香烟烟雾对雄性小鼠睾丸与附睾影响的蛋白质组学研究
发布时间:2019-07-03 17:37
【摘要】:研究背景与目的:香烟烟雾成分复杂,许多成分能够危害人体健康。流行病学调查显示,吸烟行为与男性的生育能力之间具有较高的相关性,其主要表现为精液中精子密度降低、精子运动能力减弱、精子形态异常、体外受精受孕率降低。这一现象直接导致了吸烟男性生育能力的降低,甚至因而影响到后代的健康。很多研究者试图找出吸烟行为降低男性生育能力的原因,包括彗星电泳法(comet)、末端脱氧核苷酸转移酶介导的dUTP-生物素缺口末端标记法(TUNEL)与精子染色体结构分析(SCSA)在内的各种方法证实了吸烟会导致精子DNA的断裂、染色体的结构异常以及染色体非整倍性的发生,也有研究显示吸烟会改变血清中激素的水平,从而影响精子的生成。 雄性生殖系统中,睾丸是精子生成的主要场所,而附睾是精子成熟的主要场所,这两个器官对精子与精液的质量具有非常重要的作用。我们的研究使用实验室先期建立的小鼠吸烟模型,运用蛋白质组学的方法来观察香烟烟雾处理后小鼠睾丸与附睾中蛋白质总体的表达情况,找出表达水平较没有使用香烟烟雾处理的小鼠睾丸与附睾中的相应蛋白质发生显著差异的蛋白质,结合生物信息学的方法分析这些蛋白质的功能以及它们与生育能力之间的关系,试图找出香烟烟雾处理对雄性生育能力造成影响的分子机制。 研究方法:七周龄雄性C57/BL6J小鼠每日用香烟烟雾处理两次,每次一小时,持续处理两周,对照组小鼠不用香烟烟雾处理。处理期满后取小鼠睾丸与附睾,分别提取总蛋白进行双向电泳,比较处理组小鼠与对照组小鼠相关组织的双向电泳结果,挑选两者间表达水平发生显著差异的蛋白质点切胶进行基质辅助激光解吸/电离飞行时间质谱分析,以鉴定出这些蛋白质点所代表的蛋白质。从质谱分析鉴定得到的蛋白质中随机取数个蛋白质,使用Western-blotting的方法对这些蛋白质在处理组小鼠与对照组小鼠相关组织中表达水平差异的趋势进行验证,以确定双向电泳与质谱分析的结果是否可靠,同时利用Real-time PCR的方法对这些蛋白相应的基因在相应器官中的转录水平进行检测,以观察这些基因在处理组与对照组小鼠之间转录水平的差异。 将得到验证的由双向电泳与质谱分析得到的香烟烟雾处理后小鼠睾丸与附睾组织中表达水平发生变化的蛋白质使用Blast2GO进行功能注释,同时从生物学过程、分子功能两个层次上对这些蛋白质进行基于基因本体论的归类。使用通路分析工具KEGG与IPA对这些蛋白质所涉及的通路进行分析,找出受到影响的主要通路与生物学过程。对找出的通路的下游作用标志物使用免疫组织化学的方法在组织水平上对组织切片进行检测,以验证通路分析得到的结果与推测。 研究结果:通过双向电泳,我们发现香烟烟雾处理后的小鼠睾丸与附睾组织中各自有超过1000个蛋白点的表达水平与对照组小鼠存在差异,其中睾丸组织中有27个蛋白点的表达水平差异在1.5倍以上,附睾组织中有52个蛋白点的表达水平差异在2倍以上,可以认为差异水平显著。挑选这些在香烟烟雾处理后的小鼠睾丸与附睾组织中表达水平差异显著的蛋白点进行质谱分析,睾丸与附睾组织中分别有27个蛋白点成功地得到了鉴定,它们的表达水平各自可以用来代表27种相应蛋白质的表达水平。在小鼠睾丸中,得到鉴定的27种蛋白质中的6种蛋白质表达水平上调,21种蛋白质表达水平下调;在小鼠附睾中,得到鉴定的27种蛋白质中的12种蛋白质表达水平上调,,15种蛋白质的表达水平下调。分别随机挑选睾丸组织中的5种蛋白质和附睾组织中的8种蛋白质,使用相应组织进行Western-blotting对双向电泳与质谱分析得到的结果进行验证,结果显示Western-blotting检测得到的处理组与对照组间表达差异的趋势与双向电泳和质谱分析得到的表达差异趋势基本一致,因此认为双向电泳与质谱分析的结果是可靠的。 将通过双向电泳与质谱分析得到的差异表达蛋白上传至IPA进行通路分析,发现在香烟烟雾处理后的小鼠睾丸中,表达水平与对照组小鼠具有显著差异的蛋白质中很多都能够直接或间接地与NF-κB发生作用,且根据通路分析它们的表达水平变化趋势能够抑制NF-κB的表达或激活。使用NF-κB复合体p65亚基的抗体在相应的睾丸组织切片上使用免疫组织化学的方法检测NF-κB的表达与激活情况,证实相较于对照组小鼠睾丸组织,香烟烟雾处理后的小鼠睾丸组织中生殖细胞内的NF-κB的表达或激活遭到了抑制。NF-κB在这些细胞中的正常水平的表达与激活对维持生殖细胞的正常增殖与精子的分化具有非常重要的意义,香烟烟雾抑制了这些细胞中的NF-κB的表达与激活,可能会通过影响到生殖细胞的正常增殖与精子的分化而影响到精液的质量。 同样的,将通过双向电泳与质谱分析得到的差异表达蛋白上传至KEGG进行通路分析后发现,在香烟烟雾处理后的小鼠附睾中,表达水平于对照组小鼠具有显著差异的蛋白质比较集中作用的通路有谷胱甘肽代谢通路与内质网相关降解途径(ERAD),且根据Blast2GO对这些蛋白质的注释,其中很多蛋白质均与维持机体正常的氧化还原稳态相关。这一结果暗示了香烟烟雾处理后小鼠附睾处于氧化应激状态。使用氧化应激的特异性标志物8-OHdG抗血清在相应的附睾组织切片上使用免疫组织化学的方法检测8-OHdG的存在,结果显示香烟烟雾处理后的小鼠附睾处于较为严重的氧化应激状态中。氧化应激能够引起ERAD从而影响蛋白质的正常合成,而Real-time PCR的结果显示处理组与对照组的小鼠附睾组织间差异表达蛋白在相应基因的转录水平上差异并不显著,且差异趋势与蛋白质水平的差异趋势不一致,这一点提示我们也许是氧化应激引起的ERAD导致了蛋白表达水平的差异。 此外,在对其他经过香烟烟雾处理后的小鼠睾丸与附睾组织中表达存在显著差异的蛋白经过注释与分析后我们发现,它们中的很多都能够通过不同途径影响到生育能力。 结论:香烟烟雾处理能够导致小鼠睾丸和附睾这两个重要的生殖器官中蛋白表达水平的变化,在睾丸中香烟烟雾能够通过对NF-κB途径的抑制干扰生殖细胞的正常增殖与精子的分化,在附睾中则能够通过造成氧化应激诱导ERAD的发生,从而影响蛋白质的正常合成,进一步损害附睾正常的功能。结合其他表达发生显著差异的蛋白对精子的影响,香烟烟雾最终通过这些途径降低精子的质量,从而使雄性小鼠的生育能力下降。
[Abstract]:The background and purpose of the study are that the smoke components of the cigarette are complex and many components can be harmful to the health of the human body. The epidemiological survey showed that there was a high correlation between the smoking behavior and the male fertility. The results showed that the sperm density in the semen decreased, the sperm motility was weakened, the sperm morphology was abnormal, and the in vitro fertilization was reduced. This phenomenon directly leads to a reduction in the fertility of male-smoking men, and even to the health of future generations. Many researchers have tried to find out why smoking behavior reduces male fertility, including comets electrophoresis, The method of the end-end labeling (TUNEL) of dUTP-biotin nick end-end labeling (TUNEL) and sperm chromosome structure analysis (SCSA), which is mediated by the terminal deoxynucleotiferase (TUNEL), has confirmed that smoking can lead to the breakage of the sperm DNA, the structural abnormality of the chromosome and the occurrence of the non-ploidy of the chromosome, It was also suggested that smoking would change the level of hormones in the serum, thus affecting the generation of sperm. In the male reproductive system, the testis is the main place of the generation of the sperm, and the epididymis is the main place for the maturation of the sperm. The two organs have a very important role in the quality of the sperm and the semen. Using the method of proteomics