锰对大鼠睾丸支持细胞骨架蛋白及紧密连接相关蛋白表达的抑制作用

目的研究染锰诱导的大鼠睾丸超微结构改变及支持细胞(vimentin,VM)和紧密连接Occludins、Claudin-11mRNA表达,探讨锰对支持细胞骨架蛋白和紧密连接蛋白的破坏机制。方法雄性SD大鼠随机分为空白对照组,低剂量(15 mg/kg MnCl_2)和高剂量(30 mg/kg MnCl_2)组,8只/组。实验组分别染锰4周和6周,空白对照组给予等容生理盐水,给药途径均为腹腔注射,电镜观察睾丸支持细胞及血睾屏障超微结构,免疫组织化学(SABC)法检测支持细胞VM表达,实时定量PCR反应检测血睾屏障紧密连接Occludins和Claudin-11 mRNA表达。结果 1与空白对照组比较,各染锰组支持细胞数量及VM阳性细胞率、Occludins mRNA和Claudin-11 mRNA表达均显著降低。2染锰剂量相同,6周与4周组比较,以及染锰时间相同,高剂量组与低剂量组比较,支持细胞数量及VM阳性细胞率、Occludins mRNA和Claudin-11 mRNA表达均显著降低。3各组大鼠睾丸支持细胞数量与VM阳性细胞率及Occludins mRNA和Claudin-11 mRNA表达均成正相关。结论锰可抑制大鼠睾丸支持细胞骨架蛋白及紧密连接相关蛋白表达,破坏血睾屏障,导致生精微环境改变,产生生殖毒性效应。     

细胞色素C和波形蛋白在染锰大鼠睾丸表达及对精子发生的抑制作用

目的研究氯化锰对大鼠生精细胞细胞色素C(cyto-c)和支持细胞波形蛋白(VM)表达的影响及对生精功能的抑制效应。方法雄性SD大鼠随机分为空白对照组,低剂量(15 mg/kg Mn Cl2)和高剂量(30 mg/kg Mn Cl2)组,8只/组。Mn Cl2组分别染锰4周和6周,空白对照组给予等容生理盐水,给药途径均为腹腔注射,取睾丸免疫组织化学(SABC)法检测生精细胞cyto-c和VM表达,测定睾丸脏器系数,取附睾检测精子数量和精子畸形率。结果与空白对照组比较,各染锰组生精细胞cyto-c阳性细胞率和支持细胞VM阳性细胞率及各生精功能指标均显著降低,并呈一定的时间-效应关系和剂量-效应关系。各组大鼠精子数量与cyto-c阳性细胞率和VM阳性细胞率均呈正相关。结论锰可诱导大鼠生精细胞cyto-c和支持细胞VM表达,抑制生精细胞增殖,产生生殖毒性效应。     

锌指蛋白265在大鼠睾丸Sertoli细胞中的特异性表达

目的:研究锌指蛋白265(ZNF265)在大鼠睾丸Sertoli细胞中的特异性表达.方法:分离培养SD大鼠睾丸Sertoli细胞, 以免疫荧光染色、流式细胞术(FCM)检测ZNF265在Sertoli细胞的表达, 且抽提Sertoli细胞总RNA, 用PT-PCR法检测ZNF265的表达.结果:经免疫荧光染色, 实验组Sertoli细胞核核周呈现明亮特异荧光, 对照组未见特异性荧光;FCM检测结果显示实验组Sertoli细胞ZNF265的表达较对照组明显增高;RT-PCR扩增出目的条带ZNF265.结论:大鼠睾丸Sertoli细胞特异性表达ZNF265, 且多表达于Sertoli细胞核核周胞质.     

