氧自由基—線粒體信號通路在少、弱精子癥發(fā)病中的機制研究
發(fā)布時間：2018-07-26 16:24
【摘要】：背景和目的 特發(fā)性少精子癥和弱精子癥是男性不育癥的常見類型,二者合并發(fā)生又稱為特發(fā)性少弱精子癥。三者發(fā)病率分別占男性不育的15%、30%和15.1%。雖然國內外學者在其發(fā)病機制方面進行了大量研究,但其確切機制至今仍未闡明。目前在特發(fā)性少、弱精子癥的發(fā)病學說中,基因調控異常中的凋亡學說和環(huán)境因素中的自由基損傷學說是研究的熱點。其中線粒體信號通路是一條常見的凋亡通路,但是目前對于此通路與少、弱精子癥關系的研究卻不夠深入:一方面大多停留在動物實驗,通過有機化合物、高溫、缺氧等誘導下制造少弱精子癥模型,作睪丸組織或睪丸精子的檢測,而缺乏來自人類精液的直接分析;另一方面還需要從分子層面上作進一步的探討。此外有報道:25%~40%的不育男性患者精液中可以檢出高水平的氧自由基即活性氧(ROS)。ROS對精子脂質膜、運動能力及DNA具有直接的損害作用,但是其是否作用于線粒體信號通路?目前尚未見報道。本研究通過分析少、弱及少弱精子癥和正常對照組精液中氧自由基代謝產物MDA含量、抗氧化酶T-SOD及GSH-Px活性、線粒體通路中關鍵分子(Bcl-2、Bax、Cyt C和Caspase-3)在精子中的定位和表達等,探討氧自由基-線粒體通路與少、弱精子癥的關系。繼而分別加入ROS、GSH及外源性Cyt C與精液共孵育后,研究其對氧自由基-線粒體通路部分指標的影響。最后采用化學合成的Bax-si RNA構建載體,篩選出有效干擾序列;轉染小鼠睪丸支持細胞株TM4,觀察Bax等線粒體通路基因表達的變化。材料和方法 第一部分:兩次收集精液標本,分為正常對照組、少、弱及少弱精子癥組(每次每組各30例)。應用TBA法測各組精漿MDA含量;比色法測精漿T-SOD和GSH-Px活性;流式細胞儀檢測各組精子線粒體膜電位;應用計算機輔助精液分析系統(tǒng)(CASA)測精子各項運動參數(shù);分別采用Real-time PCR、免疫細胞化學和Western blot法,檢測各組精子中Bcl-2、Bax、Cyt C和Caspase-3的m RNA水平和蛋白表達。第二部分:收集正常精液30例,分為A組(對照組)、B組(加氧化酶反應系統(tǒng))、C組(加氧化酶反應系統(tǒng)、終濃度為0.75 mmol/L的GSH)及D組(加氧化酶反應系統(tǒng)、終濃度為1.00 mmol/L的GSH)四組。經過2 h和12 h共孵育后,TBA法測各組MDA含量,比色法測T-SOD和GSH-Px活性,Real-time PCR法分別檢測各組Bcl-2、Bax、Cyt C及Caspase-3的m RNA水平,以及通過CASA檢測各組精子運動參數(shù)。第三部分:收集正常精液30例,分為A1組(對照組)、B1(加終濃度9.375mg/L外源性Cyt C)、C1(加終濃度18.75 mg/L外源性Cyt C)和D1組(加終濃度37.50 mg/L外源性Cyt C)四組;收集弱精子癥精液30例,分為A2組(對照組)、B2(加終濃度9.375 mg/L外源性Cyt C)、C2(加終濃度18.75 mg/L外源性Cyt C)、D2(加終濃度37.50 mg/L外源性Cyt C)四組。經過2 h和12 h共孵育后,TBA法測各組MDA含量,比色法測T-SOD及GSH-Px活性,通過Real-time PCR法,分別檢測各組中Caspase-3的m RNA水平,以及通過CASA檢測各組精子運動參數(shù)。第四部分:針對小鼠Bax m RNA靶序列設計四段不同的si RNA序列,構建載體,轉化感受態(tài)細胞,繼而轉染TM4細胞,設立空白對照組(未轉染)、陰性對照組、Bax-si RNA(167)、Bax-si RNA(283)、Bax-si RNA(408)、Bax-si RNA(514)組。采用Real-time PCR技術檢測轉染后各組細胞Bax m RNA的表達,確定Bax-si RNA(167)、Bax-si RNA(283)為Bax-si RNA的有效干擾序列。針對該兩序列,通過轉染TM4細胞,分別作用48 h和72 h后,Real-time PCR法測定Bax及Bcl-2、Cyt C、Caspase-3基因m RNA表達的變化。結果 第一部分1.與正常對照組相比,少、弱及少弱精子癥組精漿MDA含量顯著增加,T-SOD及GSH-Px活性顯著降低(分別為P0.05、P0.01及P0.05)。其中弱精子癥組最為顯著。2.