氧自由基—线粒体信号通路在少、弱精子症发病中的机制研究
发布时间：2018-07-26 16:24
【摘要】：背景和目的 特发性少精子症和弱精子症是男性不育症的常见类型,二者合并发生又称为特发性少弱精子症。三者发病率分别占男性不育的15%、30%和15.1%。虽然国内外学者在其发病机制方面进行了大量研究,但其确切机制至今仍未阐明。目前在特发性少、弱精子症的发病学说中,基因调控异常中的凋亡学说和环境因素中的自由基损伤学说是研究的热点。其中线粒体信号通路是一条常见的凋亡通路,但是目前对于此通路与少、弱精子症关系的研究却不够深入:一方面大多停留在动物实验,通过有机化合物、高温、缺氧等诱导下制造少弱精子症模型,作睾丸组织或睾丸精子的检测,而缺乏来自人类精液的直接分析;另一方面还需要从分子层面上作进一步的探讨。此外有报道:25%~40%的不育男性患者精液中可以检出高水平的氧自由基即活性氧(ROS)。ROS对精子脂质膜、运动能力及DNA具有直接的损害作用,但是其是否作用于线粒体信号通路?目前尚未见报道。本研究通过分析少、弱及少弱精子症和正常对照组精液中氧自由基代谢产物MDA含量、抗氧化酶T-SOD及GSH-Px活性、线粒体通路中关键分子(Bcl-2、Bax、Cyt C和Caspase-3)在精子中的定位和表达等,探讨氧自由基-线粒体通路与少、弱精子症的关系。继而分别加入ROS、GSH及外源性Cyt C与精液共孵育后,研究其对氧自由基-线粒体通路部分指标的影响。最后采用化学合成的Bax-si RNA构建载体,筛选出有效干扰序列;转染小鼠睾丸支持细胞株TM4,观察Bax等线粒体通路基因表达的变化。材料和方法 第一部分:两次收集精液标本,分为正常对照组、少、弱及少弱精子症组(每次每组各30例)。应用TBA法测各组精浆MDA含量;比色法测精浆T-SOD和GSH-Px活性;流式细胞仪检测各组精子线粒体膜电位;应用计算机辅助精液分析系统(CASA)测精子各项运动参数;分别采用Real-time PCR、免疫细胞化学和Western blot法,检测各组精子中Bcl-2、Bax、Cyt C和Caspase-3的m RNA水平和蛋白表达。第二部分:收集正常精液30例,分为A组(对照组)、B组(加氧化酶反应系统)、C组(加氧化酶反应系统、终浓度为0.75 mmol/L的GSH)及D组(加氧化酶反应系统、终浓度为1.00 mmol/L的GSH)四组。经过2 h和12 h共孵育后,TBA法测各组MDA含量,比色法测T-SOD和GSH-Px活性,Real-time PCR法分别检测各组Bcl-2、Bax、Cyt C及Caspase-3的m RNA水平,以及通过CASA检测各组精子运动参数。第三部分:收集正常精液30例,分为A1组(对照组)、B1(加终浓度9.375mg/L外源性Cyt C)、C1(加终浓度18.75 mg/L外源性Cyt C)和D1组(加终浓度37.50 mg/L外源性Cyt C)四组;收集弱精子症精液30例,分为A2组(对照组)、B2(加终浓度9.375 mg/L外源性Cyt C)、C2(加终浓度18.75 mg/L外源性Cyt C)、D2(加终浓度37.50 mg/L外源性Cyt C)四组。经过2 h和12 h共孵育后,TBA法测各组MDA含量,比色法测T-SOD及GSH-Px活性,通过Real-time PCR法,分别检测各组中Caspase-3的m RNA水平,以及通过CASA检测各组精子运动参数。第四部分:针对小鼠Bax m RNA靶序列设计四段不同的si RNA序列,构建载体,转化感受态细胞,继而转染TM4细胞,设立空白对照组(未转染)、阴性对照组、Bax-si RNA(167)、Bax-si RNA(283)、Bax-si RNA(408)、Bax-si RNA(514)组。采用Real-time PCR技术检测转染后各组细胞Bax m RNA的表达,确定Bax-si RNA(167)、Bax-si RNA(283)为Bax-si RNA的有效干扰序列。针对该两序列,通过转染TM4细胞,分别作用48 h和72 h后,Real-time PCR法测定Bax及Bcl-2、Cyt C、Caspase-3基因m RNA表达的变化。结果 第一部分1.与正常对照组相比,少、弱及少弱精子症组精浆MDA含量显著增加,T-SOD及GSH-Px活性显著降低(分别为P0.05、P0.01及P0.05)。其中弱精子症组最为显著。2.与正常对照组相比,少、弱及少弱精子症组精子的JC-1+%显著降低(均P0.01)。但三组之间无显著性差异。3.与正常对照组相比,除了少精子症组VSL、VCL及VAP无显著性差异外,其它各组各项运动参数水平均显著下降(P0.05)。4.与正常对照组相比,少、弱及少弱精子症组精子中Bcl-2 m RNA相对表达量均显著降低,Bax、Cyt C和Caspase-3 m RNA均显著增高(P0.05);少精子症组Cyt C和Caspase-3 m RNA相对表达量比弱精子症组明显增高(P0.05)。5.与正常对照组比较,Bcl-2蛋白在少、弱及少弱精子症组中的表达量均显著降低,Bax、Cyt C及Caspase-3蛋白表达量均显著增加(P0.05);与弱精子症组比较,Cyt C及Caspase-3蛋白在少精子症组中的表达量显著增高(P0.05)。第二部分1.与A组比较,B组、C组及D组MDA含量增高,T-SOD及GSH-Px活力降低(分别为P0.01,P0.05);与B组比较,C组及D组MDA含量降低,T-SOD及GSH-Px活力增高(分别为P0.05,P0.01)。2.与A组比较,B组、C组精子各项运动参数水平降低(P0.01);与B组比较,C组、D组精子各项运动参数水平增高(P0.05)。3.精子Bcl-2相对表达量:与A组比较,B组、C组降低(分别为P0.01,P0.05);D组降低,但无显著性差异。精子Bax、Cyt C及Caspase-3相对表达量:与A组比较,B组、C组增高(分别为P0.01,P0.05);D组增高,但无显著性差异。与B组比较,D组相对表达量降低(P0.05)。第三部分 1.与A1组比较,B1、C1、D1组MDA含量逐渐降低,T-SOD及GSH-Px活力逐渐增高,D1组差异显著(P0.05)。与A2组比较,B2、C2、D2组MDA含量逐渐降低,T-SOD及GSH-Px活力逐渐增高,C2、D2组差异显著(分别为P0.05,P0.01)。2.与A1组比较,B1组、C1组、D1组精子各项运动参数呈现逐渐增高的趋势,但无显著性差异。与A2组比较,B2组、C2组、D2组精子各项运动参数呈逐渐增高。C2组、D2组精子总活力及快速前向运动显著增高(P0.05)。3.与A1组比较,B1组精子Caspase-3相对表达量增高,但无显著性差异;C1组、D1组增高,差异显著(分别为P0.01,P0.05)。与A2组比较,B2组、C2组增高,但无显著性差异;D2组增高,差异具有统计学意义(P0.05)。第四部分 1.两种Bax-si RNA转染TM4细胞48 h和72 h后,与空白对照组比较,Bax、Cyt C、Caspase-3基因m RNA表达水平显著下降,Bcl-2表达水平明显升高(P0.05),而阴性对照组无明显改变。2.设计的干扰序列Bax-si RNA2(283)对目的基因Bax及线粒体通路其它基因(Bcl-2、Cyt C、Caspase-3)的干扰效果优于干扰序列Bax-si RNA1(167);转染48 h的干扰效果优于转染72 h。结论 1.特发性少、弱及少弱精子症的精液中均检测出氧自由基的增高和线粒体信号通路分子的异常。少精子症侧重于凋亡,弱精子症侧重于氧化损伤。两者存在Cyt C分子的差异。2.ROS和GSH对于精子分别具有损害和保护作用,尤其体现在运动参数的变化。ROS能够影响精子氧自由基-线粒体通路部分参数,GSH直接对抗ROS,并且呈现出一定的量效关系。3.外源性Cyt C在正常及弱精子症精液中均具有抗氧化作用,并在一定程度上提高弱精子症的精子活力。有利于弱精子症的改善。4.本实验成功筛选出针对Bax m RNA靶序列的有效干扰序列,转染TM4细胞后,能够有效干扰Bax等线粒体通路基因m RNA的表达,其中Bax-si RNA(283)转染48 h的干扰效果最好。
