雄性小鼠睾丸支持细胞中Attractin的功能研究
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摘要
第一部分Atrn基因功能缺失雄性小鼠生殖系统表型分析
目的：观察Atrn基因功能缺失雄性小鼠生殖系统表型，为研究Atrn基因在雄性生殖系统中的作用提供实验依据。
方法：以C3HeB／FeJ种系Atrn基因缺失小鼠(Atrn~(mg-3J))为实验组，同种系野生型小鼠为对照组，通过观察不同年龄段(3月龄和5月龄)Atrn基因缺失小鼠和对照组小鼠睾丸和附睾重量、形态学改变、附睾尾精子密度和活动率，并用分光光度法检测4组小鼠睾丸组织酶乳酸脱氢酶(lactate dehydrogenase，LDH)和琥珀酸脱氢酶(succinate dehydrogenase，SDH)水平，并通过与同种系野生型雌鼠交配进行交配试验和体外受精试验，评估Atrn~(mg-3J)雄鼠的生育能力。
结果：与相同月龄对照组小鼠相比，3月龄Atrn基因缺失小鼠SDH比活性降低(P＜0.05)，其他指标无明显改变。5月龄Atrn基因缺失小鼠睾丸组织切片HE染色，精曲小管结构破坏；睾丸重量下降(P＜0.05)；附睾尾精子密度和活动率下降(P＜0.05)，LDH活性和SDH比活性降低(P＜0.05)；雌鼠与5月龄Atrn基因缺失雄鼠交配后可观察到阴道栓，但每只雌鼠平均胎仔数减少(P＜0.05)；体外受精试验结果显示Atrn基因缺失小鼠附睾精子与正常卵子受精率下降(P＜0.05)。
结论：研究结果显示随着月龄的增加，Atrn基因缺失使雄性小鼠生殖系统发生退行性改变，表现为睾丸精曲小管结构改变和精子功能受损，这可能是导致Atrn基因缺失雄性小鼠生殖力下降的主要原因之一。然而受孕是一个复杂的生理过程，涉及多个因素的影响，本研究为进一步深入探讨Atrn基因在雄性生殖系统的生理作用机制奠定研究方向和实验基础。
第二部分干扰Atrn表达的小鼠睾丸支持细胞株的建立
目的：通过基因转染和RNA干扰技术，使小鼠睾丸支持细胞中内源性Atrn表达受到抑制，进一步研究Atrn在睾丸支持细胞中的作用。
方法：本实验采用Q-PCR、Western blot以及免疫荧光方法检测小鼠睾丸支持细胞株TM4细胞中Atrn mRNA和蛋白的表达。针对Atrn的编码序列中3段siRNA靶序列设计相应DNA序列，将其插入H1启动子下游，克隆到真核表达载体psiRNA-hH1neo中，转化DH5α菌株扩增，提取质粒通过酶切鉴定和DNA测序鉴定。将重组载体在阳离子脂质体Lipofectamine~(TM)2000介导下瞬时转染TM4细胞，以转染试剂组(liposome)、阴性对照组(psiRNA-hH1)、空白对照组(未转染的TM4细胞)通过Q-PCR和Western blot法检测转染第3天Atrn mRNA、蛋白表达水平，确定有效序列，用含有该序列的重组载体稳定转染细胞，G418筛选，建立稳定干扰Atrn表达的睾丸支持细胞株，并用Q-PCR和ELISA分别对其分泌产物抑制素α(Inhα)mRNA和细胞培养液中的抑制素B(Inb B)的表达进行了检测，以探讨在细胞水平抑制内源性Atrn的表达对支持细胞功能的影响。
结果：小鼠睾丸支持细胞株TM4细胞表达Atrn mRNA和蛋白。构建重组载体psiAtrn-770、psiAtrn-2273、psiAtrn-2908，酶切鉴定及DNA测序结果显示插入片断正确。将重组载体瞬时转染TM4细胞，转染后72h，以psiAtrn-2273和psiAtrn-2908转染组抑制Atrn mRNA和蛋白表达效果明显(P＜0.05)；psiAtrn-2273与psiAtrn-2908转染组之间相比，psiAtrn-2273转染组ATRN蛋白表达水平更低(P＜0.05)。