精子特异性钙通道CatSper1在精索静脉曲张机体精子中的表达及作用研究

发布时间：2018-06-16 10:11

本文选题：精索静脉曲张 + 精子特异性钙通道　；参考：《青岛大学》2016年博士论文

【摘要】：第一章精索静脉曲张患者精子Cat Sper1的差异表达目的:通过检测精索静脉曲张患者成熟精子特异性钙通道Cat Sper1的表达,探讨Cat Sper1与精索静脉曲张之间的关系,为精索静脉曲张导致不育的发病机制提供新的研究方向。方法:健康志愿者20例,精索静脉曲张患者110例。通过手淫法获取精液,进行计算机精子运动轨迹分析,应用Percoll非连续梯度离心法对精液进行优化分离,应用半定量逆转录酶-聚合酶链式反应(RT-PCR)以及蛋白质印迹法(Western Blot)分别检测精子中Cat Sper1 m RNA及蛋白表达情况。结果:与对照组比较,精索静脉曲张组精液量、液化时间无明显差异(P0.05),而精子快速前向运动以及前向运动比率(a+b(%))、精子活率以及密度均明显降低,差异具有统计学意义(P0.01)。根据精子运动活力分组,精索静脉曲张精子活力异常组(3组)Cat Sper1 m RNA及蛋白表达含量均低于对照组(1组)以及精索静脉曲张精子活力正常组(2组)(P0.01)。与1组比较,2组精子Cat Sper1 m RNA及蛋白表达轻微下降,但差异无统计学意义(P0.05)。根据精索静脉曲张临床程度分组,精索静脉曲张临床I、II、III度各组精子Cat Sper1 m RNA及蛋白表达含量差异不明显(P0.05)。精索静脉曲张单侧发病与双侧发病精子Cat Sper1 m RNA及蛋白表达含量差异不明显(P0.05)。根据精索静脉曲张患者静脉是否存在返流分组,返流组精子Cat Sper1 m RNA及蛋白表达含量均低于对照组以及无返流组(P0.01)。与对照组比较,无返流组精子Cat Sper1 m RNA及蛋白表达差异不明显(P0.05)。结论:精子特异性钙通道Cat Sper1 m RNA及蛋白在精索静脉曲张患者精子细胞中表达下降,其可能为精索静脉曲张导致不育的“二次打击”靶点之一。精子活力低下以及静脉血液返流与Cat Sper1的表达下降存在一定相关性,这将为精索静脉曲张的诊断及治疗提供新的思路。第二章CatSper1在实验性精索静脉曲张大鼠精子中的差异表达及左卡尼汀对其的保护性研究目的:检测精索静脉曲张大鼠附睾精子细胞精子特异性钙通道Cat Sper1的表达情况,观察左卡尼汀对大鼠精子Cat Sper1的影响。方法:70只雄性大鼠被随机分为7组,每组10只。分别为对照组(A组),精索静脉曲张组(B组),精索静脉曲张+生理盐水组(C组),精索静脉曲张+小、中、大剂量左卡尼汀组(D,E,F组)和F+延长喂养14d组(G组)。手术结扎部分左肾静脉制备精索静脉曲张大鼠模型。12周后,C组给予生理盐水1 ml/d,D、E、F组分别用左卡尼汀小(0.05g·kg-1·d-1)、中(0.1 g·kg-1·d-1)、大(0.2 g·kg-1·d-1)剂量灌胃35d,G组在F组基础上延长灌胃14d,处死大鼠。进行精液参数精子运动轨迹分析,应用RT-PCR以及Western Blot分别检测精液中Cat Sper1 m RNA及蛋白表达情况。结果:与A组比较,B组精子a+b(%)、精子活率及密度均不同程度下降(P0.01),B组精子Cat Sper1 m RNA及蛋白表达含量明显下降(1.44±0.67 VS 0.71±0.38;1.87±0.67 VS 0.84±0.42,P0.01)。与C组比较,应用左卡尼汀后,各组精子密度无明显增加,精子a+b(%)、精子活率以及精子Cat Sper1 m RNA及蛋白表达含量明显增高,尤其大剂量组F、G组增高更加明显(P0.01)。与F组比较,G组延长应用2周左卡尼汀,精子Cat Sper1 m RNA及蛋白表达量无明显增加(P0.05)。结论:精子特异性钙通道Cat Sper1在精索静脉曲张大鼠精子细胞中表达下降。应用左卡尼汀后,Cat Sper1的表达含量及精子a+b(%)及精子活率增加,从而提高精索静脉曲张患者的生育能力。第三章Cat Sper1对精子线粒体呼吸与能量代谢的影响目的:应用Cat Sper1多克隆抗体抑制Cat Sper1的功能,检测精子线粒体呼吸功能及能量合成能力,观察Cat Sper1对精子线粒体呼吸与能量代谢的影响。方法:健康志愿者20例,手淫法获取精液,检测精液参数均符合WHO标准。经过上游法处理后每份精液分为A、B两组,分别与Earles液以及50μg/ml抗Cat Sper1多克隆抗体共孵育。在1h,2h,4h后分别检测两组精子精液参数、细胞线粒体呼吸控制率RCR及精子细胞ATP含量。结果:与A组比较,B组精子各时间点a+b(%)尤其是a(%)均明显下降(P0.01);1h后B组精子a(%)即明显下降,2h,4h后精子下降缓慢,与1h相比无明显差异(P0.05)。与A组比较,B组精子各时间点态3值、呼吸控制率RCR以及精子细胞ATP含量均明显下降(P0.01);B组精子中,2h、4h节点精子态3值、呼吸控制率RCR以及精子细胞ATP含量与1h无明显差异(P0.05)。结论:阻断精子特异性钙通道Cat Sper1,精子细胞线粒体呼吸功能降低,精子生成ATP的能力下降,精子活力降低。这将为精索静脉曲张引起精子Cat Sper1表达下降,从而导致不育的机制寻找可能的理论依据。
[Abstract]:Chapter 1 the differential expression of Cat Sper1 in spermatozoa in patients with varicocele Objective: To explore the relationship between Cat Sper1 and varicocele by detecting the expression of specific calcium channel Cat Sper1 of spermatozoa in patients with varicocele, and to provide a new direction for the pathogenesis of sterility caused by varicocele. Method: healthy volunteers In 20 cases and 110 cases of varicocele, spermatozoa were obtained by masturbation, and the sperm movement trajectory was analyzed. The semen was optimized by Percoll discontinuous