实验性精索静脉曲张对青春期大鼠睾丸、附睾中CRES蛋白表达的影响

目的:研究青春期大鼠实验性左侧精索静脉曲张(ELV)对睾丸和附睾中胱蛋白酶抑制剂相关的附睾精子发生(CRES)蛋白表达的影响,探索精索静脉曲张导致不育的机制。方法:建立青春期雄性SD大鼠ELV模型,采用免疫组化及蛋白印迹方法检测CRES蛋白在ELV 2周组(n=8)、4周组(n=8)及其相应的对照组(n均=5)大鼠睾丸和附睾头、体、尾中的表达变化。结果:免疫组化显示,CRES蛋白在ELV实验组和对照组大鼠睾丸和附睾中均有表达。在睾丸中,CRES蛋白主要定位于圆形和长形精子细胞的胞质、精子的顶体以及残余体中,其表达与生精周期密切相关,Ⅰ~Ⅲ和Ⅸ~ⅩⅣ期表达最强,Ⅶ~Ⅷ期表达次之,Ⅳ~Ⅵ期表达减弱。在附睾中,CRES蛋白主要表达于从附睾起始段到尾部的上皮主细胞的胞质中,且以体、尾部表达最强,头部次之,管腔分泌物也呈阳性表达。实验组比对照组CRES蛋白表达增强。W estern印迹显示:实验组和对照组大鼠睾丸和附睾在相对分子质量(Mr)为19 000和14 000处均可见CRES蛋白条带,其中以Mr19 000处表达更为明显。实验组CRES蛋白的表达比对照组明显增强。对免疫组化和W estern印迹结果进行灰度值和积分吸光度值(IA)测定,并经统计学分析显示:ELV 2周组和4周组比其相对应的对照组表达增强且差异有显著性(P<0.05或P<0.01),而ELV各组间未见CRES蛋白表达有明显差异(P>0.05)。结论:CRES蛋白在大鼠睾丸和附睾中均有表达,睾丸中表达呈现生精周期特异性和细胞特异性,附睾中表达呈现附睾上皮区段和细胞特异性;CRES蛋白在青春期ELV大鼠睾丸和附睾中的表达比对照组明显增强。这些结果提示CRES蛋白在精子发生和精子成熟过程中可能起着重要的作用,并且可能与ELV引起的不育或生育力低下有关。     

精索静脉曲张与氧化应激的研究

目的 :研究精索静脉曲张 (VC)时氧化应激的损害机制。　方法 :2 8例VC不育男性 ,行精索内静脉结扎术 ,采精索内静脉血和外周静脉血 ;建立大鼠左侧VC模型 (n =12 ) ,以假手术大鼠为对照组 (n =8) ,术后 3个月取睾丸组织。采用分光光度法检测VC男性血浆及大鼠睾丸组织中一氧化氮 (NO)、一氧化氮合酶 (NOS)、黄嘌呤氧化酶(XO)、乳酸 (Lac)和乳酸脱氢酶 (LDH)含量。　结果 :VC患者精索内静脉血浆中NO、NOS、XO、Lac含量明显高于外周静脉血浆 (P <0 .0 1,P <0 .0 5 ) ,LDH明显降低 (P <0 .0 1)。实验性VC大鼠左侧睾丸组织NO、XO高于对照组 (P <0 .0 1) ,Lac明显降低 (P <0 .0 1)。　结论 :VC时 ,可由于曲张精索静脉血浆中NO、NOS和XO及睾丸组织中NO和XO产生增加 ,以及Lac和LDH含量的变化 ,而导致精子生成障碍或 /和精子活力下降 ,引起男性不育。     

强精胶囊对精索静脉曲张大鼠附睾功能性指标及精子质量的影响

目的:观察强精胶囊对精索静脉曲张大鼠附睾功能性指标及精子质量的影响.方法:100只青春期雄性SD大鼠,随机抽取10只为假手术组,90只缩窄左肾静脉建立精索静脉曲张模型,随机分为模型组、强精胶囊高中低剂量组、五子衍宗胶囊组和少腹逐瘀胶囊组.分别给予相应药物灌胃,观察大鼠附睾精子密度与活力的变化,检测附睾α-糖苷酶的含量(葡萄糖氧化酶法)和L-肉毒碱的含量(DTNB法).结果:模型组大鼠附睾精子活力与密度低下、α-糖苷酶与L-肉毒碱含量低下;强精胶囊高剂量组精子活力与密度、α-糖苷酶与L-肉毒碱含量较中、低剂量组、五子衍宗胶囊组、少腹逐瘀胶囊组明显增高(P<0.01).结论:强精胶囊可以改善模型大鼠精子密度与活力,提高附睾α-糖苷酶与L-肉毒碱的含量.     

