白藜芦醇对猪冷冻精子细胞凋亡及凋亡途径的影响

发布时间：2018-05-11 13:30

  本文选题：猪 + 精液冷冻　； 参考：《上海海洋大学》2015年硕士论文

【摘要】：低温冷冻解冻使液氮保存的猪精子细胞发生严重的凋亡,常见的凋亡途径——死亡受体外源性途径和线粒体内源性途径均可能在冻后精子中发生,介导冻融精子发生凋亡而降低精子质量。本实验在实验室之前研究基础上,通过对凋亡途径相关基因的表达情况研究探索凋亡发生的可能相关途径,并探索在冷冻保护剂中添加抗氧化剂白藜芦醇对冷冻解冻后猪精子细胞凋亡和凋亡途径的影响。研究结果:1.冷冻精子TUNEL染色后的凋亡率(80.4%)与新鲜组(9.7%)相比显著升高(P0.05);与新鲜组精子内ROS绿色荧光比率相比,冷冻组(58.2%)亦显著高于新鲜组(P0.05);与新鲜组0.926μmol/L的ATP浓度相比,冷冻组精子的ATP浓度(0.247μmol/L)则显著下降(P0.05);q RT-PCR检测结果显示,冷冻组中TNF-α、Fas、Caspase家族、P53和Bax等基因相对表达量升高,其中Fas、P53、TNF-α、caspase 8和caspase 9等相对表达量升高显著(P0.05);BCL-2、BIRC-5、Mn SOD、Cu Zn SOD和SURVIVN等基因相对表达量降低,其中BIRC-5、Mn SOD、SURVIVN降低显著(P0.05)。2.向冷冻剂中添加白藜芦醇工作液,使其在冷冻剂中的终浓度分别为12.5μM,25μM,50μM和100μM,并设置0μM作为空白对照,比较各组冻融精子活率,顶体和质膜完整情况。结果表明白藜芦醇终浓度为50μM组的精子活力,质膜完整率和顶体完整率均显著高于其余各添加组(P0.05),并显著高于0μM组的精子质量。精子各指标随着白藜芦醇浓度增大而升高,呈现浓度依赖性,但当白藜芦醇浓度过高(≥100μM)时精子质量显著下降(P0.05)。3.冻融精子经Caspase原位荧光染色后,添加白藜芦醇组的实验组冻融精子凋亡率为29.0%,与未添加白藜芦醇组的对照组(86.3%)相比显著降低(P0.05);实验组58.6%的ROS绿色荧光比率亦显著低于对照组(88.4%)(P0.05);与对照组精子m RNA各基因相比,caspase-8相对表达量降低,Fas,P53,TNF-α,Bax,caspase-3,caspase-9相对表达量显著降低(P0.05);Cu Zn SOD相对表达量升高,Bcl-2,Mn SOD,SURVIVN等显著升高(P0.05)。
[Abstract]:Cryopreservation and thawing caused severe apoptosis of porcine spermatozoa. The common apoptotic pathway, the exogenous pathway of death receptor and the endogenous pathway of mitochondria, may occur in frozen spermatozoa. Mediated apoptosis of freeze-thawed spermatozoa and reduced sperm quality. On the basis of pre-laboratory studies, this study explored the possible pathway of apoptosis through the study of the expression of genes related to the apoptosis pathway. The effects of anti-oxidant resveratrol on the apoptosis and apoptosis of porcine spermatozoa after freezing and thawing were explored. The result of the study was: 1. The apoptotic rate of frozen spermatozoa after TUNEL staining was significantly higher than that of fresh group (P 0.05), the green fluorescence ratio of ROS in frozen sperm was significantly higher than that in fresh group (P 0.05), and the concentration of ATP in fresh group was 0.926 渭 mol/L higher than that in fresh group. The ATP concentration of spermatozoa in freezing group was 0.247 渭 mol / L. The results showed that the relative expression of TNF- 伪 Fas-Caspase family p53 and Bax in frozen group was higher than that in control group. The relative expressions of Fas-P53, TNF- 伪 and caspase _ 9 increased significantly, and the relative expression of BCL-2BIRC-5Mn-SODN, Cu, Zn SOD and SURVIVN were significantly decreased, and BIRC-5Mn-SOD- SURVIVN decreased significantly (P0.05.2UVIVN). The final concentration of resveratrol was 12.5 渭 m, 25 渭 M, 50 渭 M and 100 渭 M, respectively. 0 渭 M was used as blank control to compare the viability of frozen and thawed spermatozoa and the integrity of acrosome and plasma membrane. The results showed that the sperm motility, plasma membrane integrity and acrosome integrity were significantly higher in the group of 50 渭 M resveratrol than those in the other groups, and the sperm quality was significantly higher than that in the 0 渭 M group. The sperm parameters increased with the increase of resveratrol concentration in a concentration-dependent manner, but the sperm quality decreased significantly when the concentration of resveratrol was too high (鈮，

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