【作者】 胡涛；

【导师】 陈晓春； 童强松；

【作者基本信息】 华中科技大学 ， 泌尿外科， 2010， 博士

【摘要】 第一部分TSEG-2基因真核表达载体的构建及其在细胞内的表达目的：构建睾丸特异表达基因2 (testis specific expressed gene 2, TSEG-2)的真核表达载体,探讨TSEG-2在细胞中的表达及其生物学功能。方法：以小鼠睾丸组织cDNA为模版,设计携带HindⅢ和BamHⅠ酶切位点的引物序列,聚合酶链反应扩增基因片段,克隆至携带增强型绿色荧光蛋白(EGFP)的真核表达载体pEGFP-N1;在阳离子聚合物聚乙烯亚胺(PEI)的介导下,重组质粒转染体外培养的精母细胞株GC-2spd,荧光显微镜下观察TSEG-2基因在细胞内的表达定位,MTT法测定细胞生长活性,AO/EB荧光染色法、Hoechst 33258荧光染色法、Annexin V-FITC/PI双染流式细胞术检测细胞凋亡,JC-1染色流式细胞术检测线粒体膜电位,实时定量PCR测定Fas、Bcl-2、Bax表达水平。结果：.成功构建了TSEG-2与EGFP的融合表达载体pEGFP-TSEG2,转染GC-2spd细胞后可见胞浆内绿色荧光蛋白表达。转染pEGFP-TSEG2 48h后,GC-2spd细胞生长抑制39.2%(P<0.05),出现细胞凋亡形态学改变,凋亡率为28.3%(P<0.05)；线粒体膜电位显著低于对照组(P<0.05), GC-2spd细胞中Fas和Bcl-2的表达下调,而Bax的表达上调。结论：成功构建TSEG-2基因的真核表达载体,在精母细胞株中表达并促进细胞凋亡,并提示TEGS-2可能通过内源性凋亡途径诱导细胞凋亡。为进一步在细胞和动物水平开展TSEG-2功能研究奠定了基础。第二部分TSEG-2基因在小鼠睾丸扭转复位模型中的表达特征目的探讨睾丸特异表达基因2(testis specific expressed gene 2,TSEG-2)在小鼠睾丸扭转复位模型中的表达特征。方法昆明小鼠36只,随机分组为对照组(6只)、假手术组(6只)、单侧睾丸扭转复位实验组(24只)。实验组分为2组,每组12只,左侧睾丸扭转720度维持2 h,分别于复位后1、7天取扭转侧睾丸。采用HE染色、原位末端标记技术(TUNEL)观察睾丸组织形态改变；黄嘌呤氧化酶法、硫代巴比妥酸比色法测定超氧化物歧化酶(SOD)、丙二醛(MDA)活性；原位杂交法观测TSEG-2在睾丸生精细胞内的表达定位；实时定量PCR法检测TSEG-2基因在睾丸组织中的表达水平。结果对照组和假手术组生精上皮排列规则,扭转复位后1、7天的睾丸组织内生精上皮结构松散,出现生精细胞凋亡,Johnsen’s评分分别降低23.4％、64.1％(P<0.01),SOD活性降低11.6％、22.2％(P<0.05),MDA活性升高69.6％、93.2％(P<0.01)。TSEG-2基因表达定位于小鼠睾丸生精小管的精原细胞和精母细胞。与对照组比较,扭转复位1、7天后睾丸组织内TSEG-2表达水平分别上调2.2倍、6.6倍(P<0.01)结论成功建立小鼠睾丸扭转复位模型,TSEG-2表达上调可能与抗氧化酶活性下降、生精细胞凋亡有关。第三部分睾丸特异性基因TSEG-2在隐睾模型中的表达及其在生精细胞凋亡中的作用目的进一步探讨睾丸特异表达基因2(testis specific expressed gene 2,TSEG-2)的功能及其在隐睾模型中的表达特点。方法35只BALB/C小鼠(8周龄)随机分组为单侧手术隐睾组(20只)、假手术组(10只)、空白对照组(5只)。在手术隐睾组中,将右侧睾丸固定于腹壁内侧。real-timePCR法检测TSEG-2基因在隐睾模型中的表达特点。在聚乙烯亚胺(PEI)的介导下,采用睾丸内注射方法,将重组载体pEGFP-TSEG-2(n=5)和空载体(n=5)转染至正常雄性小鼠睾丸,荧光显微镜观测转染效率,TUNEL法检测生精细胞凋亡。结果原位杂交显示TSEG-2 mRNA表达定位于小鼠睾丸生精小管的精原细胞和精母细胞。与假手术组和对照组比较,隐睾组睾丸组织内TSEG-2表达显著上调(P<0.05),生精细胞凋亡比例增高(P<0.05)。PEI能有效介导TSEG-2转染至曲精小管,转染1周后生精细胞凋亡显著增多(P<0.05)。结论这些结果提示TSEG-2可能参与了生精细胞的凋亡和隐睾的病理过程。更多还原

