小鼠睾丸引带细胞的培养及己烯雌酚对培养睾丸引带细胞的影响

目的:探讨小鼠睾丸引带细胞体外培养的有效方法,并进行形态学观察。研究经典的外源性雌激素己烯雌酚(DES)体外对小鼠睾丸引带发育的影响。方法:手术放大镜解剖出3日龄雄性昆明小鼠的睾丸引带组织并进行细胞培养。台盼蓝染色检测细胞存活率,HE染色观察细胞形态。传代培养并随机分为空白对照组、溶剂对照组(DMSO组)和实验组(DES 1~4组)共6组。溶剂对照组加入DMSO,实验组分别加入0.01、0.10、1.00以及10.00μg/ml DES。分别于培养12、24、48 h后进行细胞形态学观察,CCK-8法检测睾丸引带细胞的生长情况。结果:培养睾丸引带细胞大部分为成纤维细胞型,有少数的上皮样细胞。原代细胞的存活率为85%~90%。在不同剂量DES作用后的3个不同时间段里,细胞增殖性的检测结果存在时间-剂量效应的显著差异(P<0.01)。结论:睾丸引带细胞可经一定的方法进行体外培养,外源性雌激素对睾丸引带细胞的生长有抑制作用,且呈现一定的时间-剂量效应。对培养睾丸引带细胞影响的研究是外源性雌激素影响生殖系统发育研究的一条有效的睾丸外途径。     

己烯雌酚对小鼠睾丸引带中LGR8表达的影响

目的:通过体内实验研究己烯雌酚(DES)对小鼠睾丸引带中胰岛素样因子3受体LGR8的影响,从而探讨外源性雌激素对小鼠睾丸下降的影响。方法:8～10周龄KM孕鼠随机分为正常组、空白对照组和实验组(0.1、1.0、10、100μg/kg DES 4个剂量组),共6组,每组20只。于孕9～17 d每天分别给予实验组妊娠小鼠不同剂量的DES(0.1、1、10、100μg/kg),空白对照组给予等体积DMSO+生理盐水,正常组不给药。应用免疫组化和RT-PCR分别检测胎鼠和幼鼠睾丸引带中LGR8蛋白和mRNA的表达。结果:HE染色可见胎鼠正常组、对照组睾丸引带发育良好,中间的间叶组织和外周的肌源细胞之间分界清楚;实验组可见睾丸引带发育不良,间叶组织和肌源细胞之间无明显分界,组织结构紊乱。幼鼠正常组和实验组睾丸引带发育未见明显不同。LGR8表达于睾丸引带肌源细胞和间叶组织细胞,以肌源细胞表达为主。实验组LGR8阳性表达较正常组弱,胎鼠各实验组和幼鼠DES 1、10、100μg组与同发育阶段的正常组相比均有统计学意义(P<0.05)。DES高剂量(10、100μg)组与同发育阶段的正常组相比LGR8 mRNA表达增加(P<0.05)。各实验组PCR产物测序均未见明显突变。结论:DES可影响小鼠睾丸引带LGR8 mRNA和蛋白的表达,DES可能通过影响INSL3-LGR8信号系统干扰睾丸引带的发育,进而影响睾丸正常下降。     

新生小鼠睾丸引带形态结构及其异常的显微连续研究

目的:应用新生小鼠生殖系统整体原位固定、横断面连续组织切片显微观察的研究方法,整体展现正常及异常状态下新生小鼠睾丸引带的形态结构,以探讨应用整块组织固定、连续切片显微研究的方法在判定生殖系统形态异常中的适用性。方法:正常及孕期(9~17d)接触不同剂量外源性雌激素己烯雌酚(DES)的新生雄性昆明小鼠,切取其腹部(自膈下)固定、包埋,作连续切片、染色。显微镜下顺序摄像,分析睾丸引带的形态结构,并测算其相应位置和大小。结果:显微连续观察显示新生小鼠睾丸引带的结构清晰,形态结构各段变化较大且左右不对称,DES对睾丸引带形态结构发育的影响各段不尽相同,而且明显影响整个引带的长度。结论:孕期接触DES对新生小鼠睾丸引带的发育有明显影响,其影响是整体性的。整块组织切片显微连续研究对睾丸引带或及其它相关生殖系统器官的形态结构评价能够比较全面和精确。     