[Abstract]:Background and objective idiopathic oligozoospermia and asthenospermia are common types of male infertility. The combination of the two is called idiopathic oligoasthenospermia. The incidence of the three is 15%, 30% and 15.1%. of male infertility, respectively, although domestic and foreign scholars have done a large study on its pathogenesis, but the exact mechanism has still not been elucidated. In the pathogenesis of idiopathic oligospermia and asthenospermia, the theory of apoptosis in abnormal gene regulation and the theory of free radical damage in environmental factors are the focus of research. The mitochondrial signaling pathway is a common apoptosis pathway, but the research on the relationship between this pathway and the oligozoospermia is not deep enough: one aspect is big. More stay in animal experiments, using organic compounds, high temperature, and hypoxia to produce oligozoospermia model, test for testicular tissue or testicular sperm, but lack direct analysis from human semen; on the other hand, it needs to be further discussed at the molecular level. In addition, it is reported in the semen of 25%~40% male infertility patients. It can be detected that high levels of oxygen free radicals (ROS).ROS have direct damage to the sperm lipid membrane, exercise ability and DNA. However, it has not been reported whether it acts on the mitochondrial signaling pathway. This study has been used to analyze the MDA content of oxygen free radical metabolites in the semen of the weak and asthenospermia and the normal control group. The activity of antioxidant enzyme T-SOD and GSH-Px, the localization and expression of key molecules of mitochondrial pathway (Bcl-2, Bax, Cyt C and Caspase-3) in sperm, and the relationship between oxygen free radical mitochondrial pathway and oligozoospermia. After adding ROS, GSH and exogenous Cyt C and spermatozoa, the oxygen free radical mitochondrial pathway part was studied. In the end, the effective interference sequence was screened by Bax-si RNA, which was synthesized by chemical synthesis. Transfection of mouse testis support cell line TM4 and observing the changes of mitochondrial pathway gene expression of Bax. Part 1: two collection of semen specimens, divided into normal control group, less, weak and less asthenospermia group (each group) The content of MDA in seminal plasma of each group was measured by TBA; the activity of T-SOD and GSH-Px in seminal plasma was measured by colorimetric method; the mitochondrial membrane potential of sperm in each group was measured by flow cytometry; the motility parameters of sperm were measured by computer aided semen analysis system (CASA); Real-time PCR, immunocytochemistry and Western blot method were used to detect Bcl- of sperm in each group. 2, the m RNA level and protein expression of Bax, Cyt C and Caspase-3. Part second: collect 30 normal semen, divide into A group (control group), B group (adding oxidase reaction system), C group (adding oxidase reaction system, final concentration of 0.75 mmol/L GSH) and four groups of D group (adding oxidase reverse system, 1 final concentration 1). After that, the content of MDA was measured by TBA and the activity of T-SOD and GSH-Px was measured by colorimetric method. The Real-time PCR method was used to detect the level of Bcl-2, Bax, Cyt C and Caspase-3, and the sperm motility parameters were detected in each group. The third part: 30 cases of normal semen were collected. Four groups (18.75 mg/L exogenous Cyt C) and D1 group (adding terminal concentration 37.50 mg/L exogenous Cyt C), and 30 cases of semen of asthenospermia, divided into A2 group (control