实验性精索静脉曲张大鼠附睾分泌蛋白的鉴定研究

目的　研究精索静脉曲张对附睾合成与分泌蛋白的影响 ,鉴定病理状况下附睾特异改变的蛋白成分。　方法　部分结扎左肾静脉建立实验性左侧精索静脉曲张大鼠动物模型。用 SDS-PAGE和双向电泳分离左附睾尾匀浆上清中的蛋白质组分 ,切下稳定出现改变的蛋白质所在的条带或斑点 ,进行胶内胰蛋白酶消化 ,酶解得到的最终肽混合物进行肽质量指纹谱测定。通过检索互联网上的蛋白质数据库确定该蛋白的可能成分。　结果　与对照动物相比 ,实验性左侧精索静脉曲张大鼠左附睾尾匀浆上清中一分子量约2 0× 1 0 3、等电点约 5.5的蛋白成分明显增加。经质谱分析结合网上数据库检索表明 ,该蛋白是附睾视黄酸结合蛋白。　结论　实验性左侧精索静脉曲张导致大鼠附睾中视黄酸结合蛋白含量升高 ,这一改变对精子在附睾中成熟及精子功能的影响有待进一步研究     

瘦素及其受体在精索静脉曲张模型大鼠附睾组织的表达及意义

目的:探讨瘦素及其受体在精索静脉曲张模型大鼠附睾组织的表达及其意义。方法:将40只SD大鼠随机分为精索静脉曲张(EV)持续4、8周组(EV4、EV8组,每组12只)和相应的假手术对照组(Control 4、Control 8组,每组8只)。采用左肾静脉部分结扎的方法建立精索静脉曲张模型。采用免疫组织化学法检测大鼠附睾组织中瘦素及瘦素受体表达情况,采用实时定量PCR法检测大鼠附睾组织中瘦素及瘦素受体mRNA表达量。结果:EV4组、EV8组附睾上皮细胞瘦素及瘦素受体表达分别比Control 4组、Control 8组显著增强(P<0.01);而EV4组附睾上皮细胞瘦素及瘦素受体表达与EV8组比较无显著性差异(P>0.05)。EV4组、EV8组附睾上皮细胞瘦素及瘦素受体mRNA的表达分别比Control 4组、Control 8组显著增加(P<0.01);而EV4组瘦素及瘦素受体mRNA的表达与EV8组比较无显著性差异(P>0.05)。结论:精索静脉曲张大鼠附睾组织中瘦素及瘦素受体的表达明显增加,瘦素可能参与了精索静脉曲张引起男性不育的机制。     

精索静脉曲张大鼠附睾组织中低氧诱导因子1α和p53的表达

目的:观察精索静脉曲张(VC)大鼠附睾指数,附睾精子活率,附睾组织中低氧诱导因子1α(HIF-1α)、p53的表达及上皮组织的变化,探讨VC大鼠附睾功能降低的机制。方法:90只SD大鼠随机分为手术组、假手术组和正常对照组,每组30只。手术组建立SD大鼠左侧VC模型,采用假手术建立假手术组,正常对照组不作任何处理。各组大鼠均于手术组术后49 d断颈处死。处死前大鼠称重,处死后左侧附睾称重,收集附睾尾部精子,以计算机辅助精子分析系统行精子活率检测;应用Western印迹检测附睾组织中HIF-1α和p53的表达变化;HE染色观察附睾上皮组织的情况。结果:手术组27只大鼠建模成功。3组大鼠附睾指数差异无统计学意义[(40.53±1.76)×10-5、(43.31±1.58)×10-5、(44.10±2.62)×10-5,P0.05];手术组大鼠精子活率[(71.86±5.07)%]和前向运动精子百分率[(42.26±4.45)%]均显著低于假手术组[(78.51±4.50)%、(49.08±4.19)%]和正常对照组[(79.24±2.70)%、(52.23±2.23)%](P均0.05);手术组大鼠附睾组织HIF-1α和p53相对表达量[1.74±0.16、1.71±0.11]均显著高于假手术组[0.32±0.08、0.56±0.13]和正常对照组[0.12±0.03、0.25±0.06](P均0.01);手术组附睾管上皮细胞与另外两组相比明显疏松无序并且数量减少。结论:VC导致附睾组织低氧并可能使附睾功能降低。     