RXFP2在己烯雌酚染毒小鼠睾丸引带细胞中的作用机制研究

目的通过检测己烯雌酚(DES)对小鼠睾丸引带细胞形态与增殖性影响过程中松弛素家族肽受体2(RXFP2)的表达水平,探讨RXFP2在外源性雌激素影响小鼠睾丸引带细胞中的可能作用机制。方法小鼠睾丸引带组织进行细胞培养,传代后随机分为正常组、溶剂对照组及实验组,实验组分别加入DES终浓度为0.01、0.10、1.00、10.00μg/m L作用48 h。观察细胞形态变化,CCK-8检测其增殖情况,应用免疫荧光和Western Blot对小鼠睾丸引带细胞中RXFP2进行定位和蛋白定量分析。结果正常组细胞呈成纤维细胞型生长,同源性好。各实验组随DES剂量增加细胞数及细胞生存率均下降,细胞增殖明显受抑制,失去正常形态,胞浆收缩,胞体形态不规则。免疫荧光显示RXFP2高表达于细胞膜上。蛋白定量分析发现各实验组RXFP2水平与正常组相比表达降低,且差异有统计学意义。结论DES可影响小鼠睾丸引带细胞形态与增殖性以及RXFP2蛋白的表达,推测DES可能通过影响RXFP2信号系统介导睾丸引带的发育,进而影响睾丸的下降。     

影响睾丸引带发育的内分泌因素研究进展

睾丸引带与睾丸下降关系密切,它的分化发育和多种因素有关,特别是雄激素、降钙素基因相关肽、胰岛素样因子3、苗勒管抑制物质、表皮生长因子以及环境雌激素(EEs)等,其中降钙素基因相关肽、胰岛素样因子3和EEs对生殖系统的影响是目前研究的热点。从影响睾丸引带发育的角度探讨EEs对睾丸及生殖发育的影响很有意义。     

己烯雌酚对大鼠睾丸组织干细胞因子表达水平的影响

雌激素是精子发生过程中关键激素之一 ,其具体作用机制尚待探讨[1] 。c kit受体与其配体干细胞因子 (SCF)的相互作用 (SCF/c kit系统 )在精子生成中有重要的作用。在成年雄性大鼠睾丸中 ,编码c kit的mRNA存在于精原细胞、初级精母细胞、圆形精子细胞及睾丸间质细胞 ,但表达SCF的细胞只有支持细胞[2 ] 。所以目前认为支持细胞损伤及恢复与SCF表达水平有重要联系。本实验拟应用己烯雌酚 (DES)对 2 ,5 己二酮(支持细胞毒物 )导致的大鼠生精障碍模型进行干预 ,以探索DES与睾丸内SCF表达水平的关系。一、材料和方法1.实验动物 :健康雄…     

不同剂量己烯雌酚对新生小鼠睾丸表皮生长因子及受体的影响

目的 观察不同剂量己烯雌酚(DES)对新生小鼠睾丸组织中表皮生长因子(EGF)及其受体(EGFR)的影响,探讨DES对睾丸发育影响的机制.方法 建立小鼠DES模型.将72只怀孕的雌性昆明小鼠随机分成3组:正常组、对照组及实验组1～4(DES 10、25、50、100 μg/kg).采用免疫组织化学方法检测各组新生小鼠睾丸组织中EGF、EGFR的表达.结果 EGF和EGFR主要表达在新生小鼠睾丸的间质细胞.实验组1～4中EGF阳性细胞的累积吸光度值(IA)分别为75.43±1. 42、52.22±5.67、13.75±3.14、6.38±3.20,显著低于正常组及对照组中阳性细胞的IA值433.88±11.64、23.44±4.70;实验组EGFR阳性细胞的IA值分别为198.16±34.35、138.00±12.04、46.03±6.74、27.22±5.52,显著低于正常组及对照组中阳性细胞的IA值804.74±22.52、800.03±21.96.两者在正常、不同剂量DES实验组的两两比较差异有统计学意义(P＜0.05).随DES剂量增加,EGF和EGFR表达减弱,各组间差异有统计学意义(P＜0.05).EGF与EGFR呈强正线性相关(r=0.750,P＜0.01).结论 不同剂量DES对新生小鼠睾丸组织中EGF和EGFR表达强度均有影响,可能是影响睾丸发育机制之一.

Abstract：

Objective To investigate the effects of prenatal exposure to diethylstilbestrol (DES)with different dosages on epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in offspring mice testis, and the possible mechanism. Methods DES model was induced in mice by DES.Female Kungmiag mice were randomly divided into normal group, control group and experimental groups 1-4 ( DES 10, 25, 50, 100 μg/kg). Immunohistochemistry was applied to detect the expression of EGF and EGFR in the testicular tissue in each group. Results EGF and EGFR were expressed mainly in sffspring mice testis Leydig cells. The cumulative absorbance ( IA ) values of EGF positive cells in experimental groups 1-4 were 75. 43 ± 1.42, 52. 22 ± 5.67, 13.75 ± 3. 14, and 6. 38 ± 3.20 respectively, which were significantly lower than in normal and control groups (433.88 ± 11.64,423.44 ±4. 70 respectively).The IA values of EGFR positive cells in experimental groups 1-4 were 198. 16 ± 34. 35, 138.00 ± 12.04,46.03 ± 6. 74, 27.22 ± 5.52 respectively, which were significantly lower than in normal and control groups (804. 74 ± 22. 52,800. 03 ± 21.96 respectively). The expression levels of EGF and EGFR could be detected in each subgroup and statistically significant differences existed in the expression of EGF and EGFR between any two groups ( P ＜ 0. 05 ). With the increase of DES dosage, the expression of EGF and EGFR was decreased, with the difference being significant amond the groups ( P ＜ 0. 05 ). Conclusion DES can influence the expression of EGF and EGFR in mice testis, which might be one of possible mechanisms effecting the development of testis.     