group), B2 (plus final concentration 9.375 mg/L exogenous Cyt C), four group (plus final concentration 37.50 exogenous exogenous), after 2 and 12 The content of MDA and the activity of T-SOD and GSH-Px were measured by colorimetric method. The m RNA level of Caspase-3 in each group was detected by Real-time PCR method, and the sperm motility parameters were detected by CASA. The fourth part: to construct the carrier, transform the receptive cells, and then transfect the transfection fine to the mouse Bax m RNA target sequence. Cell, set up blank control group (untransfected), negative control group, Bax-si RNA (167), Bax-si RNA (283), Bax-si RNA (408), Bax-si RNA (514). The expression of Bax m (167) was detected by Real-time PCR technique, and the effective interference sequence was determined (167), and the two sequence was transfected by transfection. Cells, after 48 h and 72 h respectively, Real-time PCR method was used to determine the changes in Bax and Bcl-2, Cyt C, Caspase-3 gene m RNA expression. Results the first part 1. compared with the normal control group, less, weak and less asthenospermia group significantly decreased in sperm pulp MDA content. Compared with the normal control group, the JC-1+% of sperm in the less and less asthenospermia group decreased significantly (P0.01). But there was no significant difference between the three groups. Compared with the normal control group, there was no significant difference between the normal control group and the normal control group, except the VSL, VCL and VAP in the oligozoospermia group, and the levels of the transport parameters in all the other groups decreased significantly (P0.05).4. and the normal control group. The relative expression of Bcl-2 m RNA in spermatozoa of less and less asthenospermia group decreased significantly, Bax, Cyt C and Caspase-3 m RNA increased significantly (P0.05), and the relative expression of Cyt C in oligozoospermia group was significantly higher than that of the asthenospermia group. The expression of Bax, Cyt C and Caspase-3 increased significantly (P0.05). Compared with the asthenospermia group, the expression of Cyt C and Caspase-3 protein in the oligozoospermia group was significantly increased (P0.05). The second part was compared with the A group, B group, C group and decreased vitality (respectively); Compared with group B, the content of MDA in group C and D group decreased, and the activity of T-SOD and GSH-Px increased (P0.05, P0.01) in.2. and A groups, B group, and the level of sperm movement parameters in C group decreased. P0.05); D group decreased, but there was no significant difference. Sperm Bax, Cyt C and Caspase-3 relative expression: compared with A group, B group, C group increased (P0.01, P0.05), D group increased, but there was no significant difference. As compared with group A2, the content of MDA in B2, C2 and D2 group was gradually decreased, T-SOD and GSH-Px activity increased gradually, and the difference of T-SOD and GSH-Px in the group of B2, C2 and D2 was gradually higher than that of the group A2. The motility parameters of group sperm increased gradually in.C2 group, and the total activity of sperm and rapid forward movement increased significantly in group D2 (P0.05). Compared with group A1, the relative expression of Caspase-3 in group B1 was higher, but no significant difference was found in group B1, but in C1 group and D1 group, the difference was significant (P0.01, P0.05). In group D2, the difference was statistically significant (P0.05). Fourth part 1., two kinds of Bax-si RNA transfected