本文關(guān)鍵詞：脫氧膽酰酪氨酸的避孕藥效及陰道黏膜刺激研究　出處：《復(fù)旦大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 脫氧膽酰酪氨酸 殺精 避孕 陰道黏膜刺激

【摘要】：近年來,性傳播己經(jīng)成為全球艾滋病病毒(HIV)傳播的主要途徑,其中研制兼具避孕和預(yù)防AIDS/STDs的雙功能外用避孕藥特別適合我國(guó)國(guó)情。脫氧膽酰酪氨酸(deoxycholytyosine, DCT)是在保留膽酸及其衍生物可能的藥效基團(tuán)的基礎(chǔ)上,對(duì)側(cè)鏈部位用氨基酸進(jìn)行了結(jié)構(gòu)修飾,合成了新的膽酸衍生物。本研究通過避孕藥效學(xué)、殺精機(jī)制研究、陰道粘膜刺激性研究,對(duì)于DCT的成藥性初步進(jìn)行了有效性和安全性的評(píng)價(jià),也初步探討了DCT作為外用避孕藥的殺精機(jī)制。避孕藥效學(xué)研究包括體外殺精試驗(yàn)、體內(nèi)殺精試驗(yàn)與抗生育避孕試驗(yàn)。體外殺精試驗(yàn)將DCT與人類體外精子充分混合作用,觀察精子活率隨著DCT濃度及時(shí)間的變化,即劑量時(shí)間效應(yīng)試驗(yàn),并同時(shí)得出精子的運(yùn)動(dòng)參數(shù)的變化情況。然后去除DCT作用觀察精子活力恢復(fù)情況。結(jié)果顯示DCT的殺精效果具有明顯的劑量-時(shí)間效應(yīng),對(duì)精子運(yùn)動(dòng)活力參數(shù)和精子運(yùn)動(dòng)方式參數(shù)均有抑制作用,在去除藥物作用后精子活率沒有明顯改變(P0.05)。體內(nèi)殺精試驗(yàn)和家兔避孕試驗(yàn)選用發(fā)情期雌兔60只,隨機(jī)分成6組,陰道內(nèi)給藥,10min后與生育能力正常的雄兔1：1進(jìn)行交配。在交配后5 min、10 min、15 min和20 min從陰道取出精液顯微鏡下觀察精子活動(dòng)情況。結(jié)果顯示DCT gel在單次陰道給予12.50 mg和25.00 mg DCT劑量時(shí),交配后5min內(nèi)均未見活動(dòng)精子。體內(nèi)殺精試驗(yàn)約60 min后,上述各組家兔陰道給予同樣的受試物,1O-15min后再與雄兔進(jìn)行1：1交配,記錄產(chǎn)仔兔情況,并計(jì)算出避孕率。結(jié)果顯示,中、高劑量組家兔達(dá)到100%的抗生育避孕效果。殺精機(jī)制研究包括質(zhì)膜完整性試驗(yàn)、凋亡試驗(yàn)、頂體試驗(yàn)、線粒體試驗(yàn)以及利用掃描電鏡觀察精子結(jié)構(gòu)等。利用熒光染料SYBR-14/PI與熒光顯微鏡及流式細(xì)胞儀相結(jié)合定性和定量觀察DCT對(duì)精子質(zhì)膜的影響。結(jié)果顯示DCT可以明顯破壞精子質(zhì)膜,質(zhì)膜完整精子在DCT 0.25、0.5和1.0 mg/ml時(shí)質(zhì)膜完整精子比率分別為(31.26±8.35)%,(13.26±5.21)%和(0.74+0.93)%,與空白對(duì)照組具有明顯差別(P0.05),且具有明顯的劑量效應(yīng)關(guān)系。熒光顯微鏡結(jié)合FITC-PSA/PI雙染觀察精子頂體影響,結(jié)果顯示,DCT處理后精子頂體區(qū)染色暗淡切不均勻,或者根本沒有熒光染色,可見DCT對(duì)于精子頂體具有明顯的破壞作用。利用熒光染料JC-1與熒光顯微鏡及流式細(xì)胞儀相結(jié)合定性和定量觀察DCT對(duì)精子線粒體膜電位的影響。結(jié)果顯示,DCT處理后精子線粒體膜電位發(fā)生改變,精子尾部綠色熒光強(qiáng)度明顯增強(qiáng)。FCM結(jié)果顯示精子線粒體膜電位隨著DCT濃度的升高而逐漸下降,正常線粒體膜電位精子比例與空白對(duì)照組有明顯的差別(P0.05)。凋亡精子檢測(cè)顯示晚期凋亡精子比率在0.25,0.5和1.0mg/ml時(shí),分別是(11.92±1.19)%、(30.09±6.29)%和(75.55±6.30)%,具有明顯的劑量依賴性(P0.05)。DCT≤0.5 g·l-1引起精子早期凋亡率升高,但是高濃度時(shí)卻出現(xiàn)降低趨勢(shì)。電鏡結(jié)果顯示DCT處理后大部分精子的頂體脫失,質(zhì)膜不完整,被溶解、破壞；尾部出現(xiàn)穿孔甚至斷裂。陰道黏膜刺激性試驗(yàn)選用成年雌性小型豬9只,隨機(jī)分為3組,分別為基質(zhì)對(duì)照組、N-9 jelly組和DCT-gel組。在休情期每天單次陰道給藥5m1不同受試物,連續(xù)7天,給藥前和末次給藥后24h、72h收集陰道灌流液,取沉淀細(xì)胞用流式細(xì)胞儀檢測(cè)炎癥細(xì)胞及CD45+細(xì)胞比例。末次給藥后72h處死動(dòng)物,切開陰道進(jìn)行肉眼觀察,然后分離出陰道組織,分別用10%福爾馬林液固定、包埋、切片,進(jìn)行病理組織學(xué)檢查。然后利用免疫組織化學(xué)染色檢測(cè)陰道黏膜CD45表達(dá)。結(jié)果顯示,市售N-9 jelly能引起陰道粘膜組織的炎癥反應(yīng),而導(dǎo)致CVL液中的細(xì)胞總數(shù)特別是中性粒細(xì)胞及CD45+細(xì)胞增多,DCT gel組和基質(zhì)對(duì)照組與N-9jelly明顯不同,它們沒有引起小型豬CVL液中性粒細(xì)胞和CD45+細(xì)胞數(shù)量上明顯的增加。從組織學(xué)變化角度,小型豬陰道黏膜組織HE染色顯示DCT gel對(duì)黏膜上皮層、固有層、血管及周圍組織無明顯損傷(5.5±2.7),與空白膠組相比無顯著性差異(3.3±1.6),遠(yuǎn)低于N-9 jelly組(20.5±3.5,P0.05)；與空白膠組相比,DCT gel組的陰道黏膜CD45表達(dá)無顯著性差異(P0.05),N-9 jelly組CD45表達(dá)顯著增加(P0.05)。綜上所述,DCT不僅具有較強(qiáng)的體外殺精效能,并且對(duì)陰道刺激性較小,具有較好的研發(fā)價(jià)值和廣闊的應(yīng)用前景。