转染试剂组，阴性对照组与空白对照组相比，Atrn mRNA和蛋白表达差异无统计学显著意义(P＞0.05)。质粒psiAtrn-2273能有效地阻断支持细胞内源性基因的表达，以质粒psiAtrn-2273稳定转染TM4细胞获得稳定干扰Atrn表达的TM4细胞株psiAtrn-TM4，以空质粒psiRNA-hH1稳定转染后得到的细胞株psiRNA-TM4为后续实验对照，psiAtrn-TM4细胞中Atrn mRNA表达抑制率约为80.1％，蛋白表达抑制率约为72.98％。当Atrn表达受到抑制的时候，InhαmRNA和细胞培养液中的Inb B与对照组细胞相比分别上升了约34.01％和17.9％，差异有统计学显著性意义(P＜0.05)。
结论：本部分实验成功构建针对Atrn基因的特异性真核表达载体，建立了稳定干扰Atrn表达的TM4细胞株，有效地降低了支持细胞Atrn基因的表达。当内源性AtrnmRNA表达受到抑制时，小鼠睾丸支持细胞分泌产物抑制素表达水平升高。
第三部分应用蛋白组学技术分离鉴定睾丸支持细胞中ATRN功能相关的蛋白质
目的：通过蛋白组学技术分离鉴定小鼠睾丸支持细胞中Atrn基因部分缺失和完全缺失情况下差异性表达的蛋白质，为进一步研究Atrn在小鼠睾丸支持细胞中的功能提供科学依据。
方法：酶消化法原代培养小鼠睾丸支持细胞，并通过Feulgen染色、Hoechst染色、吖啶橙染色鉴定细胞纯度。以空质粒psiRNA-hH1稳定转染后得到的细胞株(psiRNA-TM4)为实验对照，采用双向凝胶电泳技术分离(two-dimensional gelelectrophoresis，2-DE)、基质辅助激光解析质谱(Matrix-assisted laserdesorption／icnization time of flight mass spectrometry，MALDI-TOF-MS)研究稳定干扰Atrn基因表达的小鼠睾丸支持细胞(psiAtrn-TM4)，Atrn基因缺失小鼠原代睾丸支持细胞(mu-Sc)细胞中与Atrn缺失相关差异表达的蛋白质，并通过Q-PCR和Western Net对部分差异表达蛋白质mRNA和蛋白表达进行验证。
结果：实验分离的培养Atrn~(mg-3J)小鼠睾丸支持细胞，纯度达95％。采用2-DE与MALDI-TOF-MS联合分离鉴定psi-RNA-TM4，psi-Atrn-TM4，mu-Sc3组细胞中差异性表达的蛋白质，鉴定出主要的14个差异表达的蛋白质，其中有8种蛋白质包括ATPSYNTHASE，PEROXIREDOXIN 2，CU／ZN SUPEROXIDE DISMUTASE 1等在Atrn基因部分缺失细胞(psiAtrn-TM4)和完全缺失细胞(mu-Sc)中表达下调，6种蛋白质包括CASPASE6，KETOHEXOKINASE等上调，其中Sod1和caspase6 mRNA和蛋白在3组细胞中的的差异表达水平得到进一步验证。
结论：Atrn基因缺失导致相关蛋白质的差异性表达提示Atrn基因对睾丸支持细胞功能的影响是通过多途径实现的，包括抗氧化应激以及调控凋亡途径。为进一步深入研究Atrn在小鼠睾丸支持细胞中的功能和Atrn缺失导致小鼠睾丸退行性变的具体作用机制提供重要了依据和线索。
PartⅠReproductive phenotype analysis in Attractin genedeficiency male mouse
Objective: The purpose of this study was to primarily explore the effect of loss-of-function of Attractin (Atrn) in the male mouse reproduction system.
