胱蛋白酶抑制剂相关的附睾精子发生基因在不同发育阶段小鼠睾丸和附睾中的表达

目的 :研究胱蛋白酶抑制剂相关的附睾精子发生 (Cres)基因在生后不同发育阶段小鼠睾丸及附睾中的表达规律。　方法 :采用半定量RT PCR方法检测CresmRNA在生后 14、2 0、2 2、2 8、35、4 9、70、4 0 0d小鼠睾丸及附睾中的表达变化。　结果 :Cres基因在 14d小鼠睾丸和附睾中呈低水平表达 ,随着小鼠的生长发育 ,CresmRNA的表达量逐渐升高。在 70d小鼠睾丸和 4 0 0d小鼠附睾中 ,CresmRNA的表达量达到峰值。　结论 :Cres基因在生后不同发育阶段小鼠睾丸及附睾中呈现明显的规律性表达 ,可能参与精子发生、成熟过程的调控。     

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。     

Bcl-2基因与大肠癌

Bcl-2家族蛋白是细胞凋亡的关键调节因子,其表达的改变不仅影响DNA损伤细胞或周期异常细胞的正常凋亡,还影响肿瘤细胞自身的凋亡,还常与肿瘤的形成及细胞对化疗药物耐药性的形成有关,Bcl-2在肿瘤细胞中有不同程度的表达。1 Bcl-2基因及蛋白的特性Bcl-2基因位于18q21,由230kb组成,含3个外显子,3′端与5′分别有5.3kb和1.4kb的非翻译片段。在特异性的染色体易位中,18号染色体上Bcl-2基因易位至14号染色体q32的免疫球蛋白     

前列腺癌向激素非依赖型转化时激素受体基因甲基化的作用

目的探讨晚期前列腺癌转为雄激素非依赖型时,雄激素受体(AR)基因启动子区域甲基化的作用。方法采用Southern杂交、PCR特异的甲基化反应、甲基化敏感的单链结构分析等技术检测大鼠激素非依赖型前列腺癌和细胞株(PLS10、20和30)的AR基因启动子区域甲基化状态。结果Northern杂交技术检测的大鼠激素非依赖型前列腺癌和细胞株中均无ARmRNA表达。RT-PCR结果显示,PLS10和30细胞株中有ARmRNA表达,而PLS20细胞株中无ARmRNA表达。Southern杂交分析AR基因启动子区域甲基化状态,PLS10细胞株中EcoRI/HpaⅡ消化的DNA出现1.0和0.7kb条带;PLS20细胞株出现1.5、l.2和0.7kb条带;PLS30出现1.3、1.2、1.0和0.7kbDNA条带,PLS20细胞株CpGs甲基化状态明显高于细胞株PLS10和30。用去甲基化剂5-aza-2-脱氧胞苷(5AC)处理后PLS20细胞恢复ARmRNA表达。CpGs位于转录起始位点上游-1和-9核苷酸。大鼠前列腺和精囊癌中均检测到CpG位点甲基化状态。结论雄激素非依赖型前列腺癌中,AR基因启动子区域异常高甲基化在AR表...     

C/EBPα基因启动子DNA超甲基化对与PPARγ相关的HDAC1抑制解除作用

[目的]探究C/EBPα在诱导鼠胚胎间充质干细胞C3H10T1/2成骨过程中的调节机制.[方法]体外构建成骨分化模型,在RNA水平和蛋白质水平检测C3H10T1/2细胞成骨分化时转录因子PPARγ(过氧化物酶体增殖物激活受体,peroxisome proliferator-activated receptor,PPAR)的mRNA和蛋白质的表达情况;通过构建干扰转录因子PPARγ表达的siRNA腺病毒载体和PPARγ转录因子的拮抗剂G3335,研究PPARγ对于CCAAT启动子结合蛋白(C/EBPα)表达的作用;染色质免疫共沉淀技术(chromatin immunoprecipitation assay,CHIP)分析PPARγ2位于C/EBPα启动子-1286bp/-1065bp区域内的结合位点,并检测PPARγ表达情况对C/EBPα启动子-1286bp/-1065bp区段DNA甲基化和组蛋白去乙酰化的影响.[结果]C3H10T1/2细胞成骨分化过程中,PPARγ通过结合C/EBPα启动子-1286bp/-1065bp区段激活/促进该基因表达.在成骨作用终末阶段/后期观察到C/EBPα启动子-1286bp/-1065bp区段的DNA发生超甲基化,从而阻抑了PPARγ与之结合,与PPARγ相关的HDAC1抑制得以解除,从而下调了-1286bp/-1065bp区段的组蛋白乙酰化.[结论]本研究通过阐述C/EBPα对PPARγ的增强调控作用,揭示间充质干细胞成骨分化的深层次分子机制;同时通过探讨DNA甲基化和组蛋白去乙酰化通过PPARγ联系的机制,从而为调控间充质干细胞分化提供分子理论模型.     

