脱氧胆酰酪氨酸的避孕药效及阴道黏膜刺激研究

发布时间：2018-01-16 21:01

本文关键词：脱氧胆酰酪氨酸的避孕药效及阴道黏膜刺激研究　出处：《复旦大学》2014年硕士论文　论文类型：学位论文

【摘要】：近年来,性传播己经成为全球艾滋病病毒(HIV)传播的主要途径,其中研制兼具避孕和预防AIDS/STDs的双功能外用避孕药特别适合我国国情。脱氧胆酰酪氨酸(deoxycholytyosine, DCT)是在保留胆酸及其衍生物可能的药效基团的基础上,对侧链部位用氨基酸进行了结构修饰,合成了新的胆酸衍生物。本研究通过避孕药效学、杀精机制研究、阴道粘膜刺激性研究,对于DCT的成药性初步进行了有效性和安全性的评价,也初步探讨了DCT作为外用避孕药的杀精机制。避孕药效学研究包括体外杀精试验、体内杀精试验与抗生育避孕试验。体外杀精试验将DCT与人类体外精子充分混合作用,观察精子活率随着DCT浓度及时间的变化,即剂量时间效应试验,并同时得出精子的运动参数的变化情况。然后去除DCT作用观察精子活力恢复情况。结果显示DCT的杀精效果具有明显的剂量-时间效应,对精子运动活力参数和精子运动方式参数均有抑制作用,在去除药物作用后精子活率没有明显改变(P0.05)。体内杀精试验和家兔避孕试验选用发情期雌兔60只,随机分成6组,阴道内给药,10min后与生育能力正常的雄兔1：1进行交配。在交配后5 min、10 min、15 min和20 min从阴道取出精液显微镜下观察精子活动情况。结果显示DCT gel在单次阴道给予12.50 mg和25.00 mg DCT剂量时,交配后5min内均未见活动精子。体内杀精试验约60 min后,上述各组家兔阴道给予同样的受试物,1O-15min后再与雄兔进行1：1交配,记录产仔兔情况,并计算出避孕率。结果显示,中、高剂量组家兔达到100%的抗生育避孕效果。杀精机制研究包括质膜完整性试验、凋亡试验、顶体试验、线粒体试验以及利用扫描电镜观察精子结构等。利用荧光染料SYBR-14/PI与荧光显微镜及流式细胞仪相结合定性和定量观察DCT对精子质膜的影响。结果显示DCT可以明显破坏精子质膜,质膜完整精子在DCT 0.25、0.5和1.0 mg/ml时质膜完整精子比率分别为(31.26±8.35)%,(13.26±5.21)%和(0.74+0.93)%,与空白对照组具有明显差别(P0.05),且具有明显的剂量效应关系。荧光显微镜结合FITC-PSA/PI双染观察精子顶体影响,结果显示,DCT处理后精子顶体区染色暗淡切不均匀,或者根本没有荧光染色,可见DCT对于精子顶体具有明显的破坏作用。利用荧光染料JC-1与荧光显微镜及流式细胞仪相结合定性和定量观察DCT对精子线粒体膜电位的影响。结果显示,DCT处理后精子线粒体膜电位发生改变,精子尾部绿色荧光强度明显增强。FCM结果显示精子线粒体膜电位随着DCT浓度的升高而逐渐下降,正常线粒体膜电位精子比例与空白对照组有明显的差别(P0.05)。凋亡精子检测显示晚期凋亡精子比率在0.25,0.5和1.0mg/ml时,分别是(11.92±1.19)%、(30.09±6.29)%和(75.55±6.30)%,具有明显的剂量依赖性(P0.05)。DCT≤0.5 g·l-1引起精子早期凋亡率升高,但是高浓度时却出现降低趋势。电镜结果显示DCT处理后大部分精子的顶体脱失,质膜不完整,被溶解、破坏；尾部出现穿孔甚至断裂。阴道黏膜刺激性试验选用成年雌性小型猪9只,随机分为3组,分别为基质对照组、N-9 jelly组和DCT-gel组。在休情期每天单次阴道给药5m1不同受试物,连续7天,给药前和末次给药后24h、72h收集阴道灌流液,取沉淀细胞用流式细胞仪检测炎症细胞及CD45+细胞比例。末次给药后72h处死动物,切开阴道进行肉眼观察,然后分离出阴道组织,分别用10%福尔马林液固定、包埋、切片,进行病理组织学检查。然后利用免疫组织化学染色检测阴道黏膜CD45表达。结果显示,市售N-9 jelly能引起阴道粘膜组织的炎症反应,而导致CVL液中的细胞总数特别是中性粒细胞及CD45+细胞增多,DCT gel组和基质对照组与N-9jelly明显不同,它们没有引起小型猪CVL液中性粒细胞和CD45+细胞数量上明显的增加。从组织学变化角度,小型猪阴道黏膜组织HE染色显示DCT gel对黏膜上皮层、固有层、血管及周围组织无明显损伤(5.5±2.7),与空白胶组相比无显著性差异(3.3±1.6),远低于N-9 jelly组(20.5±3.5,P0.05)；与空白胶组相比,DCT gel组的阴道黏膜CD45表达无显著性差异(P0.05),N-9 jelly组CD45表达显著增加(P0.05)。综上所述,DCT不仅具有较强的体外杀精效能,并且对阴道刺激性较小,具有较好的研发价值和广阔的应用前景。
[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.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

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