【Abstract】 Part 1 Construction of the eukaryotic expression vector of TSEG-2 and its expression within cultured cellsObjective To construct the eukaryotic expression vector of TSEG-2 and explore its expression and function in cultured cells.Methods TSEG-2 gene fragment with restrictive sites Hind III BamH I was cloned from mouse testis cDNA by RT-PCR, and was inserted into eukaryotic expression vector pEGFP-N1. Under the induction of polyethylenimine, the recombinant vector was transfected into cultured spermatocyte GC-2spd cells. The expression of TSEG-2 was observed under a flurosecent microscopy. The cell viabilities of GC-2spd were observed by MTT assay. Cell apoptosis was inspected by AO/EB fluorescent staining, Hoechst 33258 fluorescent staining, and Annexin V-FITC/propidium iodide staining flow cytometry. the mitochondrial membrane potential in TSEG-2-transferred GC-2spd cells was revealed by JC-1 staining flow cytometry.The mRNA levels of Fas, Bcl-2 and Bax were detected by real-time PCR.Results The fusion expression vector pEGFP-TSEG2 was constructed. Forty-eight hours post-transfetion, fusion protein expression was observed in the cytoplasm of cultured cells. Transfection of TSEG-2 into spermatocyte GC-2spd cells, resulted in decrease of cell viabilities by 39.2%(P<0.05), and obvious morphological changes of apoptosis. The apoptosis rates were 28.3%(P<0.05). Downregulation of Fas and Bcl-2, and upregulation of Bax were observed in TSEG-2-transfected cells than those in control group (P<0.05).Conclusions The eukaryotic expression vector of TSEG-2 was successfully constructed, resulting its expression and apoptosis in cultured spermatocytes, which laid the basis for subsequent research of its function at cellular and animal levels. Part 2 Expression of testis specific expressed gene 2 in mouse testicular torsion/detorsion modelObjectives:To explore the expression profiles of testis specific expressed gene 2 (TSEG-2) in mouse testicular torsion/detorsion model. Methods:Thirty six Kunming mice were randomly divided into control (n=6), sham (n=6), and experimental (n=24) groups. In experimental group, the left testes were rotated 720 degree for 2 hrs and detorsed. After 1 day and 7 days of detorsion, the testes were collected for morphological observation via HE and TUNEL staining. The activities of superoxide dismutase (SOD) and malondialdehycle (MDA) were measured by xanthine superoxidase and thiobarbiuric acid, respectively. The localization of TSEG-2 was observed by in situ hybridization. The TSEG-2 expression in testes was measured by real-time quantitative PCR. Results:Compared with the regular seminiferous tubules in control and sham groups, the seminiferous tubules of experimental group were irregular, with sparse structure and obvious apoptosis of spermatogenic cells. After 1 day and 7 days of detorsion, the Johnsen’s score was reduced by 23.4% and 64.1% (P<0.01), the SOD activities were decreased by 11.6% and 22.2%(P<0.05), with those of MDA increased by 69.6% and 93.2%(P<0.01). The TSEG-2 mRNA localized at the spermatogonia and spermatocytes of seminiferous tubules. After 1 day and 7 days of detorsion, the TSEG-2 expression was enhanced by 2.2 and 6.6 times, respectively, when compared with control group. Conclusions:The mouse testicular torsion/detorsion model was successfully established. The enhanced TSEG-2 expression may be correlated with impaired anti-oxidative activities and apoptosis of spermatogenic cells. Part 3 Expression pattern of testis-specific expressed gene 2 in cryptorchidism model and its role in apoptosis of spermatogenic cellsIn our previous study, we identified a novel testis-specific expressed gene 2 (TSEG-2) from mouse testis. To further investigate its preliminary function, thirty-five male BALB/C mice (8 weeks old) were divided into cryptorchidism group (n=20), sham group (n=10), and control group (n=5). In cryptorchidism group, the right testes were anchored to the inner lateral abdominal wall. Real-time quantitative PCR was undertaken to detect the expression of TSEG-2 gene. Meanwhile, under the mediation of polyethylenimine (PEI), the recombinant vector pEGFP-TSEG-2 (n=5) or empty vector (mock, n=5) was transfected into the testis of male mice. The transfection efficiencies were measured under a fluorescence microscope. The apoptosis of spermatogenic cells was detected by terminal deoxynuleotidyl-mediated nick end labeling (TUNEL). The results showed that the TSEG-2 transcript was significantly enhanced (P<0.05) and correlated with apoptosis of spermatogenic cells in cryptorchid testes (P<0.05),when compared with sham and control groups. PEI was efficient in mediating transfection of TSEG-2 into seminiferous tubules of testis. One week post-transfection, intratesticular injection of TSEG-2 resulted in increased apoptosis of spermatogenic cells in vivo (P<0.05). These results indicate that TSEG-2 may participate in the apoptosis of spermatogenic cells and the pathogenesis of cryptorchidism.更多还原