GAPDS在糖尿病引起的不育症中的作用及分子机制研究

Male infertility caused by diabetes is an imperative health issue that need to be resovled. Studies have shown that elevated levels of glucose inhibited the activity of the glyceraldehyde -3- phosphate dehydrogenase gene family, and lead to increased production of glycolytic intermediates and activation of oxidative stress, and hence cause cell and tissue damages which is the main cause of diabetic complications. Preliminary studies showed that glyceraldehyde -3- phosphate dehydrogenase (GAPDS) originated from the sperm and testis plays an important role in the development, movement and insemination of sperms. The activity of GAPDS enzyme in diabetic patients is closely related to the reduced sperm motility and infertility. GAPDS protein protects sperm from being damaged by high glucose level.The aim of this thesis is to investigate the function and molecular mechanisms of GAPDS-regulated reproductive cells oxidative stress damages that induced by high glucose level, through depletion and overexpression of GAPDS protein. Furthermore, by constructing an animal model of type 2- diabetic rats, we can determine the effects of GAPDS on the reproductive system in rats with diabetes and the corresponding dysfunction induced. This study will provide a new idea and theoretical basis for prevention and treatment of infertility induced by diabetes.

糖尿病引起的男性不育是当前急需解决的生殖健康问题。研究表明，高糖可抑制甘油醛-3-磷酸脱氢酶家族基因的活性，导致糖酵解中间产物增加和氧化应激途径活化，从而造成细胞和组织损伤是诱发糖尿病并发症发生的主要原因。申请人前期研究表明，睾丸和精子特异的甘油醛-3-磷酸脱氢酶（GAPDS）在精子发育、运动及受精方面起重要作用。糖尿病患者精子GAPDS酶活性与精子活力下降及不育症的发生密切相关，在精子中该蛋白具有调节高糖氧化损伤的作用。本课题拟以生殖细胞模型为基础，以RNAi和过表达等技术研究GAPDS蛋白在高糖诱导生殖细胞氧化应激损伤过程中的功能及分子机制；构建2-型糖尿病鼠动物模型，研究外源GAPDS蛋白在糖尿病诱导的生殖系统并发症中的作用及相应功能指标的变化。本课题的研究将为防治糖尿病不育提供新的思路，为基于GAPDS的治疗提供理论基础。

糖尿病引起的男性不育是当前急需解决的生殖健康问题。糖酵解中间产物增加和氧化应激途径活化，是造成细胞和组织损伤的主要原因。本项目首先对296例糖尿病患者精液标本进行分析，发现糖尿病患者精子中GAPDS蛋白活性与精子运动能力正相关（R=0.96），与不育症的发生负相关（R =-0.99）。糖尿病男性精子中GAPDS蛋白的阳性定位率显著低于非糖尿病男性精液标本。外源性GAPDS蛋白在5 ug/ml以上的GAPDS浓度下，降低精子中的ROS含量，促进精子活动。细胞实验部分，我们成功构建了TM3-GAPDS稳定表达细胞株，在35 mmol/L 葡萄糖培养条件下，TM3细胞内SOD2和过氧化氢酶的蛋白表达水平降低， ROS水平和凋亡相关蛋白质Fas-L, GRP78 and Bax/Bcl-2 增加；而TM3-GAPDS稳转株细胞内ROS水平和凋亡相关蛋白Fas-L, GRP78 and Bax/Bcl-2水平降低。在动物实验部分，我们构建了二型糖尿病大鼠模型，采用计算机辅助精子法分析，发现STZ处理组大鼠精子活力明显下降，GAPDS、LDHC和hk1的酶表达和活性明显低。免疫荧光定位显示，STZ处理后大鼠精子免疫荧光减弱，只有顶体区域才能看到免疫荧光染色。本项目证实了精子甘油醛-3-磷酸脱氢酶通过抑制细胞内ROS水平及凋亡相关蛋白Fas-L, GRP78 and Bax/Bcl-2的表达保护细胞免受高糖氧化应激损伤。