本文選題：抗菌肽 + 壬苯醇醚-9(N-9)��； 參考：《北京協(xié)和醫(yī)學(xué)院》2014年碩士論文

【摘要】：研究背景： 體外殺精劑是控制生育和避孕的有效手段,其中壬苯醇醚-9(Nonoxinol-9,N-9)因其具有強(qiáng)大的殺精效果而成為廣泛接受的表面活性劑類殺精劑,已經(jīng)在美國和其它一些國家使用超過了30年。作為一種有效的殺精避孕陰道外用生物制劑,除了擁有快速有效的殺精避孕性質(zhì)之外,安全低毒也非常重要。但近年來有研究表明,N-9存在安全隱患,長期反復(fù)使用可能會(huì)對(duì)陰道上皮細(xì)胞造成損傷,所以盡快研制新型安全的殺精避孕劑已迫在眉睫。 抗菌肽(Antimicrobial peptides)是廣泛存在于生物體內(nèi)的一類抵抗外源性病原微生物致病作用的防御性小分子多肽(分子質(zhì)量10ku),是宿主固有免疫系統(tǒng)的重要組成部分。每種抗菌肽都有其特定的抗菌譜,部分抗菌肽還兼具殺精子作用。兩棲動(dòng)物蛙類的皮膚分泌物中含有豐富的抗菌多肽,在抗菌、抗腫瘤、抗病毒和殺精等方面都展現(xiàn)出了強(qiáng)大的功能,并已在農(nóng)業(yè)及臨床等方面有所應(yīng)用。 目前,國外關(guān)于殺精抗菌肽的報(bào)道如Magainin、Dermaseptins、Nisin和Subtilosin等幾種抗菌肽都顯示出較強(qiáng)的體外殺精效果,如800～1000mg/L Magainin20s就能制動(dòng)(immobilization)猴的精子(濃度為60X106/mL-80X108/mL),值得注意的是,Magainin在最小殺精濃度時(shí),未對(duì)紅細(xì)胞和陰道粘膜細(xì)胞產(chǎn)生毒性,顯示出較高的安全性。 因此,抗菌肽成為近年來國內(nèi)外研發(fā)的熱點(diǎn)。其中線性陽離子多肽Maximin是賴仞課題組最早從中國大蹼鈴蟾(Bombina maxima)皮膚中分離提取的具有自主知識(shí)產(chǎn)權(quán)的先導(dǎo)化合物,特別是Maximin和同濃度的Magainin具有相似的精子制動(dòng)活性,故成為本實(shí)驗(yàn)殺精分析首選抗菌肽。 本文通過對(duì)19種抗菌肽Maximin衍生物(M1-M19)進(jìn)行體外殺精和細(xì)胞毒性作用的評(píng)價(jià),篩選出有望成為安全、有效的新型體外殺精劑的候選物。 研究目的： 1.通過對(duì)Maximin的19種衍生物(M1-M19)的體外殺精作用和細(xì)胞毒性分析,篩選出具有較強(qiáng)殺精效果和低細(xì)胞毒性的抗菌肽； 2.通過家兔抗生育實(shí)驗(yàn),探索篩選出的高效、低毒抗菌肽的抗生育作用并對(duì)其體內(nèi)安全性進(jìn)行初步評(píng)價(jià)； 3.初步探索殺精抗菌肽的殺精機(jī)制及其抗菌活性。 研究路線和方法： 1.精液采集及分析：精液樣品來源于北京市人類精子庫。選擇育齡、健康和有正常生育史的男性志愿者48例,年齡為25～35歲,取精前禁欲72h,手淫法獲取精液(精液量2mL),于37℃C保溫液化30mmin-。液化后進(jìn)行常規(guī)分析,嚴(yán)格按照《WHO人類精液檢查與處理實(shí)驗(yàn)室手冊(cè)》第五版推薦的標(biāo)準(zhǔn)執(zhí)行,其中精子總數(shù)60×106～100×106,精液濃度20×106/mL,精子活動(dòng)率60%。 2.體外殺精效果實(shí)驗(yàn)：按照WHO推薦的Sander-Cramer方法,精液與抗菌肽以1：5迅速混合,37℃C水浴20s,顯微鏡下隨機(jī)觀察計(jì)數(shù)5個(gè)視野,再以伊紅-苯胺黑染色技術(shù)觀察精子細(xì)胞的存活,篩選出具有較強(qiáng)殺精作用的抗菌肽衍生物。同時(shí)測(cè)定殺精抗菌肽和N-9的EC100。最后觀察測(cè)定殺精抗菌肽的時(shí)間-濃度依賴性。 3.細(xì)胞毒性研究：采用CCK-8試劑盒檢測(cè)細(xì)胞毒性,對(duì)抗菌肽衍生物和N-9進(jìn)行細(xì)胞毒性分析,評(píng)價(jià)N-9的細(xì)胞毒性并篩選出細(xì)胞毒性較低的抗菌肽衍生物,并計(jì)算它們的IC50值。 4.家兔抗生育實(shí)驗(yàn)：采用陰道內(nèi)給藥的方法向雌性育齡新西蘭大耳白兔陰道內(nèi)注入殺精劑量的抗菌肽溶液,交配后觀察統(tǒng)計(jì)兔子懷孕情況。生理鹽水為陰性對(duì)照,N-9為陽性對(duì)照。相同組分觀察陰道內(nèi)給藥后,兔紅細(xì)胞溶血情況。 5.殺精機(jī)制初探：采用HOS-EG實(shí)驗(yàn)方法了解殺精抗菌肽殺精后精子膜破壞情況。 6.抗菌實(shí)驗(yàn)：微量稀釋法檢測(cè)殺精抗菌肽候選衍生物對(duì)白色念珠菌和大腸桿菌的抗菌作用效果。 研究結(jié)果： 1.19種抗菌肽Maximin衍生物(M1-M19)中M1、M7、M1、M15和M17這五種抗菌肽在濃度為2000mg/L、作用時(shí)間為20s時(shí)就能完全制動(dòng)精子,ECloo分別是：M7=M11=2000mg/L,Ml=M15=M17=1500mg/L。N-9的EC100=100mg/L。 2.利用Hela-229細(xì)胞分析細(xì)胞毒性時(shí),M11的細(xì)胞毒性明顯低于其它四種抗菌肽,表現(xiàn)出較高的安全性。N-9具有強(qiáng)大的細(xì)胞毒性,IC50=3.01mg/L。 