to observe the overall expression of the protein in the testis and the epididymis of the mouse after the cigarette smoke treatment, using the mouse smoking model established in advance in the laboratory. The function of these proteins, as well as their correlation to fertility, was analyzed by means of bioinformatics in order to find a protein that has a significantly different level of expression than that of the corresponding proteins in the epididymis of mice that have not been treated with cigarette smoke. A molecular machine that seeks to find the effect of cigarette smoke treatment on male fertility. Methods: Seven-week-old male C57/ BL6J mice were treated with cigarette smoke twice a day, one hour for each time and two weeks for the control group, and no cigarette smoke was used in the control group. fog treatment; after the treatment period, the mouse testis and the epididymis are taken, the total protein is respectively extracted for two-dimensional electrophoresis, and the two-way electricity of the relevant tissues of the mice in the treatment group and the control group is compared and selecting the protein point cut-off glue which has a significant difference in the expression level between the two proteins to carry out matrix-assisted laser desorption/ ionization time-of-flight mass spectrum analysis to identify the protein points which are represented by the protein points, The protein was randomly selected from the protein identified by mass spectrometry, and the trend of the expression of these proteins in the treatment group mice and the control group mouse-related tissues was validated using the Western-blotting method to determine the results of the two-dimensional electrophoresis and mass spectrometry analysis. No reliable, at the same time, the transcription level of the genes corresponding to these proteins was detected in the corresponding organ using the Real-time PCR method to observe the level of transcription of these genes between the treatment group and the control group mice. The protein expressed in the testis of the mouse and the tissues of the epididymis tissues after the cigarette smoke treatment obtained by the two-dimensional electrophoresis and the mass spectrometry analysis is used for functional annotation by using the Blash2GO, and meanwhile, The biological process and the molecular function are based on the gene based on the two levels of the molecular function. Classification of the body theory. The pathways involved in these proteins were analyzed using the pathway analysis tool KEGG and IPA to identify the main pathways that were affected. Biological process. The downstream action marker of the identified pathway was tested at the tissue level using an immunohistochemical method to verify the pathway analysis. Results and speculations. The results of the study: Two-dimensional electrophoresis, we found that the expression level of more than 1000 protein points in the mouse testis and the epididymis tissues after the cigarette smoke treatment was different from that in the control group, and the expression level of 27 protein points in the testis tissue was different. at least 1.5 times, the expression level of 52 protein points in the epididymis tissue is more than 2 times, The expression level of the testis and the epididymis tissues of the mouse testis and the epididymis tissues after the cigarette smoke treatment were selected for mass spectrometry, and 27 protein spots in the testis and the epididymis tissues were respectively The level of expression can be used to represent 27 corresponding eggs. The expression level of the white matter was increased. In the mouse testis, the expression level of six proteins in the 27 proteins identified was up-regulated and the expression level of 21 proteins was down-regulated; in the epididymis of the mouse,12 of the 27 proteins identified were up-regulated,15 proteins The expression levels of 5 proteins and epididymis tissues from the testis were randomly selected. Western-blotting was used to analyze the two-dimensional electrophoresis and mass spectrometry. The results showed