gradient centrifugation. Semi quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and protein imprinting (Western Blot) were used to detect Cat Sper1 in sperm respectively. M RNA and protein expression. Results: compared with the control group, there was no significant difference in semen volume and liquefaction time in the varicocele group (P0.05), but the rate of sperm motility and forward movement (a+b (%)), sperm motility and density decreased significantly (P0.01). According to sperm motility group, spermatic vein The expression of Cat Sper1 m RNA and protein in the abnormal sperm motility group (3 groups) were lower than the control group (1 groups) and the normal group of spermatic vein varicose sperm (2 groups) (P0.01). Compared with the 1 group, the expression of Cat Sper1 m RNA and protein expression in the 2 groups decreased slightly, but the difference was not significant (P0.05). According to the clinical degree of varicocele, the group of spermatozoa was divided into groups, The difference of Cat Sper1 m RNA and protein expression in spermatozoa was not significant (P0.05) in I, II, and III degree of spermatic varicocele. The difference of M RNA and protein expression in spermatic varicocele unilateral and bilateral sperm Cat Sper1 m RNA and protein expression was not significant. The expression of R1 m RNA and protein were lower than that in the control group and no reflux group (P0.01). Compared with the control group, there was no significant difference in the expression of Cat Sper1 m RNA and protein in the non reflux group (P0.05). Conclusion: sperm specific calcium channel Cat Sper1 m and protein expression in spermatozoa of patients with varicocele may be spermatic vein One of the "two hits" target of infertility is one of the targets of "two hits". There is a certain correlation between low sperm motility and venous blood reflux and the decrease of expression of Cat Sper1. This will provide new ideas for the diagnosis and treatment of varicocele. The differential expression of CatSper1 in experimental spermatic varicocele spermatozoa and Levocarnitine in experimental spermatic vein second The purpose of the study was to detect the expression of sperm specific calcium channel Cat Sper1 in the epididymal spermatozoa of spermatic varicocele rats, and to observe the effect of levocarnitine on Cat Sper1 in rat sperm. Methods: 70 male rats were randomly divided into 7 groups, 10 in each group, respectively, in the control group (group A), the varicocele group (group B) and spermatic cord static. Pulse varicose + physiological saline group (group C), varicocele + small, medium, high dose levocarnitine group (D, E, F) and F+ lengthening 14d group (group G). After ligating part of the left renal vein to prepare the varicocele rat model of the left renal vein for.12 weeks, group C was given 1 ml/d, D, E. ), large (0.2 g. Kg-1. D-1) dose