大鼠精索静脉曲张模型附睾中丙二醛、总抗氧化物及唾液酸含量的改变及意义

目的:探讨大鼠左侧精索静脉曲张后同侧附睾组织中丙二醛(MDA)、总抗氧化物(TAC)和附睾管腔中唾液酸(SA)含量的改变及意义。方法:用8只雄性SD大鼠建立左侧精索静脉曲张模型,另取8只SD大鼠作为对照组。硫代巴比妥酸法和铁离子还原法分别检测附睾组织中MDA和TAC含量;5甲-基苯二酚法检测附睾腔液中SA含量。结果:大鼠左侧精索静脉曲张模型建立后的第7 d,同侧附睾组织中MDA和TAC含量,模型组与对照组相比,差异有显著性(P<0.005);附睾管腔液中SA含量,模型组明显低于对照组,差异有显著性(P<0.005)。结论:大鼠左侧精索静脉曲张会导致同侧附睾组织中活性氧增加,TAC含量下降,进而影响到附睾上皮细胞合成和分泌SA的功能。     

Palomo和改良Palomo曲张精索内静脉结扎术对大鼠附睾的影响

目的:探讨Palomo曲张精索内静脉结扎术(PV)和改良Palomo曲张精索内静脉结扎术(MPV)对大鼠同侧附睾的影响。方法:50只雄性青春期SD大鼠,随机分为对照组、实验性精索静脉曲张(EV)组、PV组和MPV组。部分结扎左肾静脉建立大鼠EV模型,MPV组大鼠行保留睾丸动脉的曲张精索内静脉结扎术(MPV),PV组大鼠行包含睾丸动脉的曲张精索内静脉集束结扎术(PV)。检测各组大鼠左侧附睾的显微结构、超微结构、唾液酸和肉毒碱含量。结果:EV组和PV组附睾上皮的显微结构和超微结构明显异常。对照组唾液酸和肉毒碱浓度显著高于EV组和PV组(P<0.05),与MPV组比较差异无统计学意义(P>0.05)。结论:EV可导致大鼠同侧附睾组织结构和功能的损害,MPV可以修复这些损害,而PV则进一步加重这些损害。     

实验性精索静脉曲张对青春期大鼠睾丸、附睾精子结合抗原11 mRNA及其蛋白异构体表达的影响

目的:研究实验性左侧精索静脉曲张(ELV)对青春期大鼠睾丸和附睾中精子结合抗原11(SPAG11)mRNA及其蛋白异构体SPAG11E表达的影响,并探讨其与精索静脉曲张导致男性不育的关系。方法:40只SD大鼠随机分为ELV2周组、4周组,假手术对照2周组、4周组,每组10只。ELV组行部分结扎左肾静脉建立青春期SD大鼠ELV模型,假手术对照组仅显露左肾静脉过程,但不结扎。应用RT-PCR和免疫组化法(n=5)检测SPAG11 mR-NA及SPAG11E蛋白在ELV2周组和ELV4周组及各自对照组大鼠双侧睾丸、附睾中的表达变化。结果:RT-PCR结果显示,SPAG11基因376bp的特异扩增产物仅见于大鼠附睾组织中。免疫组化结果显示,SPAG11E蛋白主要与睾丸生精上皮的圆形和长形精子细胞的顶体泡和顶体、睾丸间质细胞的胞质相结合;在附睾管上皮主细胞胞质和顶部静纤毛中表达。对RT-PCR的相对吸光度值和免疫组化的灰度值进行统计学分析显示:①左侧附睾ELV2周和4周组SPAG11 mRNA和SPAG11E蛋白的表达与各自右侧组及各自对照组比较均有显著减弱(P<0.05或P<0.01);左侧附睾ELV4周组SPAG11mRNA与SPAG11E的表达较ELV2周组显著减少(P<0.05或P<0.01);右侧附睾ELV4周组SPAG11E的表达也较ELV2周组显著减少(P<0.01);②两实验组双侧睾丸SPAG11E蛋白的表达与相应对照组比较均未见明显差异(P>0.05),且在ELV2周组和ELV4周组之间该蛋白的表达也无显著性差异(P>0.05)。结论:SPAG11是特异表达于附睾的基因,在大鼠睾丸和附睾中均可见其蛋白异构体SPAG11E免疫阳性反应,其定位及表达水平具有细胞特异性和区域特异性,而且SPAG11 mRNA及SPAG11E蛋白在ELV模型鼠附睾中的表达发生了明显变化,这提示SPAG11不仅可能在大鼠精子发生、成熟过程中发挥重要作用,还可能与精索静脉曲张所致的男性不育相关。     