that the trend of the difference between the treatment group and the control group was basically the same as that of the two-dimensional electrophoresis and mass spectrometry, and the two-dimensional electrophoresis and mass spectrometry were considered. The result is reliable. The differential expression protein obtained by the two-dimensional electrophoresis and mass spectrometry is uploaded to the IPA for path analysis, and found in the cigarette smoke. In the treated mouse's testis, the expression level was significantly different from that of the control group, and many of the proteins were able to interact directly or indirectly with the NF-antigen B, and the change trend of the expression level of the mice according to the pathway was able to inhibit the NF. -B expression or activation. The expression and activation of NF-EMAB was detected using an immunohistochemical method on the corresponding testis tissue sections using an antibody of the NF-B-B complex p65 subunit, confirming that the expression and activation of the NF-EMAB were compared to that of the Table of NF-NURB in the Germ Cells of the Mouse's Testicular Tissue after Cigarette Smoke Treatment in the Control Group Dup or activation has been inhibited. The expression and activation of the normal level of NF-B in these cells is of great importance to the normal proliferation of the maintenance germ cells and the differentiation of the sperm, and the cigarette smoke inhibits NF in these cells. -Expression and activation of the cell B, possibly by affecting the normal proliferation of the germ cells and the differentiation of the sperm The quality of the semen is affected. Similarly, the differential expression protein obtained by the two-dimensional electrophoresis and mass spectrum analysis is uploaded to the KEGG for path analysis, and is found in the cigarette In the mouse epididymis after the smoke treatment, there was a significant difference in the expression level of the expression level in the control group, and the pathway with a significant difference in the protein concentration had a glutathione metabolism pathway and the endoplasmic reticulum-related degradation pathway (ERAD), and according to Blas2 GO's comments on these proteins, many of which are all related to the maintenance of the body The normal oxidation-reduction steady-state correlation. This result implies that the cigarette smoke treatment is small The presence of 8-OHdG was detected by an immunohistochemical method on the corresponding epididymis tissue sections using the specific marker 8-OHdG antiserum of oxidative stress. The results showed that the mouse epididymis after cigarette smoke treatment was in a lower level. In a severe oxidative stress state, oxidative stress can cause ERAD to influence the normal synthesis of the protein, and the results of the Real-time PCR show that the difference in the expression of the differentially expressed protein between the treatment group and the control group on the transcription level of the corresponding gene is not significant, and the difference trend and the protein The difference in the quality level is not consistent, which suggests that we may be the ERAD induced by oxidative stress The difference in the level of protein expression was noted. In addition, after the analysis of other proteins expressed in the testis and epididymis tissues of other mice treated with cigarette smoke, we found that many of them can Conclusion: The cigarette smoke treatment can lead to the change of the expression level of the protein in the two important reproductive organs of the mouse's testis and the epididymis. The normal proliferation of germ cells is associated with the differentiation of the sperm. In the epididymis, the generation of ERAD can be induced by oxidative stress, which affects the normal protein. The synthesis and further damage to the normal function of the epididymis. The effect of a protein with a significant difference in the expression of the other expresses the effect of the protein on the sperm, which ultimately reduces the quality of the sperm through these routes.