染锰大鼠睾丸Caspase-9,Smac,Apaf-1和XIAP的表达

目的研究氯化锰导致的大鼠生精细胞caspase-9及Apaf-1表达中XIAP和Smac的调节机制,探讨锰导致的雄性不育机制。方法雄性SD大鼠随机分为对照组,氯化锰低剂量(15mg/kg MnCl_2)和氯化锰高剂量(30mg/kg MnCl_2)组,腹腔注射MnCl_24周和6周,免疫组织化学SABC法和Western blot检测生精细胞caspase-9、Apaf-1、XIAP和Smac表达。结果氯化锰组生精细胞caspase-9和Smac蛋白表达水平显著高于对照组,随时间和剂量增加而升高;而Apaf-1和XIAP蛋白表达水平则降低,并有时间和剂量依赖性。结论 caspase-9,Apaf-1,XIAP和Smac可能参与锰引起雄性生殖毒性的发病机制。     

Caspase-3mRNA与PARP在染锰大鼠生精细胞中表达变化

目的研究氯化锰对生精细胞凋亡过程半胱氨酸酶3(caspase-3)mRNA和多聚ADP核糖聚合酶(PARP)表达的影响,探讨氯化锰的生殖毒性效应及可能机制。方法雄性SD大鼠48只随机分为空白对照组,低剂量(15 mg/kg MnCl_2)和高剂量(30 mg/kg MnCl_2)组,8只/组。MnCl_2组分别染锰4周和6周,空白对照组给予等容生理盐水,给药途径均为腹腔注射,TUNEL法检测睾丸生精细胞凋亡,原位杂交法和免疫组织化学(SABC)法检测生精细胞caspase-3mRNA和PARP和表达。结果随染锰时间延长和剂量增加,各染锰组生精细胞凋亡指数(AI)及caspase-3 mRNA表达水平显著升高,PARP表达水平显著降低,凋亡指数AI分别与caspase-3 mRNA和PARP表达呈正相关和负相关。结论锰可诱导大鼠生精细胞caspase-3 mRNA表达,促进PARP分解,导致生精细胞凋亡,产生生殖毒性效应。     

锰对大鼠生精细胞Caspase-3 mRNA调控及支持细胞波形蛋白表达的影响

目的 探讨锰对大鼠生精细胞Caspase-3 mRNA调控及支持细胞波形蛋白（vimentin）表达的影响。 方法 将正常雄性SD大鼠48只,随机分为1个对照组和2个染锰组,给予各组大鼠腹腔注射生理盐水和15mg/kg、30mg/kg氯化锰。分别染锰4周和6周,随机处死各组大鼠8只。应用末端脱氧核糖核酸转移酶介导的dUTP缺口末端标记技术（TUNEL） 、原位杂交法和免疫组织化学链酶亲和素生物素过氧化物酶复合物（SABC）法进行检测研究。 结果 1. 与空白对照组比较,15mg/kg、30mg/kg染锰组生精细胞凋亡指数（AI）均升高（ P<0.05, P<0.01）,Caspase-3 mRNA阳性细胞率均显著升高（ P<0.01）,支持细胞波形蛋白（vimentin）阳性细胞率均显著降低（ P<0.01）。2. 染锰剂量相同,染锰6周组与染锰4周组比较,生精细胞AI与Caspase-3 mRNA阳性细胞率均显著升高（ P<0.01）,支持细胞vimentin阳性细胞率均显著降低（ P<0.01）。3. 染锰时间相同,30mg/kg MnCl2组与15mg/kg MnCl2组比较,生精细胞AI与Caspase-3 mRNA阳性细胞率均显著升高（ P<0.01）,支持细胞vimentin阳性细胞率均显著降低（ P<0.01）。4. 各组大鼠生精细胞AI和Caspase-3 mRNA阳性细胞率呈正相关（ r =0.842, P<0.01）,与支持细胞vimentin阳性细胞率呈负相关（ r =-0.859, P<0.01）,各组大鼠生精细胞Caspase-3 mRNA阳性细胞率和支持细胞vimentin阳性细胞率呈负相关（ r =-0.975, P<0.01）。 结论 染锰大鼠生精细胞Caspase-3 mRNA表达增加导致生精细胞凋亡增加;支持细胞vimentin表达下降;这可能是锰生殖毒性的重要分子机制之一。     