肝细胞肝癌CDH1基因启动子C/A单核苷酸多态性与蛋白表达的关系

目的 探讨原发性肝细胞肝癌CDH1基因启动子-160位点的C/A单核苷酸多态性(SNP)与其蛋白表达的关系.方法 以34例肝癌手术病人为对象,DNA直接测序法检测其血液标本中CDH1基因启动子-160位点C/A SNP,免疫组化法检测组织标本中CDH1的蛋白产物--上皮钙黏素(E-cadherin)的表达情况,比较分析C/A SNP与E-cadherin表达的关系.结果 E-cadherin高表达组18例(52.9%),低表达组16例(47.1%),两组的基因型出现率CC与CA,AA比较差异均有统计学意义(P<0.05),CA与AA间差异无统计学意义(P>0.05),A、C等位基因频率在两组差异有统计学意义(P<0.05).结论 CDH1基因启动子-160位点的C/A SNP在肝癌E-cadherin表达中可能发挥重要作用,且A等位基因的出现与E-cadherin表达下调相关.     

颅脑损伤后神经细胞凋亡的分子调控

<正> 凋亡是多细胞有机体在一定的生理或病理条件下遵循自身的程序,通过内源性DNA内切酶的激活,主动结束生命的过程。近些年来,对细胞凋亡机制的研究证明,细胞凋亡参与了颅脑损伤后继发性病理生理演变的全过程。颅脑损伤后神经细胞发生凋亡的最有力证据是关于其分子机制的研究。大量实验证实多种基因产物的主动表达与颅脑损伤后细胞凋亡之间存在密切关系。而caspase家族蛋白酶是神经细胞凋亡途径的核心成分,细胞凋亡的信号可触发由caspases参与的级联效应,导致靶细胞解体。 1 caspase家族 Caspase是eysteine-dependent aspartate-specific protease(半胱-天冬氨酸蛋白酶)的简称,是一类位点特异性蛋白水解酶。在对线虫C. elegans的基因分析显示,至少有3种基因直     

基因突变与生殖的衰竭

随着分子生物学的发展 ,许多疾病的研究已达到分子水平 ,尤其是单基因疾病 ,许多病因不清的疾患已发现有基因水平的改变。促性腺激素释放激素 ( Gn RH )基因、 Gn RH-受体( Gn RH-R)基因、促性腺激素亚单位基因、促性腺激素受体基因、传导促性腺激素信号的 G蛋白亚单位基因、编码调节促性腺激素生物合成的转录因子基因以及 Gn RH神经元或促性腺细胞正常发育所需基因发生突变 ,均可引起生殖功能异常而导致生殖的衰竭。本讲将重点介绍下列内容 :转录因子的基因缺陷导致垂体细胞分化和发育缺陷、Gn RH-R基因突变导致低促性腺激素性性腺…     

血清胱蛋白酶抑制剂C对评价狼疮性肾炎患者肾小球病理损害程度的价值

目的：探讨狼疮性肾炎患者血清胱蛋白酶抑制剂C（cystatin C）在评价肾小球病理损害程度中的价值。方法：选择狼疮性肾炎患者93例,入院后测定血清cystatin C,并行肾穿刺活检,对肾小球病理进行评分。分析cystatin C与肾小球病理评分的相关性,比较不同病理分级间cystatin C水平的差异。结果：血清cystatin C与狼疮性肾炎患者肾小球病理评分（r=0.7538,P=0.000）呈正相关,相关性具有统计学意义,肾小球病理1～3级间cystatin C水平的差异具有统计学意义（均P〈0.01）。结论：血清cystatin C水平可以客观地评估狼疮性肾炎患者肾小球病理损害程度,是检测肾小球病理及功能改变的理想血清标志物。     

附睾相关WFDC型丝氨酸蛋白酶抑制剂研究进展

精子在附睾中的成熟是通过管腔离子浓度的改变及许多糖苷酶和蛋白酶对精子表面膜的修饰过程来调控的,而这些蛋白酶的活动是受存在于附睾特定区域的蛋白酶抑制剂控制的。其中WFDC型丝氨酸蛋白酶抑制剂是一种在附睾中高度表达的蛋白,它在天然免疫与男性生育方面发挥重要作用。本文就该蛋白的结构、功能特征及其在开发男性抗生殖道感染药物、免疫避孕药物等方面的应用前景进行了综述。     

Differential effects of GnRH and androgens on Cres mRNA and protein in male mouse anterior pituitary gonadotropes

