抗菌肽衍生物的杀精效果研究

发布时间：2018-07-05 14:11

本文选题：抗菌肽 + 壬苯醇醚-9(N-9)　；参考：《北京协和医学院》2014年硕士论文

【摘要】：研究背景：体外杀精剂是控制生育和避孕的有效手段,其中壬苯醇醚-9(Nonoxinol-9,N-9)因其具有强大的杀精效果而成为广泛接受的表面活性剂类杀精剂,已经在美国和其它一些国家使用超过了30年。作为一种有效的杀精避孕阴道外用生物制剂,除了拥有快速有效的杀精避孕性质之外,安全低毒也非常重要。但近年来有研究表明,N-9存在安全隐患,长期反复使用可能会对阴道上皮细胞造成损伤,所以尽快研制新型安全的杀精避孕剂已迫在眉睫。抗菌肽(Antimicrobial peptides)是广泛存在于生物体内的一类抵抗外源性病原微生物致病作用的防御性小分子多肽(分子质量10ku),是宿主固有免疫系统的重要组成部分。每种抗菌肽都有其特定的抗菌谱,部分抗菌肽还兼具杀精子作用。两栖动物蛙类的皮肤分泌物中含有丰富的抗菌多肽,在抗菌、抗肿瘤、抗病毒和杀精等方面都展现出了强大的功能,并已在农业及临床等方面有所应用。目前,国外关于杀精抗菌肽的报道如Magainin、Dermaseptins、Nisin和Subtilosin等几种抗菌肽都显示出较强的体外杀精效果,如800～1000mg/L Magainin20s就能制动(immobilization)猴的精子(浓度为60X106/mL-80X108/mL),值得注意的是,Magainin在最小杀精浓度时,未对红细胞和阴道粘膜细胞产生毒性,显示出较高的安全性。因此,抗菌肽成为近年来国内外研发的热点。其中线性阳离子多肽Maximin是赖仞课题组最早从中国大蹼铃蟾(Bombina maxima)皮肤中分离提取的具有自主知识产权的先导化合物,特别是Maximin和同浓度的Magainin具有相似的精子制动活性,故成为本实验杀精分析首选抗菌肽。本文通过对19种抗菌肽Maximin衍生物(M1-M19)进行体外杀精和细胞毒性作用的评价,筛选出有望成为安全、有效的新型体外杀精剂的候选物。研究目的： 1.通过对Maximin的19种衍生物(M1-M19)的体外杀精作用和细胞毒性分析,筛选出具有较强杀精效果和低细胞毒性的抗菌肽； 2.通过家兔抗生育实验,探索筛选出的高效、低毒抗菌肽的抗生育作用并对其体内安全性进行初步评价； 3.初步探索杀精抗菌肽的杀精机制及其抗菌活性。研究路线和方法： 1.精液采集及分析：精液样品来源于北京市人类精子库。选择育龄、健康和有正常生育史的男性志愿者48例,年龄为25～35岁,取精前禁欲72h,手淫法获取精液(精液量2mL),于37℃C保温液化30mmin-。液化后进行常规分析,严格按照《WHO人类精液检查与处理实验室手册》第五版推荐的标准执行,其中精子总数60×106～100×106,精液浓度20×106/mL,精子活动率60%。 2.体外杀精效果实验：按照WHO推荐的Sander-Cramer方法,精液与抗菌肽以1：5迅速混合,37℃C水浴20s,显微镜下随机观察计数5个视野,再以伊红-苯胺黑染色技术观察精子细胞的存活,筛选出具有较强杀精作用的抗菌肽衍生物。同时测定杀精抗菌肽和N-9的EC100。最后观察测定杀精抗菌肽的时间-浓度依赖性。 3.细胞毒性研究：采用CCK-8试剂盒检测细胞毒性,对抗菌肽衍生物和N-9进行细胞毒性分析,评价N-9的细胞毒性并筛选出细胞毒性较低的抗菌肽衍生物,并计算它们的IC50值。 4.家兔抗生育实验：采用阴道内给药的方法向雌性育龄新西兰大耳白兔阴道内注入杀精剂量的抗菌肽溶液,交配后观察统计兔子怀孕情况。生理盐水为阴性对照,N-9为阳性对照。相同组分观察阴道内给药后,兔红细胞溶血情况。 5.杀精机制初探：采用HOS-EG实验方法了解杀精抗菌肽杀精后精子膜破坏情况。 6.抗菌实验：微量稀释法检测杀精抗菌肽候选衍生物对白色念珠菌和大肠杆菌的抗菌作用效果。研究结果： 1.19种抗菌肽Maximin衍生物(M1-M19)中M1、M7、M1、M15和M17这五种抗菌肽在浓度为2000mg/L、作用时间为20s时就能完全制动精子,ECloo分别是：M7=M11=2000mg/L,Ml=M15=M17=1500mg/L。N-9的EC100=100mg/L。 2.利用Hela-229细胞分析细胞毒性时,M11的细胞毒性明显低于其它四种抗菌肽,表现出较高的安全性。N-9具有强大的细胞毒性,IC50=3.01mg/L。 3.杀精抗菌肽衍生物M11的家兔抗生育实验结果显示M11药物组的活胎数和避孕率与阴性对照组相对比均无统计学意义。 4.HOS-EG实验结果显示,杀精抗菌肽和N-9对精子的细胞膜完全破坏；M11对精子的破坏主要集中的精子尾部,而M15和M17对精子的损伤基本上都是头尾共同破坏,这种作用方式类似于N-9。 5.微量稀释法分析杀精抗菌肽衍生物M11的抗菌活性,结果在浓度50mg/L时并未对大肠杆菌和白色念珠菌产生杀菌作用。结论： 1.19种抗菌肽Maximin衍生物(M1-M19)中M1、M7、M1、M15和M17这五种抗菌肽具有较强的体外杀精效果； 2.细胞毒性实验表明M1、M7、M11、M15和M17这五种抗菌肽的细胞毒性大小分别是：M11M15M17M7M1。其中抗菌肽M11表现出几乎无细胞毒性的特点,安全性最高； 3.杀精抗菌肽(M11、M15和M17)具有明显的杀精作用时间-浓度依赖性特点；HOS-EG实验表明,抗菌肽杀精作用是通过破坏精子细胞膜而实现的。 4.杀精抗菌肽衍生物M11未能实现对家兔抗生育的目的。 5.杀精抗菌肽衍生物M11对白色念珠菌和大肠杆菌的抑菌实验中未观察到抑制现象。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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