Loss of the tight junction protein ZO-1 in dextran sulfate sodium induced colitis

BACKGROUND: Inflammatory bowel disease (IBD) is associated with increased intestinal permeability and decreased expression of tight junction (TJ) proteins in the inflamed mucosa. Whether this alteration in TJ expression is a prerequisite for the development of intestinal inflammation or a secondary result of that inflammation is unknown. This study looked at the expression of the TJ protein ZO-1 and the corresponding permeability changes in dextran sulfate sodium (DSS) induced colitis in a mouse model. MATERIALS AND METHODS: BALB/c mice were fed 3% DSS or water for 1, 3, 5, or 7 days. The animals were weighed, stool was checked for blood, and the colon length measured. Segments of the colon were used for histology, immunohistochemistry for ZO-1, or Western blot for TJ proteins. Colonic permeability was measured using Evan's Blue dye. RESULTS: DSS treated animals had heme positive stools, colitis by histology, significant weight loss, and colon shortening. There was an absence of ZO-1 by Western blot in the 7-day DSS treated animals, double the amount of claudin-1 and normal cytokeratin. The loss of ZO-1 started after 1 d of DSS treatment and was followed by a significant increase in permeability to Evan's blue by day 3. CONCLUSIONS: The loss of ZO-1 and increased permeability preceded the development of significant intestinal inflammation suggesting that in DSS colitis alterations in the TJ complex occur before the intestinal inflammation and not as a consequence of it. These changes in the TJ complex may facilitate the development of the inflammatory infiltrate seen in colitis.     

紧密连接蛋白ZO-1在肾癌中的表达及其临床意义

目的 探讨闭锁小带蛋白(sonula occluden-1,ZO-1)这种紧密连接相关蛋白在肾癌中的表达情况及其临床意义。方法 采用免疫组织化学法检测55例肾癌患者手术切除的肾脏病理标本中正常肾组织以及肾癌组织中ZO-1蛋白的表达,并分析肾癌组织中ZO-1的表达与肿瘤的侵袭和转移之间的关系。结果 ZO-1蛋白在正常肾组织和肾癌组织中的阳性表达率分别为74.5%和32.7%。 两组间进行比较显示,ZO-1蛋白在肾癌组织中的表达水平明显低于正常肾组织,组间具有差异性（P<0.05）。在肾癌组织中,ZO-1蛋白的表达水平与癌组织的临床分期、分化程度以及淋巴结转移有关。结论 人肾癌组织中ZO-1蛋白的表达较正常肾组织减少,并且其可能参与肾癌肿瘤细胞的侵袭和转移。     

热射病对大鼠小肠上皮紧密连接occludin蛋白的影响

目的 观察热射病大鼠小肠上皮屏障通透性的改变,并通过观察肠上皮细胞紧密连接形态及肠上皮细胞紧密连接蛋白occludin蛋白表达的变化,初步探讨热射病对肠上皮屏障通透性的影响及机制.方法 将SD大鼠随机分为两组,每组10只(n=10),热射病组及常温对照组,制备热射病大鼠模型,动物麻醉后放入42 ℃恒温箱热暴露50 mi...     