與正常對照組相比,少、弱及少弱精子癥組精子的JC-1+%顯著降低(均P0.01)。但三組之間無顯著性差異。3.與正常對照組相比,除了少精子癥組VSL、VCL及VAP無顯著性差異外,其它各組各項運動參數(shù)水平均顯著下降(P0.05)。4.與正常對照組相比,少、弱及少弱精子癥組精子中Bcl-2 m RNA相對表達量均顯著降低,Bax、Cyt C和Caspase-3 m RNA均顯著增高(P0.05);少精子癥組Cyt C和Caspase-3 m RNA相對表達量比弱精子癥組明顯增高(P0.05)。5.與正常對照組比較,Bcl-2蛋白在少、弱及少弱精子癥組中的表達量均顯著降低,Bax、Cyt C及Caspase-3蛋白表達量均顯著增加(P0.05);與弱精子癥組比較,Cyt C及Caspase-3蛋白在少精子癥組中的表達量顯著增高(P0.05)。第二部分1.與A組比較,B組、C組及D組MDA含量增高,T-SOD及GSH-Px活力降低(分別為P0.01,P0.05);與B組比較,C組及D組MDA含量降低,T-SOD及GSH-Px活力增高(分別為P0.05,P0.01)。2.與A組比較,B組、C組精子各項運動參數(shù)水平降低(P0.01);與B組比較,C組、D組精子各項運動參數(shù)水平增高(P0.05)。3.精子Bcl-2相對表達量:與A組比較,B組、C組降低(分別為P0.01,P0.05);D組降低,但無顯著性差異。精子Bax、Cyt C及Caspase-3相對表達量:與A組比較,B組、C組增高(分別為P0.01,P0.05);D組增高,但無顯著性差異。與B組比較,D組相對表達量降低(P0.05)。第三部分 1.與A1組比較,B1、C1、D1組MDA含量逐漸降低,T-SOD及GSH-Px活力逐漸增高,D1組差異顯著(P0.05)。與A2組比較,B2、C2、D2組MDA含量逐漸降低,T-SOD及GSH-Px活力逐漸增高,C2、D2組差異顯著(分別為P0.05,P0.01)。2.與A1組比較,B1組、C1組、D1組精子各項運動參數(shù)呈現(xiàn)逐漸增高的趨勢,但無顯著性差異。與A2組比較,B2組、C2組、D2組精子各項運動參數(shù)呈逐漸增高。C2組、D2組精子總活力及快速前向運動顯著增高(P0.05)。3.與A1組比較,B1組精子Caspase-3相對表達量增高,但無顯著性差異;C1組、D1組增高,差異顯著(分別為P0.01,P0.05)。與A2組比較,B2組、C2組增高,但無顯著性差異;D2組增高,差異具有統(tǒng)計學意義(P0.05)。第四部分 1.兩種Bax-si RNA轉染TM4細胞48 h和72 h后,與空白對照組比較,Bax、Cyt C、Caspase-3基因m RNA表達水平顯著下降,Bcl-2表達水平明顯升高(P0.05),而陰性對照組無明顯改變。2.設計的干擾序列Bax-si RNA2(283)對目的基因Bax及線粒體通路其它基因(Bcl-2、Cyt C、Caspase-3)的干擾效果優(yōu)于干擾序列Bax-si RNA1(167);轉染48 h的干擾效果優(yōu)于轉染72 h。結論 1.特發(fā)性少、弱及少弱精子癥的精液中均檢測出氧自由基的增高和線粒體信號通路分子的異常。少精子癥側重于凋亡,弱精子癥側重于氧化損傷。兩者存在Cyt C分子的差異。2.ROS和GSH對于精子分別具有損害和保護作用,尤其體現(xiàn)在運動參數(shù)的變化。ROS能夠影響精子氧自由基-線粒體通路部分參數(shù),GSH直接對抗ROS,并且呈現(xiàn)出一定的量效關系。3.外源性Cyt C在正常及弱精子癥精液中均具有抗氧化作用,并在一定程度上提高弱精子癥的精子活力。有利于弱精子癥的改善。4.本實驗成功篩選出針對Bax m RNA靶序列的有效干擾序列,轉染TM4細胞后,能夠有效干擾Bax等線粒體通路基因m RNA的表達,其中Bax-si RNA(283)轉染48 h的干擾效果最好。
[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.
【學位授予單位】：鄭州大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R698.2
本文編號：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.
【學位授予單位】：鄭州大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R698.2
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