gavage 35d. Group G extended 14d in group F on the basis of group F and killed rats. The sperm motility trajectory of semen was analyzed. RT-PCR and Western Blot were used to detect Cat Sper1 and protein expression in semen. (P0.01) the expression of Cat Sper1 m RNA and protein in group B decreased significantly (1.44 + 0.67 VS 0.71 + 0.38; 1.87 + 0.67 VS 0.84 + 0.42, P0.01). Compared with the C group, the sperm density was not significantly increased, sperm a+b (%), sperm viability, and spermatozoon Cat and protein expression levels were significantly increased, especially in large dose groups The increase in the G group was more obvious (P0.01). Compared with the F group, the expression of Cat Sper1 m RNA and protein in the sperm was not significantly increased in the G group for 2 weeks. Conclusion: the expression of the sperm specific calcium channel Cat Sper1 in the spermatozoa of the varicocele rat was decreased. The sperm viability increased, thus improving the fertility of the patients with varicocele. Third the effects of Cat Sper1 on the mitochondrial respiration and energy metabolism of spermatozoa aim: to use the Cat Sper1 polyclonal antibody to inhibit the function of Cat Sper1, to detect the mitochondrial respiratory function and energy synthesis ability, and to observe the mitochondrial respiration and energy of the sperm of Cat Sper1. Methods: 20 healthy volunteers, 20 cases of masturbation, the semen were obtained and the semen parameters were all conformed to the standard of WHO. After the upstream process, each semen was divided into A, B two, and Earles solution and 50 mu g/ml anti Cat Sper1 polyclonal antibody respectively. After 1h, 2h and 4H respectively, the parameters of sperm semen were detected in two groups, and the cell mitochondrial respiration was detected respectively. Control rate RCR and sperm cell ATP content. Results: compared with group A, a+b (%), especially a (%) in each time point of group B decreased significantly (P0.01), and a (%) of sperm in group B decreased obviously after 1h, 2h, and the sperm decreased slowly after 4H. Compared with the group, the 3 value of each time point of sperm, respiratory control rate and sperm cells were compared with those of the group. The content of TP decreased significantly (P0.01), in group B, the sperm state of 2h, 4H node 3, respiratory control rate RCR and sperm cell ATP content were not significantly different from 1H (P0.05). Conclusion: blocking spermatozoa specific calcium channel Cat Sper1, sperm cell mitochondrial respiratory function decreased, spermatogenesis ATP capacity decreased, sperm vitality decreased. This will be spermatocele. The mechanism of Cat Sper1 expression in the spermatozoa decreased, resulting in the mechanism of infertility.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R697.24

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