[Abstract]:In recent years, sexual transmission has become the global AIDS virus (HIV) the main route of transmission, which is developed with double function topical contraceptive pill and prevention of AIDS/STDs is especially suitable for the situation of China. Deoxycholyltyosine (deoxycholytyosine, DCT) is based on the pharmacophore may retain cholic acid and its derivatives on the side chain site structure was modified by amino acid, cholic acid derivatives were synthesized. The new study through the contraceptive effect, study the spermicidal mechanism, irritation of the vaginal mucosa, the medicine DCT a preliminary evaluation of efficacy and safety, also discussed the DCT as spermicidal contraceptive mechanism research. Including in vitro spermicidal contraceptive efficacy test, in vivo spermicidal test and anti contraceptive test. In vitro spermicidal test DCT and human sperm in vitro fully mixing, observe the sperm survival rate with DCT Changes in concentration and time, the dose time effect test, and draws the changes of sperm motility parameters at the same time. Then the removal of DCT to observe the effect of sperm motility recovery. The results showed that DCT of the spermicidal effect has obvious dose time effect, inhibition of sperm motility parameters and sperm motion parameters are in the removal of drug effects after sperm motility rate did not change significantly (P0.05). In vivo spermicidal test and rabbit contraceptive test selected female estrus 60 rabbits were randomly divided into 6 groups, intravaginal administration, 10min and fertility of normal male rabbits were 1:1 mating. After mating, 5 min, 10 min, 15 and min 20 min removed under the microscope of semen sperm motility from the vagina. The results showed that DCT gel received 12.50 mg and 25 mg DCT in a single dose of vagina, 5min after mating showed no activity of sperm. In vivo spermicidal test about 60 min After the rabbits were given the same vaginal sample, after 1O-15min and 1:1 of male rabbit mating pups was recorded and calculated the contraception rate. The results showed that in the high dose group of rabbits reached 100% of the antifertility contraceptive effect. The spermicidal mechanism research including plasma membrane integrity test, apoptosis test. The top body test, test and observation of sperm mitochondrial structure by scanning electron microscopy. The use of fluorescent dye SYBR-14/PI and fluorescence microscopy and flow cytometry qualitative and quantitative observation of the effects of DCT on sperm membrane. The results show that DCT can significantly damage the sperm plasma membrane, membrane integrity of sperm plasma membrane in DCT 0.25,0.5 and 1 mg/ml when the ratio of intact sperm respectively (31.26 + 8.35)% and (13.26 + 5.21)% and (0.74+0.93)%, and the control group with significant difference (P0.05), and has an obvious dose-response relationship. Fluorescence microscopy combined with FITC-PSA/PI Double staining of sperm acrosome, results showed that after DCT treatment the acrosome region gloomy staining must not uniform, or no visible fluorescence staining, DCT for sperm acrosome has obvious destructive effect. The influence of combination of qualitative and quantitative observation of DCT on sperm mitochondrial membrane