Methods: Firstly, the morphological changes of testis and epdidymis between Atrnmutant (Atrn~(mg-3J)) and wild-type mice at 3months and 5months were studied. Also, theweight difference in testis and epdidymis, as well as the sperm dencity and motilitybetween mutants and controls were investigated. Further, the testicular lactatedehydrogenase (LDH), succinate dehydrogenase (SDH) among these animals weremeasured, and the fertility of their sperm in vivo and vitro were observed.
Results: In comparison with the control group, no obviously pathological modify wasdetected in 3-month-old Atrn~(mg-3J) mouse testis except reduced SDH activity. In 5-month-oldmutants, the morphology of seminiferous tubule was disorder, the density and motility ofsperm were decreased in epididymis, the sperm fertility was impaired and testicular enzymeactivity were also descent.
Conclusion: The data showed the age-related Atrn gene progressively loss-of-functionmight result in the degeneration in male Atrn~(mg-3J)mouse, representated as the disordermorphology of seminiferous tubule and impaired sperm function, which would be one ofmain reasons to damage male reproductive ability (P＜0.05).
PartⅡThe establishing of mouse sertoli cells targeting gene Atrnby RNA interference
Objective: To stable suppress the Atrn gene expression in mouse Sertoli cells usingsiRNA eukaryotic expression vector and explore the function of Atrn gene in these cells.
Methods: The expression of Atrn in TM4 was examined by Q-PCR, Western blot andimmunocytochemistry.The siRNAs were designed according to the coding the cDNAsequence of Atrn gene, and cloned into the downstream of H1 promoter of psiRNA-hH1neo.Then, the vector was cloned by transforming into DH5αstrain and identified byrestriction endonuclease digestion and DNA sequencing. Three recombinant vectors and thepsiRNA-hH1 were transfected into TM4 cells by Lipofectamine~(TM)2000. The mRNA andprotein expression of Atrn in TM4 cells were investigated by Q-PCR and Western blot afer72h. Then, the TM4 cells were transfected with the most effective Atrn-specific siRNAexpression vectors and psiRNA-hH1 respectively, selection media with G418 was addedon the third day after transfection. Cultures were maintained in selective media for two tothree weeks until the mostly cells of the blank control group were dead. The G418-selectedtransformed cells were grown and expanded further. At last, the analysis of inhibin alphamRNA and inbibin B expression in G418-selected transformed cells was carried out byRT-PCR and ELISA.
Results: The Atrn mRNA and protein were both expressed in TM4 cells. Theconstructed psiAtrn-770, psiAtrn-2273, psiAtrn-2908 digested with restrictionendonuclease Ase I was linearized. The sequencing results confirmed that the sequence ofinserted fragment was correct. After transient transfection, the effect of suppression of theexpression of Atrn in the cells with the psiAtrn-2273 and psiAtrn-2908 were obviously (P＜0.05). Especially, the expression of ATRN protein of psiAtrn-2273 was more lower thanthe psiAtrn-2908 (P＜0.05). Then the psiAtrn-TM4 and psiRNA-TM4 cells wereestablished by stable transformed by psiAtrn-2273 and psiRNA-hH1. Compared with the psiRNA-TM4 cells, the expression of Atrn was suppressed obviously and the level of InhamRNA and Inh B was significantly higher in the psiAtrn-TM4 cells (P＜0.05)
Conclusion: The eukaryotic expression vector of siRNA targeting Atrn gene wassuccessfully constructed, and the Atrn knock-down in mouse sertoli cells TM4(psiAtrn-TM4) was successfully established by stable RNA interference. When theendogenous expression of Atrn was suppressed, the level of inhibin secreted by Sertoli cellswas increased.