to TM4 cells 48 h and 72 h, compared with the blank control group, Bax, Cyt C, Caspase-3 gene expressed a significant decrease in the expression level, and the negative control group had no significant changes in the interference sequence of the design. (283) The interference effect on the target gene Bax and the other genes of the mitochondrial pathway (Bcl-2, Cyt C, Caspase-3) was better than the interference sequence Bax-si RNA1 (167); the interference effect of the transfection of 48 h was better than that of the transfection of 72 h. CONCLUSION 1., and the increase of oxygen free radicals and the abnormality of the mitochondrial signaling pathway were detected in the semen of weak and oligozoospermia. Children's disease focuses on apoptosis, and asthenospermia focuses on oxidative damage. There is a difference between Cyt and C molecules:.2.ROS and GSH have damage and protection to spermatozoa respectively, especially in the change of movement parameters,.ROS can affect the parameters of sperm oxygen free radical mitochondrial pathway, GSH directly antagonism ROS, and presents a certain dose effect. .3. exogenous Cyt C has antioxidant effect in both normal and asthenospermia semen, and improves sperm motility of asthenospermia to a certain extent. It is beneficial to the improvement of asthenospermia. The effective interference sequence of Bax m RNA target sequence is successfully screened by.4. experiment. After transfection of TM4 cells, it can effectively interfere with the mitochondrial pathway of Bax and so on. Because of the expression of M RNA, the interference effect of Bax-si RNA (283) transfected with 48 h was the best.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R698.2
本文编号：2146588
[Abstract]:Background and objective idiopathic oligozoospermia and asthenospermia are common types of male infertility. The combination of the two is called idiopathic oligoasthenospermia. The incidence of the three is 15%, 30% and 15.1%. of male infertility, respectively, although domestic and foreign scholars have done a large study on its pathogenesis, but the exact mechanism has still not been elucidated. In the pathogenesis of idiopathic oligospermia and asthenospermia, the theory of apoptosis in abnormal gene regulation and the theory of free radical damage in environmental factors are the focus of research. The mitochondrial signaling pathway is a common apoptosis pathway, but the research on the relationship between this pathway and the oligozoospermia is not deep enough: one aspect is big. More stay in animal experiments, using organic compounds, high temperature, and hypoxia to produce oligozoospermia model, test for testicular tissue or testicular sperm, but lack direct analysis from human semen; on the other hand, it needs to be further discussed at the molecular level. In addition, it is reported in the semen of 25%~40% male infertility patients. It can be detected that high levels of oxygen free radicals (ROS).ROS have direct damage to the sperm lipid membrane, exercise ability and DNA. However, it has not been reported whether it acts on the mitochondrial signaling pathway. This study has been used to analyze the MDA content of oxygen free radical metabolites in the semen of the weak and asthenospermia and the normal control group. The activity of antioxidant enzyme T-SOD and GSH-Px, the localization and expression of key molecules of mitochondrial pathway (Bcl-2, Bax, Cyt C and Caspase-3) in sperm, and the relationship between oxygen free radical mitochondrial pathway and oligozoospermia. After adding ROS, GSH