胰岛素样激素3与睾丸引带发育和睾丸下降

睾丸下降是生殖系统发育过程中的重要阶段,其机制尚未阐明。睾丸引带发育与睾丸下降关系密切。睾丸下降不全常常伴随睾丸引带发育异常,它们除了可引起隐睾和睾丸发育不良等疾病外,还是生殖系统先天畸形以及以后生殖功能、性功能异常和高比例生殖系统肿瘤发生的原因。胰岛素样激素3是近年来倍受关注的影响睾丸引带发育和睾丸下降的重要因素,本文就相关进展进行综述。     

邻苯二甲酸二(2-乙基)己酯对小鼠胎鼠睾丸及睾丸引带的影响

目的:研究邻苯二甲酸二(2-乙基)己酯(DEHP)对小鼠胎鼠睾丸与睾丸引带形态结构及功能的影响,探讨隐睾发生的可能机制。方法:30只健康KM孕鼠随机均分为3组:空白对照组、玉米油对照组、DEHP组。DEHP组以剂量500mg/(kg.d)的DEHP灌胃作用于怀孕12～19d(GD12～GD19)的KM母鼠,观察DEHP对每胎胎鼠数、雌雄性胎鼠比例、雄性胎鼠体重、睾丸重量、睾丸引带形态结构、位置、睾丸到膀胱颈之间的相对距离(TBD)、颅侧悬韧带残留情况、引带内雄激素受体(AR)、雌激素受体(ER)、肌动蛋白、增殖细胞核抗原(PCNA)表达水平的影响。结果:DEHP对每胎胎鼠数、雌雄性胎鼠比例、雄性胎鼠体重无明显影响;DEHP可影响胎鼠睾丸重量及TBD;DEHP组睾丸均有一定程度的下降不全,睾丸引带形态结构无明显差异;光镜下见DEHP组睾丸生精小管、支持细胞存在明显的发育障碍,睾丸Leydig细胞明显增生;DEHP组睾丸引带AR阳性表达率降低(P<0.01)。结论:DEHP可通过抗雄激素效应导致睾丸引带功能障碍,使睾丸下降发生异常而诱导小鼠隐睾;同时造成睾丸Ser-toli细胞、睾丸Leydig细胞和生精细胞的发育障碍和功能改变。     

腹横纹切口保留睾丸引带阴囊皮下睾丸固定术治疗隐睾症的研究

为了研究治疗先天性隐睾症的最佳术式，以提高隐睾症的治疗水平，采用腹横纹切口保留睾丸引带明囊皮下睾丸固定术（研究组）治疗隐睾症96例110枚，获得随访者94例108枚，同时采用患倒下腹斜切口肉膜囊睾丸固定术（对照组）治疗隐睾症50例62枚。结果研究组睾丸大小及硬度化94枚，占87．0％，良9枚，占8．3％，差5枚，占4．6％；睾丸位置优93枚，占86．1％，良9枚，占8．3％，差6枚，占5．6％；无睾丸萎缩及回缩，外表美观。对照组睾丸大小及硬度优41枚，占66．1％，良7枚，占11．3％，差14枚，占22．6％；睾丸位置优43枚，占69．4％，良8枚，占12．9％，差11枚，占17．7％。经统计学处理，两组睾丸大小及硬度方面比较有极显著性差异（P＜0．01），睾丸位置比较也有显著性差异（P＜0．05）。认为腹横纹切口保留睾丸引带阴囊皮下睾丸固定术损伤小，外表美观，明显降低了睾丸萎缩及回缩等并发症，符合生理要求，疗效满意。     

放射治疗对小鼠肾癌细胞凋亡和Bcl-2与Bax蛋白表达的影响

目的:观察放射治疗对BALB/c小鼠肾癌的抑制作用及对Bcl-2和Bax蛋白表达的影响.方法:建立BALB/c小鼠的肾癌细胞移植瘤放射治疗模型;HE染色测定肿瘤细胞死亡面积,采用免疫印迹法(western blot法)检测肿瘤组织的凋亡相关蛋白表达.结果:放疗组小鼠移植瘤生长速度较肿瘤对照组明显减慢.放疗组小鼠肿瘤组织中抗凋亡蛋白Bcl-2表达下调,促凋亡蛋白Bax表达上调.结论:经放射治疗后,小鼠肿瘤生长受到明显抑制,可能与放疗引发肿瘤细胞凋亡相关蛋白表达变化相关.     