3.殺精抗菌肽衍生物M11的家兔抗生育實(shí)驗(yàn)結(jié)果顯示M11藥物組的活胎數(shù)和避孕率與陰性對(duì)照組相對(duì)比均無統(tǒng)計(jì)學(xué)意義。 4.HOS-EG實(shí)驗(yàn)結(jié)果顯示,殺精抗菌肽和N-9對(duì)精子的細(xì)胞膜完全破壞；M11對(duì)精子的破壞主要集中的精子尾部,而M15和M17對(duì)精子的損傷基本上都是頭尾共同破壞,這種作用方式類似于N-9。 5.微量稀釋法分析殺精抗菌肽衍生物M11的抗菌活性,結(jié)果在濃度50mg/L時(shí)并未對(duì)大腸桿菌和白色念珠菌產(chǎn)生殺菌作用。 結(jié)論： 1.19種抗菌肽Maximin衍生物(M1-M19)中M1、M7、M1、M15和M17這五種抗菌肽具有較強(qiáng)的體外殺精效果； 2.細(xì)胞毒性實(shí)驗(yàn)表明M1、M7、M11、M15和M17這五種抗菌肽的細(xì)胞毒性大小分別是：M11M15M17M7M1。其中抗菌肽M11表現(xiàn)出幾乎無細(xì)胞毒性的特點(diǎn),安全性最高； 3.殺精抗菌肽(M11、M15和M17)具有明顯的殺精作用時(shí)間-濃度依賴性特點(diǎn)；HOS-EG實(shí)驗(yàn)表明,抗菌肽殺精作用是通過破壞精子細(xì)胞膜而實(shí)現(xiàn)的。 4.殺精抗菌肽衍生物M11未能實(shí)現(xiàn)對(duì)家兔抗生育的目的。 5.殺精抗菌肽衍生物M11對(duì)白色念珠菌和大腸桿菌的抑菌實(shí)驗(yàn)中未觀察到抑制現(xiàn)象。
[Abstract]:Research background:
In vitro sperm killer is an effective means to control fertility and contraception, in which nonyl alcohol ether -9 (Nonoxinol-9, N-9) has become a widely accepted surface active agent because of its strong sperm killing effect. It has been used in the United States and some other countries for more than 30 years. In recent years, studies have shown that N-9 has a potential safety hazard, and long-term repeated use may cause damage to the epithelial cells of the vagina, so the development of a new type of safe contraceptive as soon as possible has been forced in the eyebrow.
Antibacterial peptide (Antimicrobial peptides) is a kind of defensive small molecular polypeptide (molecular mass 10KU) that widely exists in organism to resist the pathogenic effect of exogenous pathogenic microorganism. It is an important part of the inherent immune system of host. Each antibacterial peptide has its specific antimicrobial spectrum, and some antimicrobial peptides also have spermatozoon effect. Amphibian The skin secretions of animal frogs contain abundant antibacterial peptides, which have shown powerful functions in antibacterial, anti-tumor, antiviral and sperm killing, and have been applied in agriculture and clinical fields.
At present, reports of antibioppeptides in foreign countries such as Magainin, Dermaseptins, Nisin and Subtilosin have shown strong in vitro spermatozoon effect, such as 800 ~ 1000mg/L Magainin20s to be able to brake (immobilization) monkey sperm (concentration 60X106/mL-80X108/ mL). It is worth noting that Magainin is at the minimum concentration of spermatozoa. Toxicity to red blood cells and vaginal mucosa cells showed high safety.