PartⅢIdentification of differential proteins in TM4 mouse Sertoli cellswith Atrn silence by proteome analysis
Objective: To separate and identify the differential proteins in TM4 mouse Sertoli cellswith Atrn partly or completely loss-of-function by proteome analysis, and provide ascientific basis to clarify the effort of Atrn in mouse Sertoli cells.
Methods: The Atrn~(mg-3J) mouse primary sertoli cells was isolated and cultured byenzymatic treatments and identified through several methods. Atrn knock-down in mousesertoli cells TM4 (psiAtrn-TM4) was established by stable RNA interference. Comparedproteomic changes among the psiAtrn-TM4, primary cultures of testis Sertoli cells ofAtrn~(mg-3J) (mu-Sc) and control gourp (psiRNA-TM4) using two-dimensional gelelectrophoresis (2-DE) and Matrix-assisted laser desorption/ionization time of flight massspectrometry (MALDI-TOF-MS). At last, the expression levels of some of these differentialproteins were further measured by Q-PCR and Western blot.
Results: Atrn~(mg3-J) mouse primary sertoli cells, with 95% purity,was established. Fifteendifferentially expression protein spots between the three cells were identified byMALDI-TOF-MS and the NCBI proteins database. Except the decreased expression ofSOD, others may be novel proteins associated with ATRN function such as down regulatedof ATP SYNTHASE, PEROXIREDOXINⅡand upregulated CASPASE6,KETOHEXOKINASE in psiAtrn-TM4 and mu-Sc. Futher, the mRNA and proteinexpression levels of SOD and CASPASE6 in the these cells were confirmed.
Conclusion: These dates suggested that these differential protein may be associated withthe function of Atrn in Sertoli cells involving multi-pathways,including antioxidative stressand antiapoptosis pathway, which provided new clue to interpret the the function of Atrngene in mouse Sertoli cells and the mechanism of degeneration of testis in Atrn mutants.
目的：观察Atrn基因功能缺失雄性小鼠生殖系统表型，为研究Atrn基因在雄性生殖系统中的作用提供实验依据。
方法：以C3HeB／FeJ种系Atrn基因缺失小鼠(Atrn~(mg-3J))为实验组，同种系野生型小鼠为对照组，通过观察不同年龄段(3月龄和5月龄)Atrn基因缺失小鼠和对照组小鼠睾丸和附睾重量、形态学改变、附睾尾精子密度和活动率，并用分光光度法检测4组小鼠睾丸组织酶乳酸脱氢酶(lactate dehydrogenase，LDH)和琥珀酸脱氢酶(succinate dehydrogenase，SDH)水平，并通过与同种系野生型雌鼠交配进行交配试验和体外受精试验，评估Atrn~(mg-3J)雄鼠的生育能力。
结果：与相同月龄对照组小鼠相比，3月龄Atrn基因缺失小鼠SDH比活性降低(P＜0.05)，其他指标无明显改变。5月龄Atrn基因缺失小鼠睾丸组织切片HE染色，精曲小管结构破坏；睾丸重量下降(P＜0.05)；附睾尾精子密度和活动率下降(P＜0.05)，LDH活性和SDH比活性降低(P＜0.05)；雌鼠与5月龄Atrn基因缺失雄鼠交配后可观察到阴道栓，但每只雌鼠平均胎仔数减少(P＜0.05)；体外受精试验结果显示Atrn基因缺失小鼠附睾精子与正常卵子受精率下降(P＜0.05)。