Gonadoblastoma in dysgenetic testis causing male pseudohermaphroditism in newborn

A newborn with dysgenetic male pseudohermaphroditism associated with a gonadoblastoma is presented. The importance of differentiating this syndrome from other forms of male pseudohermaphroditism is emphasized.     

Leptin regulates gallbladder genes related to gallstone pathogenesis in leptin-deficient mice

BACKGROUND: Little is known about the genetic factors that cause alterations in gallbladder motility, cholesterol crystal nucleation, biliary lipids, and, ultimately, cholesterol gallstones. Obese, leptin-deficient (Lep(ob)) mice have large gallbladder volumes with decreased contraction in vitro and are predisposed to cholesterol crystal formation. Leptin administration to these mice causes weight loss and restores gallbladder function. We hypothesize that administration of leptin to Lep(ob) mice would cause weight loss, decrease gallbladder volume, and change gallbladder genes related to gallbladder motility, nucleating factors, and lipid metabolism. STUDY DESIGN: Twenty-four 8-week-old Lep(ob) mice were fed a nonlithogenic diet for 4 weeks. Twelve mice received daily IP saline injections, and 12 received 5 mug/g recombinant leptin. Gallbladder mRNA was pooled and analyzed on murine genome microarray chips. Selected genes were confirmed by real-time polymerase chain reaction (PCR) in a second group of mice treated by the same protocol. RESULTS: Leptin-deficient mice given leptin had significant weight loss and reductions in gallbladder volume. These mice had upregulation of the leptin receptor (p = 0.007; PCR = 1.1-fold increase) but downregulation of leptin (p = 0.003; PCR = 13.5-fold decrease). Leptin upregulated the cholecystokinin A receptor (p < 0.001; PCR = 3.1-fold increase), acetylcholine beta2 receptor (p = 0.005), and the Ca+-calmodulin-dependent protein kinase (p = 0.002) genes. Leptin also altered immunoglobulin heavy chain 4 (p = 0.005; PCR = 17.7-fold increase), mucin 3 (p = 0.006), and carboxylesterase (p = 0.016; PCR = 2.5-fold decrease) genes. Leptin downregulated 3-hydroxy 3-methylglutaryl coenzyme A reductase (p = 0.006; PCR = 2.5-fold decrease) and LDL receptor (p = 0.003). CONCLUSIONS: Leptin modulates obesity and regulates gallbladder genes related to cholesterol gallstone pathogenesis.     

抗精子抗体对小鼠睾丸超微结构的影响

目的研究免疫性不育雄性小鼠的睾丸超微结构的特点,探讨抗精子抗体（AsAb）对睾丸生精微环境的影响。方法用同种精子及福氏佐剂免疫昆明种雄性小鼠。ELISA法检测AsAb,以此验证获得免疫性不育动物模型,同时设立对照组;解剖小鼠取其精子观察精子质量差别,透射电镜观察各组睾丸的超微结构。结果人工免疫后的雄鼠血清AsAb均为阳性。正常对照组均为阴性;模型组小鼠精子质量与对照组比较显著下降,生精上皮超微结构受损严重。结论用同种精子及福氏佐剂免疫动物,可成功制作抗精子抗体介导的免疫性不育动物模型;AsAb可通过攻击睾丸生精微环境影响雄性小鼠的生育力。     

Validation of reference genes in human testis and ejaculate

Beta-actin (ACTB), glyceraldehyde-3-phosphate-dehydrogenase (GAPD), Heat Shock Protein 1, beta (HSPCB) and Adenosine Triphosphate subunit 5 beta (ATP5B) with distinct functional characteristics and expression patterns were investigated as suitable references for gene expression studies. We determined the expression stability of the four reference genes in ejaculates, cryopreserved as well as fixed and paraffin-embedded testicular tissue (from fertile and subfertile men) applying real-time qRT-PCR and statistical analysis. The mean gene expressions (mean Ct value) were compared for each gene between the fertile and subfertile donors by using the Wilcoxon-Mann-Whitney test. We did not observe significant statistical differences between variability of genes. To detect random effects, we used the two-way analysis of variance with a hierarchical model. The results show no significant statistical differences between proband and repetition within the probands. Taken together, we concluded that ACTB, GAPD, HSPCB and ATP5B have a variable expression within these samples, but this variability is not statistically significant. This finding demonstrated that all these genes could be appropriated for further studies on gene expression in ejaculate and testis tissue. Therefore, the selection of the suitable reference genes is highly specific for a particular experimental model and validation for each situation, on an individual basis, is a crucial requirement.