and exogenous Cyt C and spermatozoa, the oxygen free radical mitochondrial pathway part was studied. In the end, the effective interference sequence was screened by Bax-si RNA, which was synthesized by chemical synthesis. Transfection of mouse testis support cell line TM4 and observing the changes of mitochondrial pathway gene expression of Bax. Part 1: two collection of semen specimens, divided into normal control group, less, weak and less asthenospermia group (each group) The content of MDA in seminal plasma of each group was measured by TBA; the activity of T-SOD and GSH-Px in seminal plasma was measured by colorimetric method; the mitochondrial membrane potential of sperm in each group was measured by flow cytometry; the motility parameters of sperm were measured by computer aided semen analysis system (CASA); Real-time PCR, immunocytochemistry and Western blot method were used to detect Bcl- of sperm in each group. 2, the m RNA level and protein expression of Bax, Cyt C and Caspase-3. Part second: collect 30 normal semen, divide into A group (control group), B group (adding oxidase reaction system), C group (adding oxidase reaction system, final concentration of 0.75 mmol/L GSH) and four groups of D group (adding oxidase reverse system, 1 final concentration 1). After that, the content of MDA was measured by TBA and the activity of T-SOD and GSH-Px was measured by colorimetric method. The Real-time PCR method was used to detect the level of Bcl-2, Bax, Cyt C and Caspase-3, and the sperm motility parameters were detected in each group. The third part: 30 cases of normal semen were collected. Four groups (18.75 mg/L exogenous Cyt C) and D1 group (adding terminal concentration 37.50 mg/L exogenous Cyt C), and 30 cases of semen of asthenospermia, divided into A2 group (control group), B2 (plus final concentration 9.375 mg/L exogenous Cyt C), four group (plus final concentration 37.50 exogenous exogenous), after 2 and 12 The content of MDA and the activity of T-SOD and GSH-Px were measured by colorimetric method. The m RNA level of Caspase-3 in each group was detected by Real-time PCR method, and the sperm motility parameters were detected by CASA. The fourth part: to construct the carrier, transform the receptive cells, and then transfect the transfection fine to the mouse Bax m RNA target sequence. Cell, set up blank control group (untransfected), negative control group, Bax-si RNA (167), Bax-si RNA (283), Bax-si RNA (408), Bax-si RNA (514). The expression of Bax m (167) was detected by Real-time PCR technique, and the effective interference sequence was determined (167), and the two sequence was transfected by transfection. Cells, after 48 h and 72 h respectively, Real-time PCR method was used to determine the changes in Bax and Bcl-2, Cyt C, Caspase-3 gene m RNA expression. Results the first part 1. compared with the normal control group, less, weak and less asthenospermia group significantly decreased in sperm pulp MDA content. Compared with the normal control group, the JC-1+% of sperm in the less and less asthenospermia group decreased significantly (P0.01). But there was no significant difference between the three groups. Compared with the normal control group, there was no significant difference between the normal control group and the normal control group, except the VSL, VCL and VAP in the oligozoospermia group, and the levels of the transport parameters in all the other groups decreased significantly (P0.05).4. and the normal control group. The relative expression of Bcl-2 m RNA in spermatozoa of less and less