大鼠睾丸支持细胞发育的形态学研究

对７０例不同胎龄及生后不同发育阶段Ｗｉｓｔａｒ大鼠睾丸支持细胞的发育进行了光镜、电镜研究。结果：１．依形态学特点，将支持细胞的发育过程初步分为４期：分化前期（胚胎１１～１５ｄ）、分化期（胚胎１５ｄ～生后１至２周）、成熟前期（生后２周～青春期前）、成熟期（青春期后）。２．胚胎及新生时，在支持细胞间见到两侧胞膜紧密相贴及不典型的桥粒连接等；自生后４周，见到紧密连接。３．自胚胎１７ｄ至成熟以后，在支持细胞与其周围的生殖细胞间见到大量类似桥粒和类似中间连接等不典型的细胞连接。４．胚胎１９ｄ及新生期，睾丸索内除支持细胞及生殖细胞外，尚可见一种特殊的细胞。     

大鼠睾丸支持细胞的分离纯化与鉴定

目的　简化大鼠睾丸支持细胞的分离纯化方法 ,通过不同方法对Sertoli细胞进行形态学观察鉴定。方法　取鼠龄 16～ 2 2天的雄性Wistar大鼠睾丸 ,采用酶次第消化 ,培养过程中纯化Sertoli细胞 ,并用多种方法对Sertoli细胞进行鉴定。结果　大鼠睾丸支持细胞占培养细胞总数的 90 %以上。HE染色 ,Sertoli细胞突起很多 ,核仁清晰 ,在胞质中可见吞噬物和大小不等的空泡 ;甲基绿 派洛宁染色 ,Sertoli细胞富含RNA ;Feulgen染色和透射电镜 ,核仁周围可见卫星核小体。结论　本实验培养方法可获得更多、纯度高的Sertoli细胞 ,HE染色、甲基绿 派洛宁染色、Feulgen染色和透射电镜是鉴定Sertoli细胞的有效方法     

大鼠睾丸支持细胞的分离、纯化和鉴定

目的 培养高纯度的大鼠睾丸支持细胞,并应用检测ABP mRNA原位杂交方法加以鉴定.方法 选用18～22d龄SD雄性大鼠睾丸,采用0.25%胰蛋白酶、0.1%透明质酸酶、 0.1%胶原酶三酶依次消化法分离支持细胞,置于32℃ 5% CO2的培养箱培养,48h后用20mmol/L Tris-HCl低渗处理培养细胞.培养1周后应用伊红染色、吖啶橙荧光染色、Feulgen染色对所培养的支持细胞进行鉴定,同时应用原位杂交检测ABP mRNA方法鉴定分离培养的支持细胞.结果 培养1周后所获培养的支持细胞纯度达95%以上.分离培养的支持细胞ABP mRNA表达阳性,其形态结构特征与用其他方法鉴定为支持细胞的形态结构特征一致.结论 采用三酶连续消化及低渗处理法分离培养的支持细胞纯度高,而且应用原位杂交方法检测ABP mRNA是一种新的、特异、有效的鉴定支持细胞及其功能的方法.     

睾丸波形蛋白表达与生精细胞凋亡的关系

睾丸的生精上皮具有高度增殖能力,生精过程中细胞增殖与凋亡的动态平衡,对于维持精子的生成的数量和质量具有重要的作用。外来化学毒物可以直接引起睾丸损伤,导致生精细胞过量凋亡,从而导致精子生成减少。生精细胞凋亡时细胞骨架蛋白改变的研究成为一个热点,睾丸波形蛋白表达发生了异常改变,但睾丸波形蛋白表达与生精细胞凋亡的相关性还需进一步研究。     