The Cres gene defines a new subgroup in the family 2 cystatins of cysteine protease inhibitors. However, unlike typical cystatins, CRES does not inhibit cysteine proteases but rather inhibits the serine protease prohormone convertase 2, an enzyme with roles in proprotein processing in the neuroendocrine system. Cres is expressed in the gonadotropes and colocalizes with LHbeta, suggesting a role in the regulation of gonadotrope secretion. Our present studies were carried out to examine the regulation of Cres mRNA and protein expression by GnRH and steroid hormones, thus providing clues regarding its role in gonadotropes. Castration profoundly reduced Cres mRNA, while replacement with estradiol (E(2)), testosterone (T), or dihydrotestosterone (DHT) further decreased Cres, suggesting negative regulation by GnRH or steroid hormones. The administration of Antide, a GnRH antagonist, resulted in a 3-fold increase in Cres mRNA, supporting a negative regulation by GnRH. Because all hormonal manipulations in vivo resulted in alterations in steroid hormones, organ culture was used to assess the effects of GnRH independent of steroids. Mouse pituitaries cultured in the absence of GnRH or steroids showed high Cres mRNA levels, while DHT or E(2) resulted in decreases of 25% and 68%, respectively. However, an 85% decrease in Cres mRNA occurred following the administration of GnRH, demonstrating that GnRH, and to a lesser degree E(2), negatively regulate Cres mRNA in gonadotropes. Examination of CRES protein by immunohistochemistry showed that levels were profoundly reduced following castration, while DHT and in part T, but not E(2), restored CRES levels. Castrated mice treated with Antide showed little effect. However, castrated mice treated with Antide + DHT showed a dramatic recovery of CRES, suggesting that androgens act directly at the level of the gonadotrope to regulate CRES protein. Together, our studies suggest that Cres mRNA and protein are low at peak gonadotrope secretory activity, possibly as a means to allow proprotein processing events to occur that are integral to gonadotrope function.     

Alterations in the testis and epididymis associated with loss of function of the cystatin-related epididymal spermatogenic (CRES) protein

Cystatin-related epididymal spermatogenic protein (CRES) or cystatin 8 (Cst8 gene) is a member of the cystatin superfamily of cysteine protease inhibitors. It differs from typical cystatins because it lacks consensus sites for cysteine protease inhibition and exhibits reproductive-specific expression. In the present study, we examined CRES expression within the testes, efferent ducts, and epididymides of normal mice by light microscope immunolocalization. Alterations to these tissues in male mice lacking the Cst8 gene (Cst8(-/-2)) were also characterized by histomorphometry and electron microscopy. In the normal testis, CRES was localized exclusively in mid and late elongating spermatids. In the efferent ducts, CRES was localized to the apical region of the epithelial cells suggestive of localization in the endosomes. In the initial segment of the epididymis, principal cells showed supranuclear and luminal reactions. In the cauda region, CRES was present exclusively as aggregates in the lumen and was detected in clear cells. Compared with wild-type mice (Cst8(+/+)), older (10-12 months) Cst8(-/-) mice had modest but statistically significant reductions in tubular, epithelial, and/or luminal profile areas in the testis and epididymis. By electron microscopy, some Cst8(-/-) tubules in the testis were normal in appearance, but others showed a vacuolated seminiferous epithelium, degenerating germ cells, and alterations to ectoplasmic specializations. In the epididymal lumen, abnormally shaped sperm heads and tails were noted along with immature germ cells. In addition, principal cells contained numerous large irregularly shaped lysosomes suggestive of disrupted lysosomal functions. In both the testis and epididymis, however, these abnormalities were not apparent in younger mice (4 months), only in the older (10-12 months) Cst8(-/-) mice. These findings suggest that the altered testicular and epididymal histology reflects a cumulative effect of the loss of CRES and support a role for CRES in maintaining the normal integrity and function of the testis and epididymis.     

Extracellular quality control in the epididymis

The epididymal lumen represents a unique extracellular environment because of the active sperm maturation process that takes place within its confines. Although much focus has been placed on the interaction of epididymal secretory proteins with spermatozoa in the lumen, very little is known regarding how the complex epididymal milieu as a whole is maintained, including mechanisms to prevent or control proteins that may not stay in their native folded state following secretion. Because some misfolded proteins can form cytotoxic aggregate structures known as amyloid, it is likely that control/surveillance mechanisms exist within the epididymis to protect against this process and allow sperm maturation to occur. To study protein aggregation and to identify extracellular quality control mechanisms in the epididymis, we used the cystatin family of cysteine protease inhibitors, including cystatin-related epididymal spermatogenic and cystatin C as molecular models because both proteins have inherent properties to aggregate and form amyloid. In this chapter, we present a brief summary of protein aggregation by the amyloid pathway based on what is known from other organ systems and describe quality control mechanisms that exist intracellularly to control protein misfolding and aggregation. We then present a summary of our studies of cystatinrelated epididymal spermatogenic （CRES） oligomerization within the epididymal lumen, including studies suggesting that transglutaminase cross-linking may be one mechanism of extracellular quality control within the epididymis. （Asian J Androl 2007 July; 9： 500-507）     