跨膜结合蛋白occludin在血吸虫病肝硬化小鼠结肠上皮细胞中的表达及其意义

目的:探讨跨膜结合蛋白occludin在血吸虫病肝硬化小鼠结肠上皮细胞中的表达变化及其意义。方法:将40只健康小鼠随机分成对照组(N组)和模型组(M组)各20只,M组采用腹部敷贴法制作血吸虫病模型;造模后120 d处死动物,用鲎试验法测定门静脉血内毒素水平;免疫组化和半定量Western印迹方法测定结肠上皮紧密连接蛋白occludin的表达;透射电镜观察结肠上皮细胞的超微结构。结果:M组门静脉血内毒素水平高于N组(P0.01);M组结肠上皮occludin蛋白的表达水平低于N组(P0.01);两者之间呈显著正相关。结论:血吸虫病肝硬化可发生肠源性内毒素易位、内毒素血症,可能与紧密连接蛋白occludin表达下降和紧密连接蛋白结构异常有关。     

Structure and function of epididymal protein cysteine-rich secretory protein-1

Cysteine-rich secretory protein-1 （CRISP-1） is a glycoprotein secreted by the epididymal epithelium. It is a member of a large family of proteins characterized by two conserved domains and a set of 16 conserved cysteine residues. In mammals, CRISP-1 inhibits sperm-egg fusion and can suppress sperm capacitation. The molecular mechanism of action of the mammalian CRISP proteins remains unknown, but certain non-mammalian CRISP proteins can block ion channels. In the rat, CRISP-1 comprises two forms referred to as Proteins D and E. Recent work in our laboratory demonstrates that the D form of CRISP-1 associates transiently with the sperm surface, whereas the E form binds tightly. When the spermatozoa are washed, the E form of CRISP-1 persists on the sperm surface after all D form has dissociated. Cross-linking studies demonstrate different protein-protein interaction patterns for D and E, although no binding partners for either protein have yet been identified. Mass spectrometric analyses revealed a potential post-translational modification on the E form that is not present on the D form. This is the only discernable difference between Proteins D and E, and presumably is responsible for the difference in behavior of these two forms of rat CRISP- 1. These studies demonstrate that the more abundant D form interacts with spermatozoa transiently, possibly with a specific receptor on the sperm surface, consistent with a capacitation-suppressing function during sperm transit and storage in the epididymis, and also confirm a tightly bound population of the E form that could act in the female reproductive tract. （Asian J Androl 2007 July; 9： 508-514）     

Oxidative stress and vascular damage in hypertension

Oxidative stress, a state of excessive reactive oxidative species activity, is associated with vascular disease states such as hypertension. In this review, we discuss the recent advances in the field of reactive oxidative species-mediated vascular damage in hypertension. These include the identification of redox-sensitive tyrosine kinases, the characterization of enzymatic sources of superoxide production in human blood vessels, and their relationship with vascular damage in atherosclerosis and hypertension. Finally, recent developments in the search for strategies to attenuate vascular oxidative stress are reviewed.     

bFGF ameliorates intestinal mucosal permeability and barrier function through tight junction proteins in burn injury rats

BackgroudTo investigate the protective effect of exogenous basic fibroblast growth factor (bFGF) treatment on the intestinal mucosa in scalded rats.MethodsThirty-six SD rats were randomly divided into 3 groups (n = 12): sham group, scald group and bFGF group (0.5 mg/kg). Intestinal barrier dysfunction was evaluated by permeability of intestinal mucosa to fluorescein isothiocyanate (FITC)-dextran and Chiu’s grading system. H&E staining was used to detect the morphological changes of intestinal mucosa. Immunohistochemistry was used to observe zonula occludens-1 (ZO-1) and occludin. Western blot assay was used to detect the expression of ZO-1, Claudin-1, occludin and myosin light-chain kinase (MLCK).ResultsThe results demonstrated that following bFGF treatment, permeability of the intestinal epithelium barrier of was significantly decreased compared to scald group. H&E staining and Chiu’s grading were consistent with previous result. The expression of ZO-1, Claudin-1, occludin in bFGF group were significantly increased compared to scald group, while MLCK protein was decreased.ConclusionsbFGF ameliorates permeability of intestinal mucosa after burns. The possible mechanism may be relate to bFGF could increase the expression level of tight junction proteins (TJPs).