miR-122-5p介导的occludin对支持细胞间紧密连接的调控机制研究

Low sperm count is one of the important reasons for male infertility. Abnormal expression of tight junction in blood testosterone barrier can affect the sperm count. We have constructed the miRNA and mRNA expression profiles of sperm, and found that occludin regulated by miR-122-5p was worth exploring which is a new method affecting the sperm count. First, the expressional level of miR-122-5p and occludin in plasma and semen in patients with oligozoospermia were analyzed to determine its effect on sperm count; Then the miR-122-5p overexpression and inhibition vector, occludin RNAi vector, occ-1 and occ-2 mutant vector were constructed and transfected into Sertoli cell. The development of Sertoli cell TJs and their response to treatment was assessed quantitatively daily by TER, using a Millipore Millicell-electrical resistance system (Millipore), so as to study on tight junction of blood-testis barrier mechanism regulated by miR-122-5p mediated occluding in vitro. Finally, in vivo mice models with TJ damaged by closed peptide injection and mice model with occludin gene knockout were used to analyze the regulatory mechanism of TJ. This project can enrich the regulatory mechanism of spermatogenesis, and provide new research perspectives and methods for the treatment of male infertility due to oligozoospermia.

精子数量少是男性不育的重要原因之一，血睾屏障中紧密连接异常表达会影响精子数量。项目组前期发现miR-122-5p介导的occludin可能会通过调节紧密连接的功能来影响精子数量。本项目首先通过检测miR-122-5p和occludin在寡精症患者血浆及精液支持细胞中的表达水平来确定其对精子数量的影响；再采用体外BTB实验模型，通过构建miR-122-5p的过表达及沉默载体、occludin RNAi载体、occludin的第一胞外环和第二胞外环基因突变载体，以TER作为衡量TJ功能的检测指标，来体外研究miR-122-5p介导的occludin对TJ的调控机制；最后以封闭肽睾丸注射造成TJ破坏的体内实验动物模型和occludin的条件性基因敲除小鼠模型来研究occludin对TJ的调控机理。从而丰富精子发生的调控机制，并为寡精症导致的男性不育的诊疗提供新策略。

全球约有15-20%的夫妻存在不育不孕问题，其中男性原因导致的不育占一半左右。精子数量少是男性不育的重要原因之一。关于寡精子的发病机理一直是国内外男性生殖领域的研究热点，但目前对寡精症发病机制的了解还不是很充分，因此进一步探讨精子数量过少的发生机制并为寡精症提供新的治疗靶标具有重要的意义。本项目拟：首先通过检测miR-122-5p和occludin在寡精症患者血浆内及精液内支持细胞中的表达水平来确定miR-122-5p及occludin对精子数量的影响；再采用睾丸支持细胞原代双室培养法制备的体外实验模型，通过构建miR-122-5p的过表达及沉默载体、构建occludin RNAi载体、构建occludin的第一胞外环（Occ-1）和第二胞外环（Occ-1）基因突变载体，以睾丸支持细胞跨上皮电阻（TER）作为衡量TJ功能的检测指标， 来体外研究miR-122-5p介导的occludin对TJ的调控机制；最后以封闭肽注射造成TJ破坏的体内实验模型和occludin的基因敲除小鼠模型，通过对miR-122-5p和occludin动态检测来研究BTB内TJ的调控机制。