结论：研究结果显示随着月龄的增加，Atrn基因缺失使雄性小鼠生殖系统发生退行性改变，表现为睾丸精曲小管结构改变和精子功能受损，这可能是导致Atrn基因缺失雄性小鼠生殖力下降的主要原因之一。然而受孕是一个复杂的生理过程，涉及多个因素的影响，本研究为进一步深入探讨Atrn基因在雄性生殖系统的生理作用机制奠定研究方向和实验基础。
第二部分干扰Atrn表达的小鼠睾丸支持细胞株的建立
目的：通过基因转染和RNA干扰技术，使小鼠睾丸支持细胞中内源性Atrn表达受到抑制，进一步研究Atrn在睾丸支持细胞中的作用。
方法：本实验采用Q-PCR、Western blot以及免疫荧光方法检测小鼠睾丸支持细胞株TM4细胞中Atrn mRNA和蛋白的表达。针对Atrn的编码序列中3段siRNA靶序列设计相应DNA序列，将其插入H1启动子下游，克隆到真核表达载体psiRNA-hH1neo中，转化DH5α菌株扩增，提取质粒通过酶切鉴定和DNA测序鉴定。将重组载体在阳离子脂质体Lipofectamine~(TM)2000介导下瞬时转染TM4细胞，以转染试剂组(liposome)、阴性对照组(psiRNA-hH1)、空白对照组(未转染的TM4细胞)通过Q-PCR和Western blot法检测转染第3天Atrn mRNA、蛋白表达水平，确定有效序列，用含有该序列的重组载体稳定转染细胞，G418筛选，建立稳定干扰Atrn表达的睾丸支持细胞株，并用Q-PCR和ELISA分别对其分泌产物抑制素α(Inhα)mRNA和细胞培养液中的抑制素B(Inb B)的表达进行了检测，以探讨在细胞水平抑制内源性Atrn的表达对支持细胞功能的影响。
结果：小鼠睾丸支持细胞株TM4细胞表达Atrn mRNA和蛋白。构建重组载体psiAtrn-770、psiAtrn-2273、psiAtrn-2908，酶切鉴定及DNA测序结果显示插入片断正确。将重组载体瞬时转染TM4细胞，转染后72h，以psiAtrn-2273和psiAtrn-2908转染组抑制Atrn mRNA和蛋白表达效果明显(P＜0.05)；psiAtrn-2273与psiAtrn-2908转染组之间相比，psiAtrn-2273转染组ATRN蛋白表达水平更低(P＜0.05)。转染试剂组，阴性对照组与空白对照组相比，Atrn mRNA和蛋白表达差异无统计学显著意义(P＞0.05)。质粒psiAtrn-2273能有效地阻断支持细胞内源性基因的表达，以质粒psiAtrn-2273稳定转染TM4细胞获得稳定干扰Atrn表达的TM4细胞株psiAtrn-TM4，以空质粒psiRNA-hH1稳定转染后得到的细胞株psiRNA-TM4为后续实验对照，psiAtrn-TM4细胞中Atrn mRNA表达抑制率约为80.1％，蛋白表达抑制率约为72.98％。当Atrn表达受到抑制的时候，InhαmRNA和细胞培养液中的Inb B与对照组细胞相比分别上升了约34.01％和17.9％，差异有统计学显著性意义(P＜0.05)。
结论：本部分实验成功构建针对Atrn基因的特异性真核表达载体，建立了稳定干扰Atrn表达的TM4细胞株，有效地降低了支持细胞Atrn基因的表达。当内源性AtrnmRNA表达受到抑制时，小鼠睾丸支持细胞分泌产物抑制素表达水平升高。
第三部分应用蛋白组学技术分离鉴定睾丸支持细胞中ATRN功能相关的蛋白质
目的：通过蛋白组学技术分离鉴定小鼠睾丸支持细胞中Atrn基因部分缺失和完全缺失情况下差异性表达的蛋白质，为进一步研究Atrn在小鼠睾丸支持细胞中的功能提供科学依据。
方法：酶消化法原代培养小鼠睾丸支持细胞，并通过Feulgen染色、Hoechst染色、吖啶橙染色鉴定细胞纯度。以空质粒psiRNA-hH1稳定转染后得到的细胞株(psiRNA-TM4)为实验对照，采用双向凝胶电泳技术分离(two-dimensional gelelectrophoresis，2-DE)、基质辅助激光解析质谱(Matrix-assisted laserdesorption／icnization time of flight mass spectrometry，MALDI-TOF-MS)研究稳定干扰Atrn基因表达的小鼠睾丸支持细胞(psiAtrn-TM4)，Atrn基因缺失小鼠原代睾丸支持细胞(mu-Sc)细胞中与Atrn缺失相关差异表达的蛋白质，并通过Q-PCR和Western Net对部分差异表达蛋白质mRNA和蛋白表达进行验证。
结果：实验分离的培养Atrn~(mg-3J)小鼠睾丸支持细胞，纯度达95％。采用2-DE与MALDI-TOF-MS联合分离鉴定psi-RNA-TM4，psi-Atrn-TM4，mu-Sc3组细胞中差异性表达的蛋白质，鉴定出主要的14个差异表达的蛋白质，其中有8种蛋白质包括ATPSYNTHASE，PEROXIREDOXIN 2，CU／ZN SUPEROXIDE DISMUTASE 1等在Atrn基因部分缺失细胞(psiAtrn-TM4)和完全缺失细胞(mu-Sc)中表达下调，6种蛋白质包括CASPASE6，KETOHEXOKINASE等上调，其中Sod1和caspase6 mRNA和蛋白在3组细胞中的的差异表达水平得到进一步验证。
结论：Atrn基因缺失导致相关蛋白质的差异性表达提示Atrn基因对睾丸支持细胞功能的影响是通过多途径实现的，包括抗氧化应激以及调控凋亡途径。为进一步深入研究Atrn在小鼠睾丸支持细胞中的功能和Atrn缺失导致小鼠睾丸退行性变的具体作用机制提供重要了依据和线索。
PartⅠReproductive phenotype analysis in Attractin genedeficiency male mouse
Objective: The purpose of this study was to primarily explore the effect of loss-of-function of Attractin (Atrn) in the male mouse reproduction system.
