是17BIPHE2,一种对蛋白酶敏感性低的工程导管素抗菌肽,宫颈阴道液(CVF)中比其亲本肽更好的杀精子剂,LL-37和GF-17?
在相同的质量浓度下,17BIPHE2对重悬于含CVF的培养基中的人精子表现出最高的杀精活性。
LL-37及其截短的肽GF-17具有杀精子和杀微生物活性,尽管它们容易在体液中蛋白水解降解。
在小鼠和人类精子中体外评估了17BIPHE2的杀精剂活性,两者都重新悬浮在培养基中,然后在含有CVF的培养基中孵育的人精子上;在后一种情况下,比较了17BIPHE2与LL-37和GF-17在CVF中的杀精活性和肽稳定性。然后在小鼠中评估17BIPHE2的体内避孕作用及其可逆性。最后,测定了17BIPHE2对淋病奈瑟菌的体外杀菌效果。
通过视频显微镜检查和排除SytoxGreen,一种不透膜的荧光染料,分别。成功的体外受精(IVF)是通过与未处理或17BIPHE2处理的精子共温育后卵母细胞中两个原核的存在来确定的。将单独或与17BIPHE2一起的精子经子宫颈注射到雌性小鼠中,注射后42小时形成双细胞胚胎表明体内受精成功,并在21-25天后通过幼仔分娩怀孕。通过免疫印迹和HPLC评估肽完整性。通过恢复雌性小鼠的妊娠来评估17BIPHE2的避孕作用的可逆性,经宫颈前注射17BIPHE2,与可育雄性自然交配。通过微量稀释肉汤测定获得17BIPHE2对淋病奈瑟菌的最低抑制/杀菌浓度。
在相同的质量浓度下,17BIPHE2是一种比LL-37或GF-17更有效的杀精子剂,杀精子浓度为32.4µM。这主要是由于17BIPHE2对CVF蛋白酶的敏感性较低。重要的是,用32.4µM17BIPHE2治疗3次的雌性小鼠生殖道保持正常,在停止17BIPHE2治疗后其繁殖力恢复.
出于道德原因,17BIPHE2对受精和妊娠的抑制作用目前不能在女性中进行。此外,虽然我们的研究已经证明了17BIPHE2在体外作为小鼠和人类精子的杀精子剂的有效性,仍然需要开发17BIPHE2的剂量制剂(例如,在水凝胶中),以允许17BIPHE2保留在阴道/子宫腔中,同时控制释放其杀精子作用。
由于17BIPHE2在其杀精浓度下也对淋病奈瑟菌具有杀菌活性,它是一个有希望被开发成阴道多用途预防技术剂的候选人,从而增强妇女免受意外怀孕和性传播感染的能力。
这项工作得到了加拿大卫生研究院(N.T.PJT173268)的支持。没有竞争的利益可以宣布。
不适用。
Is 17BIPHE2, an engineered cathelicidin antimicrobial peptide with low susceptibility to proteases, a better spermicide in cervicovaginal fluid (CVF) than its parental peptides, LL-37 and GF-17?
At the same mass concentration, 17BIPHE2 exhibited the highest spermicidal activity on human sperm resuspended in CVF-containing medium.
LL-37 and its truncated peptide GF-17 exert both spermicidal and microbicidal activities, although they are prone to proteolytic degradation in body fluids.
Spermicidal activities of 17BIPHE2 were evaluated in vitro in mouse and human sperm, both resuspended in medium, and then on human sperm incubated in CVF-containing medium; in the latter condition, the spermicidal activity and peptide stability in CVF of 17BIPHE2 were compared with that of LL-37 and GF-17. The in vivo contraceptive effects of 17BIPHE2 and the reversibility thereof were then assessed in mice. Finally, in vitro microbicidal effects of 17BIPHE2 on Neisseria gonorrhoeae were determined.
Sperm motility and plasma membrane integrity were assessed by videomicroscopy and exclusion of Sytox Green, a membrane-impermeable fluorescent dye, respectively. Successful in vitro fertilization (IVF) was determined by the presence of two pronuclei in oocytes following their coincubation with capacitated untreated or 17BIPHE2-treated sperm. Sperm alone or with 17BIPHE2 were transcervically injected into female mice and successful in vivo fertilization was indicated by the formation of two-cell embryos 42-h postinjection, and by pregnancy through pup delivery 21-25 days afterwards. Peptide intactness was assessed by immunoblotting and HPLC. Reversibility of the contraceptive effects of 17BIPHE2 was evaluated by resumption of pregnancy of the female mice, pretranscervically injected with 17BIPHE2, following natural mating with fertile males. Minimum inhibitory/bactericidal concentrations of 17BIPHE2 on N. gonorrhoeae were obtained through microdilution broth assay.
At the same mass concentration, 17BIPHE2 was a more effective spermicide than LL-37 or GF-17 on human sperm resuspended in CVF-containing medium, with the spermicidal concentration of 32.4 µM. This was mainly due to lower susceptibility of 17BIPHE2 to CVF proteases. Importantly, the reproductive tract of mouse females treated three times with 32.4 µM 17BIPHE2 remained normal and their fecundity resumed after stopping 17BIPHE2 treatment.
For ethical reasons, the inhibitory effects of 17BIPHE2 on fertilization and pregnancy cannot presently be performed in women. Also, while our study has proven the effectiveness of 17BIPHE2 as a spermicide for mouse and human sperm in vitro, dosage formulation (e.g. in hydrogel) of 17BIPHE2 still needs to be developed to allow 17BIPHE2 to remain in the vagina/uterine cavity with controlled release for its spermicidal action.
Since 17BIPHE2 also exerted bactericidal activity against N. gonorrhoeae at its spermicidal concentration, it is a promising candidate to be developed into a vaginal multipurpose prevention technology agent, thus empowering women against unplanned pregnancies and sexually transmitted infections.
This work was supported by the Canadian Institutes of Health Research (PJT 173268 to N.T.). There are no competing interests to declare.
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