体外热处理阻断精子垂直传播HIV方法研究： 表观遗传学和子代安全性评估

HIV-positive men are increasing year by year, and the HIV can be vertically transmitted to their offspring through sperm. However, until now, there are no effective methods to prevent such transmission. We recently reported that mouse sperm were highly resistant to heat treatment, and sperm treated with 80 degree for 30 minutes could support full-term embryonic development after ICSI. On the other hand, HIV viruses are highly sensitive to heat and could be inactivated after 56 degree for 30minutes. Considering the fact that mammalian spermatozoa have a high degree similar structure, we propose to establish human sperm in-vitro heat treatment procedures to inactivate HIV virus, then to obtain HIV-free offspring by assisted reproduction technology. Based on our preliminary studies, we aim to improve the heat procedures. And the epigenetics of embryos derived from heated sperm would be detected by immunofluorescence and Western blot method. The mice offsprings'safety is evaluated by their physical, developmental, behavioral phenotype. The project will contribute to establish a method on interrupting transmission of the HIV virus by sperm and fulfill the reproduction needs of male HIV infector.

目前男性HIV感染者逐年上升，其携带的病毒可以通过精子垂直传播给下一代，然而国内外均无有效的方法阻断HIV病毒经精子传播。本项目申请人首次以动物实验模型证明小鼠精子具有高度的热耐受性，80度30分钟的热处理后仍可受精且生育出健康小鼠。而HIV病毒对热具有高度的敏感性，56度30分钟即可被灭活。由于哺乳动物的精子结构具有高度的相似性，在此基础上我们提出一种体外建立人精子热处理的程序，灭活HIV病毒，再通过辅助生殖技术获得无HIV病毒健康后代的研究设想。本项目在前期研究的基础上，进一步完善体外热处理精子的程序和温度；同时，对热处理精子受精后产生胚胎应用免疫荧光，蛋白印迹等方法对重要的表观遗传学位点进行检测；及对出生的子代小鼠生理、发育、行为等重要表型进行子代安全性评估。本项目的研究将有助于建立切断经精子传播HIV病毒的方法，满足HIV男性感染者生育需求。

本项目旨在研究人类精子的是否可以耐受灭火HIV病毒高温的热处理后保持受精能力支持胚胎发育的能力，以及从表观遗传学和动物实验证实该方法的的子代安全性，为建立一种阻断经精子传播HIV途径提供理论基础和前期初步探索。本项目证实来源于加热小鼠精子行单精子卵母细胞浆内注射后产生子代小鼠的生长发育及社会行为学与正常对照组相比无统计学差异。人类精子加热后，精子几个关键基因热应激蛋白HSP70、HSP90及基因修复功能基因DDB1均比对照组升高，证实精子受热后，保护性基因和修复功能基因表达升高，从而初步证实受热精子的安全性。对人类精子加热后对卵子激活也进行了初步探索，证实使用60度30分钟处理人类精子行单精子卵母细胞浆内注射后，经卵子激活后仍具有支持胚胎发育到囊胚的潜能，从而证实该方案具有初步可行性。