再注射1000μmol/l 辣椒素。結果實驗一: 前列腺內注射肉毒桿菌素A 七天後，前列腺產生萎縮現象。並且前列腺內注射肉毒桿菌素A 5U、10U 和20U 七天後，前列腺細胞凋亡的數目分別有意義地增加12 倍、16 倍和22 倍，細胞增生的數目分別有意義地減少38%、77%和80%，而a1A 腎上腺素接受器表現量分別有意義地減少13%、80%和81%，雄性激素接受器的蛋白質表現量則無顯著地改變。注射肉毒桿菌素A 十四天後上述改變則明顯減緩。實驗二: 辣椒素誘發大白鼠疼痛有劑量依隨效應(dose dependent)，並產生行為改變，包括閉眼、活動力降低等。辣椒素誘發發炎反應，包括產生發炎細胞、COX2表現，以及急性期之血漿滲漏。注射辣椒素七天後，上述發炎情況會消失。大白鼠前列腺先給予肉毒桿菌素A 七天後，由辣椒素引起的疼痛和發炎反應較為減緩，並有劑量依隨效應。大白鼠前列腺先注射肉毒桿菌素A 20U，再由辣椒素引起的發炎細胞數量會減少82.1%、COX2 的表現量會減少83.0%和Evans blue 滲漏量會減少50.4%。並減緩疼痛的行為改變，包括66.7% 閉眼指數、46.5% 活動力指數。結論一: 前列腺內注射肉毒桿菌素A 會誘發細胞凋亡、抑制細胞增生、負調節a1A 腎上腺素接受器而改變細胞動態平衡。因此肉毒桿菌素A 可應用於改善良性前列腺肥大於細胞動力學的動態和靜態部分(dynamic and static components)的作用。結論二: 研究結果並且支持肉毒桿菌素A 具抗發炎和止痛的效果。大白鼠前列腺內注射辣椒素誘發神經性前列腺炎和前列腺疼痛的實驗動物模式，可應用於臨床上研究前列腺炎。

Intraprostatic injection of BTX-A has demonstrated clinical improvement in men with bladder out let obstruction. Firstly, we investigated the mechanisms of action of BTX-A on the prostate. Secondly, an animal model for nonbacterial prostatitis in rats was developed using intraprostatic injection of capsaicin, an agent thought to excite c-afferent fibers and cause neurogenic inflammation. The analgesic and anti-inflammatory properties of Botulinum toxin type A (BoNT-A) was tested in this model. (1) Adult male Spragu-Dawley rats were injected with varying doses of BTX-A into the prostate, and the prostates were harvested after 1 or 2 weeks. The effects of BTX-A on prostate histology, and the proliferative and apoptotic indexes were determined using hematoxylin and eosin staining, proliferative cell nuclear antigen staining and TUNEL staining, respectively. Changes in a1A adrenergic receptor and androgen receptor were evaluated by Western blotting.
(2) Adult male Spragu-Dawley rats were injected with varying doses of capsaicin into
the prostate. The nociceptive effects of capsaicin were evaluated for 30 min by using a behavior approach; the prostate was removed for histology and cyclooxygenase (COX) 2 concentration measurement. Evans blue (50mg/kg) was also injected intravenously to assess for plasma protein extravasation. The other set of animals were injected with up to 20U of BoNT-A into the prostates 1 wk prior to intraprostatic injection of 1000umol/l capsaicin. (1) One week after BTX-A injection generalized prostate atrophy was observed. There was a significant increase in apoptotic cells (12, 16 and 22-fold), and decrease in proliferative cells (38%, 77% and 80%) and a1A adrenergic receptor (13%, 80% and 81%) for 5U, 10U and 20U, respectively. There was no significant change in androgen receptors. The effects were decreased 2 weeks after BTX-A treatment. (2) Capsaicin dose dependently induced modifications in pain behavior closing of the eyes, hypolocomotion, and inflammatory changes: increase of inflammatory cell accumulation, COX2 expression, and plasma extravasation at the acute stage, but completely recovered at 1 wk. BoNT-A pretreatment dose dependently reversed pain behavior and inflammation. BoNT-A 20U significantly decreased inflammatory cell accumulation, COX2 expression, and Evans blue extraction (82.1%, 93.0% and 50.4, respectively), and reduced pain behavior (66.% for eye score and 46.5% for locomotion score). Conclusion (1): BTX-A injection into the prostate alters cellular dynamics by inducing apoptosis, inhibiting proliferation and down-regulating a1A adrenergic receptors. BTX-A may potentially be the drug that has dual actions on the static and dynamic components of benign prostatic hyperplasia. (2): Intraprostatic capsaicin injection induced neurogenic prostatitis and prostatic pain, and may be a useful research model. BoNT-A produced anti-inflammatory and analgesic effects, and support clinical evaluation in prostatitis.

Abbreviation ………………………………………… ……………………………3
中文摘要 …..…………………………………………………………………………4
Abstract ………………………………………………………… ……………………6
文獻回顧 ……………………………………………………………………………..8
一、 良性前列腺肥大 …………………………………………………………..8
二、 慢性非細菌性前列腺炎/慢性盆腔疼痛綜合徵(CP/CPPS) ………………8
三、 肉毒桿菌素A(BTX-A) ………….…………………………………………9
四、 辣椒素(Capsaicin) ……...………………………………………….……..10
五、 臨床治療前列腺疾病之現況 …...……………………………………….11
六、 肉毒桿菌素於前列腺之作用 …..………………………………………..12
研究目的 ……………………………………………………………………………14
實驗設計 ……..……………………………………………………………………..15
實驗一:評估肉毒桿菌素A對大白鼠前列腺的影響 …..……………………15
實驗二:
A. 以辣椒素建立大白鼠前列腺炎動物模式 …...……………………….15
B. 評估肉毒桿菌素A對大白鼠前列腺炎的影響 …..………………….16
材料方法 …..………………………………………………………………………..17
實驗一:評估肉毒桿菌素A對大白鼠前列腺的影響 …..……………………17
實驗二:
A. 以辣椒素建立大白鼠前列腺炎動物模式 …...……………………….19
B. 評估肉毒桿菌素A對大白鼠前列腺炎的影響 …..………………….22
結果 …..……………………………………………………………………………..24
實驗一:評估肉毒桿菌素A對大白鼠前列腺的影響 …..……………………24
實驗二:
A. 以辣椒素建立大白鼠前列腺炎動物模式 ……...…………………….26
B. 評估肉毒桿菌素A對大白鼠前列腺炎的影響 …..………………….28
討論 …...…………………………………………………………………………….29
前列腺內注射肉毒桿菌素A對於腺組織及受器之影響 ……………………29
辣椒素引發大白鼠前列腺之發炎反應 ………………………………………31
肉毒桿菌素對大白鼠前列腺炎的影響 ...…………………………………….33
結論 ……..…………………………………………………………………………..34
參考文獻 ……………………………………………………………………………35
圖與表 ………………………………………………………………………………42

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