Methods: Firstly, the morphological changes of testis and epdidymis between Atrnmutant (Atrn~(mg-3J)) and wild-type mice at 3months and 5months were studied. Also, theweight difference in testis and epdidymis, as well as the sperm dencity and motilitybetween mutants and controls were investigated. Further, the testicular lactatedehydrogenase (LDH), succinate dehydrogenase (SDH) among these animals weremeasured, and the fertility of their sperm in vivo and vitro were observed.
Results: In comparison with the control group, no obviously pathological modify wasdetected in 3-month-old Atrn~(mg-3J) mouse testis except reduced SDH activity. In 5-month-oldmutants, the morphology of seminiferous tubule was disorder, the density and motility ofsperm were decreased in epididymis, the sperm fertility was impaired and testicular enzymeactivity were also descent.
Conclusion: The data showed the age-related Atrn gene progressively loss-of-functionmight result in the degeneration in male Atrn~(mg-3J)mouse, representated as the disordermorphology of seminiferous tubule and impaired sperm function, which would be one ofmain reasons to damage male reproductive ability (P＜0.05).
PartⅡThe establishing of mouse sertoli cells targeting gene Atrnby RNA interference
Objective: To stable suppress the Atrn gene expression in mouse Sertoli cells usingsiRNA eukaryotic expression vector and explore the function of Atrn gene in these cells.
Methods: The expression of Atrn in TM4 was examined by Q-PCR, Western blot andimmunocytochemistry.The siRNAs were designed according to the coding the cDNAsequence of Atrn gene, and cloned into the downstream of H1 promoter of psiRNA-hH1neo.Then, the vector was cloned by transforming into DH5αstrain and identified byrestriction endonuclease digestion and DNA sequencing. Three recombinant vectors and thepsiRNA-hH1 were transfected into TM4 cells by Lipofectamine~(TM)2000. The mRNA andprotein expression of Atrn in TM4 cells were investigated by Q-PCR and Western blot afer72h. Then, the TM4 cells were transfected with the most effective Atrn-specific siRNAexpression vectors and psiRNA-hH1 respectively, selection media with G418 was addedon the third day after transfection. Cultures were maintained in selective media for two tothree weeks until the mostly cells of the blank control group were dead. The G418-selectedtransformed cells were grown and expanded further. At last, the analysis of inhibin alphamRNA and inbibin B expression in G418-selected transformed cells was carried out byRT-PCR and ELISA.