Somatostatin regulates tight junction proteins expression in colitis mice

Tight junction plays a critical role in intestinal defence. The alteration and perturbation of tight junction proteins could induce intestine barrier damage, and lead to the malabsorption of electrolytes and water. Previous studies had showed that colonic infection and inflammation could lead to the alteration of tight junction function, and somatostatin could protect intestinal epithelia. Thus, this study could explore that whether somatostatin could regulate tight junction in colitis mice. Colitis mice with diarrhea were induced by Citrobacter rodentium (CR) and Dextran sulfate sodium (DSS). In CR infected model, cladudin-1 and claudin-3 expression significantly decreased compared with the control mice (P < 0.05); after octreotide treatment, claudin-1 and claudin-3 expression significantly increased compared with untreated CR infected mice (P < 0.05). In DSS colitis model, occludin and claudin-3 expression significantly decreased compared with the control mice (P < 0.05); and octreotide treatment could only significantly upregulate claudin-3 expression compared with untreated DSS colitis mice (P < 0.05). To testify our results in vivo, we repeated the models in caco-2 cells by exposed with enteropathogenic Escherichia coli (E. Coli) and Tumor necrosis factor α (TNF-α). The results in vitro were consistent with in vivo study. The results suggested that somatostatin play a role in intestinal barrier protection by modulating tight junction proteins expression.     

Role of stretch on tight junction structure in alveolar epithelial cells.

Previous studies have demonstrated that high tidal volumes can cause interstitial and alveolar edema, with degradation of pulmonary epithelial barrier integrity. Separate studies have shown that F-actin disruption and decreased intracellular ATP (ATP(i)) levels in the nonpulmonary epithelium can increase tight junction (TJ) permeability. We hypothesized that large epithelial stretch perturbs ATP(i) and actin architecture, each of which adversely affects TJ structure, and thus increases TJ permeability. Primary alveolar epithelial cells were subjected to a uniform 25% or 37% change in surface area (DeltaSA), cyclic biaxial stretch (15 cycles/min) for 1 h, or treated with either glycolytic metabolic inhibitors or cytoskeletal disrupting agents. Unstretched, untreated cells served as controls. Changes in the TJ proteins occludin and ZO-1 were determined by immunocytochemical evaluation. A stretch amplitude of 25% DeltaSA did not produce any significant cytologic changes compared with controls, but an amplitude of 37% DeltaSA stretch resulted in significant decreases in the intensity of the peripheral occludin band, the degree of cell-cell attachment (CCA), and total cellular occludin content. ATP depletion significantly diminished the occludin band intensity and decreased CCA. Actin disruption did not affect TJ protein band intensities (although the occludin distribution became punctate) but altered CCA. Untreated cells stretched cyclically at 25% or 50% DeltaSA for 1 h had significantly decreased ATP(i) compared with unstretched controls. These results suggest that stretch-induced ATP(i) reduction and actin perturbation disrupt TJ structure and CCA, which may lead to the alveolar flooding associated with high tidal volumes.     

Tissue-specific expression of the tight junction proteins claudins and occludin in the rat salivary glands

Tight junctions (TJs) are essential features of endothelial barrier membranes and of fluid-secreting epithelial cells, such as in the salivary glands. Novel integral membrane proteins have been identified as components of TJs, namely claudins and occludin. The aim of the present study was to determine the distribution of occludin and claudins in the large salivary glands of the rat. The parotid, submandibular and sublingual salivary glands were harvested from adult Sprague-Dawley rats and cryostat sections were stained using immunoperoxidase and immunofluorescence methods. Claudin-1 was expressed in endothelial cells of microvessels and in short selected segments of the duct system. Claudin-3 was expressed principally in the acinar cells and intercalated ducts, while claudin-4 was principally expressed by the striated and interlobular ducts. Claudin-5 was specific to endothelial cells of microvessels. Occludin was ubiquitously detected in the duct system. Double labelling and confocal microscopy showed some co-localization of claudin-3 with claudin-4, and minimal co-localization of occludin with claudin-4, in the striated ducts. Claudin 2 was not detected in any of the salivary glands. The results indicate specificity of the chemical composition of tight junctions in the rat salivary glands, and may reflect different physiological roles for TJs in the glandular and duct epithelial cells, and in endothelial cells of salivary gland microvessels.     

IL-8对血管内皮细胞紧密连接的影响

目的 研究白细胞介素-8(IL-8)对血管内皮细胞紧密连接的影响.方法 采用免疫荧光染色检测经不同浓度和时间IL-8处理后EA.hy926细胞的3种紧密连接蛋白occludin、claudin-5和ZO-1的形态和分布;逆转录PCR(RT-PCR)检测3种蛋白mRNA的表达水平.结果 IL-8可改变内皮细胞紧密连接蛋白...     