potential using the fluorescent dye JC-1 with fluorescence microscope and flow the combination of cytometry. The results showed that the change of sperm mitochondrial membrane potential occurred after DCT treatment, sperm tail green fluorescence intensity significantly enhanced.FCM showed that the sperm mitochondrial membrane potential increased with the concentration of DCT decreased gradually, normal sperm mitochondrial membrane potential and the proportion of blank control group had significant difference (P0.05). The apoptosis of sperm detection late apoptotic sperm ratio in 0.25,0.5 and 1.0mg/ml, respectively (11.92 + 1.19)% and (30.09 + 6.29)% and (75.55 + 6.30)%, with significant dose dependent (P0.05).DCT = 0 .5 G - L-1 induced sperm early apoptosis rate increased, but decreased at high concentration. The results of SEM showed that most of the sperm acrosome loss after DCT treatment, the plasma membrane is not complete, was dissolved and destroyed; tail perforation and fracture. The vaginal mucosa irritation into female 9 pigs were randomly divided into test. For the 3 groups, respectively for the matrix control group, N-9 jelly group and DCT-gel group. In anestrous every single vaginal administration of 5m1 in different subjects, for 7 consecutive days, administered before and after the last administration of 24h, 72h collected vaginal lavage fluid, and inflammatory cells and CD45+ cells to detect the proportion of precipitation cells with flow cytometry. 72h after the last administration were animal, open vagina eye, then separated the vaginal tissue, were treated with 10% formalin fixation, embedding, sectioning, histopathological examination was performed. Then using immunohistochemical staining to detect vaginal adhesion The membrane expression of CD45. The results show that the commercially available N-9 jelly can cause inflammation of vaginal mucosa, resulting in the total number of CVL cells in liquid especially neutrophils and CD45+ cells increased, DCT gel and N-9jelly matrix group and the control group were significantly different. They did not cause significantly increased number of small pig neutrophils CVL solution and CD45+ cells. The perspective changes from the organization, a small group of pig vaginal mucosa HE staining showed that the DCT gel on the epithelial layer, lamina propria, no obvious vascular injury and surrounding tissues (5.5 + 2.7), compared with the blank gel group showed no significant difference (3.3 + 1.6), jelly group (far less than N-9 20.5 + 3.5, P0.05); compared with the blank gel group, the expression of CD45 DCT in group gel vaginal mucosa had no significant difference (P0.05), significantly increased expression of N-9 jelly CD45 (P0.05). In conclusion, DCT not only has strong spermicidal potency, and irritation of the vagina It is small, and has good R & D value and broad application prospect.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R965

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本文編號(hào)：1434782