Age-dependent expression of the cystatin-related epididymal spermatogenic （Cres） gene in mouse testis and epididymis

Aim： To investigate the spatial and temporal expression of the cystatin-related epididymal spermatogenic （Cres） gene in mouse testis and epididymis during postnatal development. Methods： The QuantiGene assay and indirect immunofluorescence technique were used to examine the Cres mRNA and Cres protein level in mouse testis and epididymis on postnatal days 14, 20, 22, 28, 35, 49, 70 and 420. Results： （1） In both the testis and epididymis, Cres mRNA was fast detected on day 20, then it increased gradually from day 20 to day 70, and the high expression level maintained till day 420. （2） In the testis, the Cres protein was exclusively localized to the elongating spermatids and was first detected on day 22. The number of Cres-positive spermatids increased progressively till day 49. From day 49 to day 420, the number of Cres-positive cells was almost stable. （3） The Cres protein was first detected on day 20 in the proximal caput epididymal epithelium. By day 35, the expression level of the Cres protein increased dramatically and the high level was maintained till day 420. Moreover, the luminal fluid of the midcaput epididymis was also stained Cres-positive from day 35 on. No Cres-positive staining was observed in distal caput, corpus and cauda epididymis throughout. Conclusion： The Cres gene displays a specific age-dependent expression pattern in mouse testis and epididymis on both the mRNA and protein level.     

Cystatin C deficiency is associated with the progression of small abdominal aortic aneurysms.

BACKGROUND: The cysteine protease inhibitor cystatin C may play a role in the development and progression of abdominal aortic aneurysms (AAAs). METHODS: From a mass screening trial of men aged 65-73 years, 151 small AAAs were followed for a mean of 2.9 years. Of these patients, 142 had serum samples taken to determine the levels of cystatin C, creatinine and C-reactive protein (CRP). RESULTS: Serum cystatin C concentration correlated negatively with AAA size (r = - 0.22 (95 per cent confidence interval (c.i.) - 0.59 to - 0.02)) and annual expansion rate (r = - 0.24 (95 per cent c.i. - 0.75 to - 0.05)), persisting after adjustment for renal function, smoking, diastolic blood pressure, CRP, age and AAA size. Creatinine clearance and CRP did not correlate with size or expansion rate. Thirty-one AAAs had expanded to over 50 mm, when operation was recommended. The serum level of cystatin C was a significant predictor of this occurrence, with a sensitivity and specificity of 61 and 57 per cent respectively. However, initial AAA size had the optimal sensitivity and specificity (both 81 per cent) in this regard. CONCLUSION: Deficiency of cystatin C was associated with increased aneurysm size and expansion rate, possibly due to lack of inhibition of cysteine proteases.     

Aberrant expression of cystatin C in prostate cancer is associated with neuroendocrine differentiation

OBJECTIVE: To investigate the expression of cystatin C and the relationship with neuroendocrine differentiation and proliferation in benign and malignant prostatic tissues, as cystatin C, the most important inhibitor of human lysosomal cysteine proteases, is considered to be a major regulator of pathological protein degradation in inflammatory and neoplastic diseases. MATERIALS AND METHODS: Immunoreactivity for cystatin C, prostate-specific antigen, Ki-67 and the neuroendocrine marker chromogranin A was examined in whole-mount radical prostatectomy specimens and using tissue microarrays. Cystatin C in tissue homogenates was analysed by Western blotting and enzyme-linked immunosorbent assay (ELISA). The expression and relative levels of cystatin C mRNA were assessed by in situ hybridization and quantitative real-time polymerase chain reaction (QRT-PCR). RESULTS: The intensity of cystatin C immunostaining in Gleason grade 2 and 3 prostate cancer was significantly higher than in benign prostatic tissues, but decreased significantly with increasing Gleason grades. There was strong expression of cystatin C in neuroendocrine-like cells, which increased significantly with increasing Gleason grades. The Ki-67 immunoreactivity also increased significantly during de-differentiation. In situ hybridization showed staining patterns in concordance with the immunohistochemical results. ELISA showed high concentrations of cystatin C in benign and malignant tissue extracts and QRT-PCR further corroborated that the cystatin C gene is highly expressed in both benign and malignant prostatic tissues. CONCLUSIONS: There was a significant decrease in the immunohistochemical expression of cystatin C in non-neuroendocrine prostate cancer cells, concomitant with increasing Gleason grades. That there were more strongly cystatin C-positive neuroendocrine-like cells in prostate cancer than in benign prostatic tissue suggests a connection between cystatin C and neuroendocrine differentiation in prostate cancer progression.