Emerging roles of transmembrane-type tight junction proteins in cancers

Tight junctions (TJs) are the most apical components of the cell–cell adhesion machinery in epithelial and endothelial cells and they play essential roles in homeostasis. Recent studies have revealed that aberrant expression of tight junction proteins (TJPs) is frequently observed in various type of cancers. Here we review cancer-associated aberrant expression of TJPs with focus on transmembrane-type TJPs including claudins, junctional adhesion molecule-A (JAM-A), and occludin. Some transmembrane-type TJPs are upregulated at the early neoplastic stage and their expression persists during dedifferentiation. Aberrant expression of TJPs contributes to proliferation, invasion, and dysregulated signaling of cancer cells. In addition to an increase in their expression level, their localization is altered from a TJ-restricted pattern to distribution throughout the whole cell membrane, making them suitable as therapeutic targets. Extracellular domains of transmembrane-type TJPs can be approached by target drugs not only from the lumen side (apical side) but also from the extracellular matrix side (basal side), including blood vessels. Aberrantly expressed TJPs are potential useful diagnostic markers as well as therapeutic targets for cancers.     

Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria

Penetration of the gut mucosa by pathogens expressing invasion genes is believed to occur mainly through specialized epithelial cells, called M cells, that are located in Peyer's patches. However, Salmonella typhimurium that are deficient in invasion genes encoded by Salmonella pathogenicity island 1 (SPI1) are still able to reach the spleen after oral administration. This suggests the existence of an alternative route for bacterial invasion, one that is independent of M cells. We report here a new mechanism for bacterial uptake in the mucosa tissues that is mediated by dendritic cells (DCs). DCs open the tight junctions between epithelial cells, send dendrites outside the epithelium and directly sample bacteria. In addition, because DCs express tight-junction proteins such as occludin, claudin 1 and zonula occludens 1, the integrity of the epithelial barrier is preserved.     

Segment-specific expression of tight junction proteins, claudin-2 and -10, in the rat epididymal epithelium

Tight junctions are known to be related to the variance by segment of the luminal fluid microenvironment of the epididymis. We examined the expression of claudins that regulate the paracellular permeability of ions, solutes, and water through tight junctions in the rat epididymis. RT-PCR analysis showed that claudin-2 and -10 were expressed in a segment-specific manner among fifteen claudins examined. Immunofluorescence microscopy demonstrated that both claudins showed developmental stage- and segment-specific subcellular localizations in the epididymal epithelium. Claudin-2 was detected principally in the apical junctional region in the initial segment at postnatal day 7 and week 10 but lost its expression in all the distal segments including the caput, corpus, and cauda. Claudin-10 was detected principally in the apical junctional region in all segments on postnatal day 7 but only in the initial segment--especially along the entire lateral membrane--at 10 weeks. In freeze fracture electron microscopy, well-developed intramembranous particle strands were observed on the P face and complementary grooves with a few particles on the E face at 10 weeks. Many particles were occasionally observed on the grooves in the E face in the initial segment and distal caput. Tight junction strands were continuous and impermeable to lanthanum nitrate on postnatal day 7, suggesting the establishment of functional tight junctions. These segment-specific expressions of claudin-2 and -10 may contribute to creating the specific luminal fluid microenvironment which is necessary for the transport, maturation, and storage of spermatozoa.     

细胞紧密连接的结构组成及其调控的研究进展

紧密连接(tight junction)位于相邻细胞间隙的顶端侧面,对于维持表皮和内皮细胞的选择渗透性屏障功能是非常关键的。紧密连接有两个主要功能:渗透性调节功能、维持细胞极性。本文综述了最新研究的紧密连接的蛋白质组成、紧密连接组装的信号调节、组成蛋白间的相互作用调控机制以及在生命活动中意义,为寻找某些疾病的治疗方案提供一定的理论基础。