asthenospermia group decreased significantly, Bax, Cyt C and Caspase-3 m RNA increased significantly (P0.05), and the relative expression of Cyt C in oligozoospermia group was significantly higher than that of the asthenospermia group. The expression of Bax, Cyt C and Caspase-3 increased significantly (P0.05). Compared with the asthenospermia group, the expression of Cyt C and Caspase-3 protein in the oligozoospermia group was significantly increased (P0.05). The second part was compared with the A group, B group, C group and decreased vitality (respectively); Compared with group B, the content of MDA in group C and D group decreased, and the activity of T-SOD and GSH-Px increased (P0.05, P0.01) in.2. and A groups, B group, and the level of sperm movement parameters in C group decreased. P0.05); D group decreased, but there was no significant difference. Sperm Bax, Cyt C and Caspase-3 relative expression: compared with A group, B group, C group increased (P0.01, P0.05), D group increased, but there was no significant difference. As compared with group A2, the content of MDA in B2, C2 and D2 group was gradually decreased, T-SOD and GSH-Px activity increased gradually, and the difference of T-SOD and GSH-Px in the group of B2, C2 and D2 was gradually higher than that of the group A2. The motility parameters of group sperm increased gradually in.C2 group, and the total activity of sperm and rapid forward movement increased significantly in group D2 (P0.05). Compared with group A1, the relative expression of Caspase-3 in group B1 was higher, but no significant difference was found in group B1, but in C1 group and D1 group, the difference was significant (P0.01, P0.05). In group D2, the difference was statistically significant (P0.05). Fourth part 1., two kinds of Bax-si RNA transfected to TM4 cells 48 h and 72 h, compared with the blank control group, Bax, Cyt C, Caspase-3 gene expressed a significant decrease in the expression level, and the negative control group had no significant changes in the interference sequence of the design. (283) The interference effect on the target gene Bax and the other genes of the mitochondrial pathway (Bcl-2, Cyt C, Caspase-3) was better than the interference sequence Bax-si RNA1 (167); the interference effect of the transfection of 48 h was better than that of the transfection of 72 h. CONCLUSION 1., and the increase of oxygen free radicals and the abnormality of the mitochondrial signaling pathway were detected in the semen of weak and oligozoospermia. Children's disease focuses on apoptosis, and asthenospermia focuses on oxidative damage. There is a difference between Cyt and C molecules:.2.ROS and GSH have damage and protection to spermatozoa respectively, especially in the change of movement parameters,.ROS can affect the parameters of sperm oxygen free radical mitochondrial pathway, GSH directly antagonism ROS, and presents a certain dose effect. .3. exogenous Cyt C has antioxidant effect in both normal and asthenospermia semen, and improves sperm motility of asthenospermia to a certain extent. It is beneficial to the improvement of asthenospermia. The effective interference sequence of Bax m RNA target sequence is successfully screened by.4. experiment. After transfection of TM4 cells, it can effectively interfere with the mitochondrial pathway of Bax and so on. Because of the expression of M RNA, the interference effect of Bax-si RNA (283) transfected with 48 h was the best.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R698.2
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