基于“肾主生殖”理论的金匮肾气丸对微波辐射损伤小鼠精原干细胞自我更新与分化的微环境调控研究

Microwave radiation can damage testicular spermatogenic cells and lead to male infertility. Spermatogonial stem cells (SSCs) have tolerance for microwave, so regulating the self-renewal and differentiation of uninjured spermatogonial stem cells is the key to treatment. However, they are not clear that how the change of spermatagonial stem cell niche in the injured testicular affects SSCs self-renewal and differentiation and how to treat. Our preliminary study showed that Golden Coffer Kidney Qi Pill could increase the spermatogenic cells of infertility mice caused by microwave radiation and repair the injured sertoli cells and atrophic testis, so we deduce that the injured niche can decrease spermatogenic cells and the treatment on injured niche can regulate the self-renewal and differentiation of SSCs. In this project, using male mice as the research object, we will establish the infertility model caused by microwave radiation and treat with Golden Coffer Kidney Qi Pill. We will test sperm parameters with CASA, observe the change of all kinds of cell structures in the mice testis with confocal microscopy and test inflammatory factor, lipid peroxidation and sex hormones of testicular tissue so that we can study the effect of the injured niche on reproduction and the curative effect of Golden Coffer Kidney Qi Pill. Using SSCs and sertoli cells of mice through in vitro culture as the research project, we will study the injured niche effect on the SSCs self-renewal and differentiation and the influence of Golden Coffer Kidney Qi Pill. We will thereby reveal the mechanism of "kidney governing reproduction" and provide theoretical supports for the clinical application of Golden Coffer Kidney Qi Pill and the traditional Chinese medicine modernization.

微波辐射可损伤睾丸生精细胞而致不育。因精原干细胞（SSCs）对微波耐受，故调控尚未受损SSCs自我更新与分化成为治疗关键，但受损睾丸中SSCs微环境改变对SSCs自我更新与分化有何影响及如何治疗尚不清楚。我们前期研究发现，金匮肾气丸可使微波辐射不育小鼠生精细胞增多、受损支持细胞及萎缩睾丸得以修复，故推断：受损微环境可使生精细胞减少，治疗受损微环境可调控SSCs自我更新及分化。本项目以雄小鼠为研究对象，建立微波辐射不育模型，用金匮肾气丸治疗，CASA检测精子参数，共聚焦显微镜观察小鼠睾丸各种细胞结构变化，检测睾丸组织炎性因子、脂质过氧化物及性激素，研究受损微环境对生殖的影响及金匮肾气丸疗效；以小鼠SSCs及支持细胞为研究对象，通过体外共同培养，研究受损微环境对SSCs自我更新及分化的影响及金匮肾气丸干预。从而揭示“肾主生殖”机制，为金匮肾气丸临床应用及中医现代化提供理论支撑。

微波辐射可损伤睾丸生精细胞而致不育，金匮肾气丸有良好的治疗作用。精原干细胞（SSCs）对微波耐受，但SSCs自我更新与分化容易受微波辐射损伤，已知这种损伤与微波直接作用有关，但微波辐射损伤的SSCs微环境是否也对SSCs自我更新与分化产生损伤，目前尚不知晓。.本项目以雄小鼠为研究对象，建立微波辐射不育模型，通过精液参数测定研究小鼠生殖水平改变；通过支持细胞、曲细精管基膜、管周肌样细胞、间质细胞等形态学观察研究SSCs微环境结构变化，通过组织炎性因子、脂质过氧化物及性激素检查研究SSCs微环境的生化环境，并进行金匮肾气丸干预。.研究结果表明，微波辐射损伤SSCs微环境。微波辐射可使睾丸重量减轻，管周肌样细胞结构异常；睾丸曲细精管上皮排列疏松，管腔断裂，管腔内可见蛋白水肿液和脱落细胞，曲细精管基膜不完整；支持细胞变性、坏死、脱落，间质细胞结构异常；各类生精细胞排列不整齐，层次不清楚，精母细胞、精子细胞变性、坏死脱落，精细胞数目明显减少，甚则曲细精管内无精子细胞存在。金匮肾气丸治疗后，小鼠睾丸的管周肌样细胞、曲细精管界膜、支持细胞及间质细胞结构正常；曲细精管内可见成熟精子，各类生精细胞排列整齐，层次清楚。.微波损伤的SSCs微环境中CAT明显降低，表明损伤与过氧化反应有关；微波损伤的SSCs微环境中IL-2、IL-6明显升高，表明损伤与炎性相关。微波损伤的SSCs微环境中T、LH、FSH水平没有改变，但金匮肾气干预后损伤的SSCs微环境T、LH、FSH水平明显升高；微波辐射使小鼠血T水平降低，金匮肾气可以使之恢复正常。.上述实验表明，微波辐射可导致SSCs微环境炎症反应及氧化应激损伤，可导致SSCs微环境性激素水平改变。金匮肾气丸可以提高睾酮水平，修复SSCs微环境的微波辐射损伤，治疗微波辐射损伤性男性不育症。.中医称睾丸为肾，SSCs及其微环境与生育的关系揭示了“肾主生殖”的现代生物学本质；微波辐射损伤SSCs及其微环境导致男性不育症，揭示了“肾虚精亏”的现代生物学本质