【学位授予单位】:上海交通大学
【学位级别】:博士
【学位授予年份】:2013
【分类号】:R114
本文编号:2509558
[Abstract]:The background and purpose of the study are that the smoke components of the cigarette are complex and many components can be harmful to the health of the human body. The epidemiological survey showed that there was a high correlation between the smoking behavior and the male fertility. The results showed that the sperm density in the semen decreased, the sperm motility was weakened, the sperm morphology was abnormal, and the in vitro fertilization was reduced. This phenomenon directly leads to a reduction in the fertility of male-smoking men, and even to the health of future generations. Many researchers have tried to find out why smoking behavior reduces male fertility, including comets electrophoresis, The method of the end-end labeling (TUNEL) of dUTP-biotin nick end-end labeling (TUNEL) and sperm chromosome structure analysis (SCSA), which is mediated by the terminal deoxynucleotiferase (TUNEL), has confirmed that smoking can lead to the breakage of the sperm DNA, the structural abnormality of the chromosome and the occurrence of the non-ploidy of the chromosome, It was also suggested that smoking would change the level of hormones in the serum, thus affecting the generation of sperm. In the male reproductive system, the testis is the main place of the generation of the sperm, and the epididymis is the main place for the maturation of the sperm. The two organs have a very important role in the quality of the sperm and the semen. Using the method of proteomics to observe the overall expression of the protein in the testis and the epididymis of the mouse after the cigarette smoke treatment, using the mouse smoking model established in advance in the laboratory. The function of these proteins, as well as their correlation to fertility, was analyzed by means of bioinformatics in order to find a protein that has a significantly different level of expression than that of the corresponding proteins in the epididymis of mice that have not been treated with cigarette smoke. A molecular machine that seeks to find the effect of cigarette smoke treatment on male fertility. Methods: Seven-week-old male C57/ BL6J mice were treated with cigarette smoke twice a day, one hour for each time and two weeks for the control group, and no cigarette smoke was used in the control group. fog treatment; after the treatment period, the mouse testis and the epididymis are taken, the total protein is respectively extracted for two-dimensional electrophoresis, and the two-way electricity of the relevant tissues of the mice in the treatment group and the control group is compared and selecting the protein point cut-off glue which has a significant difference in the expression level between the two proteins to carry out matrix-assisted laser desorption/ ionization time-of-flight mass spectrum analysis to identify the protein points which are represented by the protein points, The protein was randomly selected from the protein identified by mass spectrometry, and the trend of the expression of these proteins in the treatment group mice and the control group mouse-related tissues was validated using the Western-blotting method to determine the results of the two-dimensional electrophoresis and mass spectrometry analysis. No reliable, at the same time, the transcription level of the genes corresponding to these proteins was detected in the corresponding organ using the Real-time PCR method to observe the level of transcription of these genes between the treatment group and the control group mice. The protein expressed in the testis of the mouse and the tissues of the epididymis tissues after the cigarette smoke treatment obtained by the two-dimensional electrophoresis and the mass spectrometry analysis is used for functional annotation by using the Blash2GO, and meanwhile, The biological process and the molecular function are based on the gene based on the two levels of the molecular function. Classification of the body theory. The pathways involved in these proteins were analyzed using the pathway analysis tool KEGG and IPA to identify the main pathways that were affected. Biological process. The downstream action marker of the identified pathway was tested at the tissue level using an immunohistochemical method to verify the pathway analysis. Results and speculations. The results of the study: Two-dimensional electrophoresis, we found that the expression level of more than 1000 protein points in the mouse testis and the epididymis tissues after the cigarette smoke treatment was different from that in the control group, and the expression level of 27 protein points in the testis tissue was different. at least 1.5 times, the expression level of 52 protein points in the epididymis tissue is more than 2 times, The expression level of the testis and the epididymis tissues of the mouse testis and the epididymis tissues after the cigarette smoke treatment were selected for mass spectrometry, and 27 