Results: The Atrn mRNA and protein were both expressed in TM4 cells. Theconstructed psiAtrn-770, psiAtrn-2273, psiAtrn-2908 digested with restrictionendonuclease Ase I was linearized. The sequencing results confirmed that the sequence ofinserted fragment was correct. After transient transfection, the effect of suppression of theexpression of Atrn in the cells with the psiAtrn-2273 and psiAtrn-2908 were obviously (P＜0.05). Especially, the expression of ATRN protein of psiAtrn-2273 was more lower thanthe psiAtrn-2908 (P＜0.05). Then the psiAtrn-TM4 and psiRNA-TM4 cells wereestablished by stable transformed by psiAtrn-2273 and psiRNA-hH1. Compared with the psiRNA-TM4 cells, the expression of Atrn was suppressed obviously and the level of InhamRNA and Inh B was significantly higher in the psiAtrn-TM4 cells (P＜0.05)
Conclusion: The eukaryotic expression vector of siRNA targeting Atrn gene wassuccessfully constructed, and the Atrn knock-down in mouse sertoli cells TM4(psiAtrn-TM4) was successfully established by stable RNA interference. When theendogenous expression of Atrn was suppressed, the level of inhibin secreted by Sertoli cellswas increased.
PartⅢIdentification of differential proteins in TM4 mouse Sertoli cellswith Atrn silence by proteome analysis
Objective: To separate and identify the differential proteins in TM4 mouse Sertoli cellswith Atrn partly or completely loss-of-function by proteome analysis, and provide ascientific basis to clarify the effort of Atrn in mouse Sertoli cells.
Methods: The Atrn~(mg-3J) mouse primary sertoli cells was isolated and cultured byenzymatic treatments and identified through several methods. Atrn knock-down in mousesertoli cells TM4 (psiAtrn-TM4) was established by stable RNA interference. Comparedproteomic changes among the psiAtrn-TM4, primary cultures of testis Sertoli cells ofAtrn~(mg-3J) (mu-Sc) and control gourp (psiRNA-TM4) using two-dimensional gelelectrophoresis (2-DE) and Matrix-assisted laser desorption/ionization time of flight massspectrometry (MALDI-TOF-MS). At last, the expression levels of some of these differentialproteins were further measured by Q-PCR and Western blot.
Results: Atrn~(mg3-J) mouse primary sertoli cells, with 95% purity,was established. Fifteendifferentially expression protein spots between the three cells were identified byMALDI-TOF-MS and the NCBI proteins database. Except the decreased expression ofSOD, others may be novel proteins associated with ATRN function such as down regulatedof ATP SYNTHASE, PEROXIREDOXINⅡand upregulated CASPASE6,KETOHEXOKINASE in psiAtrn-TM4 and mu-Sc. Futher, the mRNA and proteinexpression levels of SOD and CASPASE6 in the these cells were confirmed.
Conclusion: These dates suggested that these differential protein may be associated withthe function of Atrn in Sertoli cells involving multi-pathways,including antioxidative stressand antiapoptosis pathway, which provided new clue to interpret the the function of Atrngene in mouse Sertoli cells and the mechanism of degeneration of testis in Atrn mutants.
引文
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33．刘娟，熊承良．Attractin蛋白在大鼠睾丸和附睾中的免疫组织化学定位．中华男科学杂志，2004，10(7)：515-517，520．
34．隋聪，熊承良，沈士亮．敲除Attractin或Mahoganoid基因对雄性小鼠生殖内分泌激素的影响．中国计划生育学杂志，2007，15(12)：729-731．