Therefore, antibacterial peptide has become a hot spot at home and abroad in recent years, in which linear cationic polypeptide Maximin is the first derivative compound extracted from the skin of Bombina maxima of Chinese webbed bell (maxima), especially Maximin and the same concentration of Magainin have similar sperm braking activity. Antiseptic peptide is the first choice for the analysis of sperm killing in this experiment.
In this paper, 19 kinds of antibacterial peptide Maximin derivatives (M1-M19) were evaluated for sperm killing and cytotoxicity in vitro, and a candidate for a new in vitro sperm killing agent, which was expected to be a safe and effective, was screened.
The purpose of the study is:
1. through the analysis of spermatogenesis and cytotoxicity of 19 derivatives of Maximin (M1-M19) in vitro, the antimicrobial peptides with strong spermatogenesis and low cytotoxicity were screened.
2. through the Rabbit anti fertility experiment, we explored the antifertility effect of the highly effective and low toxic antimicrobial peptide and evaluated its safety in vivo.
3. preliminarily explore the sperm killing mechanism and antibacterial activity of antibacterial peptides.
Research routes and methods:
1. semen collection and analysis: the semen samples were derived from the human sperm bank of Beijing city. 48 male volunteers with reproductive age, healthy and normal birth history were selected. The age was 25~35 years old. The age was 25~35 years old. The pre sperm abstinence was 72h, the masturbation method was used to obtain semen (semen 2mL). The routine analysis was carried out after the 37 C C liquefaction of 30mmin-., and the 2. in vitro spermatide effect experiment: according to the Sander-Cramer method recommended by WHO, semen and antibacterial peptide were mixed with 1:5 quickly, C water bath was 20s at 37 C. Under microscope, 5 fields were observed and counted randomly. The survival of spermatozoa was observed by eosinosaniline black staining technique, and antibacterial peptide derivatives with strong spermatogenesis were screened and spermatogenesis was determined. Antimicrobial peptides and EC100. of N-9 were finally observed to determine the time concentration dependence of antimicrobial peptides.
3. cytotoxicity study: detection of cytotoxicity by CCK-8 kit, cytotoxicity analysis against bacteriocin derivatives and N-9, evaluation of cytotoxicity of N-9 and screening out the lower cytotoxic peptide derivatives, and calculating their IC50 values.
4. anti fertility experiment of rabbit: using the method of intravaginal administration, the antiseptic peptide solution was injected into the vagina of the female child-bearing old Zealand white rabbit. After mating, the condition of the rabbit pregnancy was observed. The normal saline was negative control and N-9 was the positive control. The hemolysis of rabbit red blood cells was observed after the same component in the vagina.
5. spermatogenesis mechanism: HOS-EG experiment was used to understand spermatozoa membrane destruction after spermatozoa killing spermatozoa.
6. antibacterial experiment: microdilution method was used to detect the antibacterial effect of the antibacterial peptides extracted from Candida albicans and E. coli.
The results of the study:
The five antibacterial peptides of 1.19 antimicrobial peptides Maximin derivatives (M1-M19), M1, M7, M1, M15 and M17, can completely immobilize spermatozoa when the concentration is 2000mg/L and the action time is 20s. ECloo is M7=M11=2000mg/L, Ml=M15=M17=1500mg/L.N-9
2. when Hela-229 cells were used to analyze cytotoxicity, the cytotoxicity of M11 was significantly lower than that of the other four kinds of antimicrobial peptides, showing a high safety.N-9 with strong cytotoxicity, IC50=3.01mg/L.
3. the anti fertility test of M11, a sperm killing antibacterial peptide derivative, showed that the number of live births and contraceptive rates in the M11 group were not significantly different from those in the negative control group.
The results of 4.HOS-EG experiment showed that spermatozoon and N-9 destroyed the cell membrane of spermatozoa completely; the destruction of sperm by M11 was mainly concentrated on the sperm tail, and the damage of M15 and M17 to sperm was basically the joint destruction of the head and tail, which was similar to that of N-9..
5. the microdilution method was used to analyze the antibacterial activity of the M11, which results in the killing effect of 50mg/L on E. coli and Candida albicans.
Conclusion:
Among the 1.19 antimicrobial peptide Maximin derivatives (M1-M19), these five antimicrobial peptides M1, M7, M1, M15 and M17 have strong sperm killing effect in vitro.
2. cytotoxicity tests showed that the cytotoxic size of five antimicrobial peptides, M1, M7, M11, M15 and M17, were respectively M11M15M17M7M1. of which antibacterial peptide M11 showed almost no cytotoxicity, and the safety was the highest.
3. spermatid antibacterial peptides (M11, M15 and M17) have obvious time concentration dependence of spermatogenesis, and HOS-EG experiments show that the antiseptic effect of antibacterial peptides is realized by destroying the sperm cell membrane.
4. M11, a sperm killing antibacterial peptide derivative, failed to achieve the purpose of anti fertility in rabbits.
5. inhibitory effect of M11, a bacteriostasis peptide derivative, on Candida albicans and Escherichia coli was not observed.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96

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本文編號(hào)：2100475

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