protein spots in the testis and the epididymis tissues were respectively The level of expression can be used to represent 27 corresponding eggs. The expression level of the white matter was increased. In the mouse testis, the expression level of six proteins in the 27 proteins identified was up-regulated and the expression level of 21 proteins was down-regulated; in the epididymis of the mouse,12 of the 27 proteins identified were up-regulated,15 proteins The expression levels of 5 proteins and epididymis tissues from the testis were randomly selected. Western-blotting was used to analyze the two-dimensional electrophoresis and mass spectrometry. The results showed that the trend of the difference between the treatment group and the control group was basically the same as that of the two-dimensional electrophoresis and mass spectrometry, and the two-dimensional electrophoresis and mass spectrometry were considered. The result is reliable. The differential expression protein obtained by the two-dimensional electrophoresis and mass spectrometry is uploaded to the IPA for path analysis, and found in the cigarette smoke. In the treated mouse's testis, the expression level was significantly different from that of the control group, and many of the proteins were able to interact directly or indirectly with the NF-antigen B, and the change trend of the expression level of the mice according to the pathway was able to inhibit the NF. -B expression or activation. The expression and activation of NF-EMAB was detected using an immunohistochemical method on the corresponding testis tissue sections using an antibody of the NF-B-B complex p65 subunit, confirming that the expression and activation of the NF-EMAB were compared to that of the Table of NF-NURB in the Germ Cells of the Mouse's Testicular Tissue after Cigarette Smoke Treatment in the Control Group Dup or activation has been inhibited. The expression and activation of the normal level of NF-B in these cells is of great importance to the normal proliferation of the maintenance germ cells and the differentiation of the sperm, and the cigarette smoke inhibits NF in these cells. -Expression and activation of the cell B, possibly by affecting the normal proliferation of the germ cells and the differentiation of the sperm The quality of the semen is affected. Similarly, the differential expression protein obtained by the two-dimensional electrophoresis and mass spectrum analysis is uploaded to the KEGG for path analysis, and is found in the cigarette In the mouse epididymis after the smoke treatment, there was a significant difference in the expression level of the expression level in the control group, and the pathway with a significant difference in the protein concentration had a glutathione metabolism pathway and the endoplasmic reticulum-related degradation pathway (ERAD), and according to Blas2 GO's comments on these proteins, many of which are all related to the maintenance of the body The normal oxidation-reduction steady-state correlation. This result implies that the cigarette smoke treatment is small The presence of 8-OHdG was detected by an immunohistochemical method on the corresponding epididymis tissue sections using the specific marker 8-OHdG antiserum of oxidative stress. The results showed that the mouse epididymis after cigarette smoke treatment was in a lower level. In a severe oxidative stress state, oxidative stress can cause ERAD to influence the normal synthesis of the protein, and the results of the Real-time PCR show that the difference in the expression of the differentially expressed protein between the treatment group and the control group on the transcription level of the corresponding gene is not significant, and the difference trend and the protein The difference in the quality level is not consistent, which suggests that we may be the ERAD induced by oxidative stress The difference in the level of protein expression was noted. In addition, after the analysis of other proteins expressed in the testis and epididymis tissues of other mice treated with cigarette smoke, we found that many of them can Conclusion: The cigarette smoke treatment can lead to the change of the expression level of the protein in the two important reproductive organs of the mouse's testis and the epididymis. The normal proliferation of germ cells is associated with the differentiation of the sperm. In the epididymis, the generation of ERAD can be induced by oxidative stress, which affects the normal protein. The synthesis and further damage to the normal function of the epididymis. The effect of a protein with a significant difference in the expression of the other expresses the effect of the protein on the sperm, which ultimately reduces the quality of the sperm through these routes.
【学位授予单位】:上海交通大学
【学位级别】:博士
【学位授予年份】:2013
【分类号】:R114
【参考文献】
相关期刊论文 前1条
1 古洪元;唐茂萍;方鹏;朱子珏;徐汪节;王朝霞;王莲芸;乔中东;;香烟烟雾对雄性小鼠生育能力的影响[J];上海交通大学学报(农业科学版);2012年03期
本文编号:2509558
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