人类趋化素样因子超家族在非小细胞肺癌中的表达

目的 探讨人类趋化素样因子超家族(CKLFSF)成员CKLF1、CMTM1、CMTM2和CMTM4在非小细胞肺癌(NSCLC)组织中的表达及其临床意义.方法 应用组织微阵列和免疫组织化学方法 检测50例NSCLC组织和癌旁正常肺组织中CKLF1、CMTM1、CMTM2和CMTM4蛋白的表达及差异.结果 CKLF1、CMTM1、CMTM2和CMTM4蛋白在癌旁正常肺组织中的阳性表达率均为100%,在NSCLC组织中的阳性表达率分别为12%、2%、14%和26%,差异有统计学意义(P＜0.01).性别、病理分化程度、病理分期与CKLF1、CMTM1、CMTM2和CMTM4蛋白在NSCLC组织中的表达程度之间无明显相关(P＞0.05).肺腺癌组织中CMTM1、CMTM4蛋白的表达明显高于其在鳞癌组织中的表达(PcmTm1=0.011,PcmTm4＜0.01).结论 位于人类染色体同一基因簇内的趋化素样因子超家族成员CKLF1、CMTM1、CMTM2和CMTM4可能是潜在的NSCLC抑癌基因.     

趋化素样因子超家族6与程序性死亡配体1在胶质瘤中的表达及其临床意义

目的:检测趋化素样因子超家族6(CMTM6)及程序性死亡配体1(PD-L1)在胶质瘤组织中的表达,并分析两种蛋白的表达相关性及其对预后的影响。方法:收集郑州大学第五附属医院神经外科收治的胶质瘤患者76例与同时期脑外伤开颅患者(对照组)40例,留取术中组织标本,通过免疫组织化学法(IHC)和蛋白质印迹法(Western ...     

cklfsf 2a在小鼠睾丸发育中的基因表达和蛋白定位

目的研究趋化素样因子2a（cklfsf 2a）在小鼠睾丸中的基因调控和蛋白定位。方法采用RT-PCR方法检测cklfsf 2a基因在不同周龄小鼠睾丸组织中的表达情况，制备cklfsf 2a多克隆抗体，分别采用免疫组织化学和原位杂交方法检测cklfsf 2a蛋白和mRNA在小鼠睾丸组织中的定位。结果cklfsf 2a mRNA在出生后14d内出现，表达量逐渐增加，在成年前达到高峰并持续表达。cklfsf 2a蛋白和mRNA均特异性的表达于睾丸初级精母细胞。结论cklfsf 2a基因存在发育的表达调控，随着睾丸发育的日趋成熟，表达量逐渐增加，其作用可能和精母细胞的发育有关。     

Ccdc70基因在小鼠睾丸精子发生过程中的表达特征

目的:探讨Ccdc70(Coiled-coil domain containing 70)基因在小鼠睾丸中的表达特征并分析其在生精过程中的潜在功能。方法:经表达谱芯片筛选出小鼠睾丸特异性基因Ccdc70,通过RT-PCR、real-time PCR、Western印迹及免疫组化检测Ccdc70基因在成年小鼠睾丸中表达特征,并对该蛋白做相关的生物信息学分析。结果:RT-PCR、real-time PCR和Western印迹结果表明Ccdc70基因在小鼠睾丸中高表达,在附睾中低表达;免疫组化结果表明Ccdc70蛋白在睾丸中主要表达于小鼠精母细胞和圆形精子胞质,在附睾中主要表达在附睾管上皮细胞。生物信息学分析显示该蛋白存在一个CCDC结构域,并在哺乳动物进化过程中高度保守。结论:Ccdc70为小鼠睾丸高表达基因,且主要表达于精母细胞、圆形精子及附睾管上皮细胞,提示其可能参与调控小鼠精子发生过程及附睾精子的进一步成熟。     

去泛素化酶24基因在小鼠睾丸精子发生过程中的表达特征

目的:研究去泛素化酶24(USP24)基因在小鼠睾丸精子发生过程中的表达特征,初步探讨其在生精过程中的作用。方法:通过实时荧光定量PCR(qPCR)和免疫荧光等方法检测USP24在不同周龄野生型小鼠睾丸组织以及成年雄激素受体敲除(ARKO)小鼠睾丸中的表达特征;采用双荧光素酶报告基因实验检测USP24启动子转录活性。结果:qPCR和免疫荧光结果表明,USP24基因在出生1周时表达水平较低,3周时急剧升高,随后维持在相似水平至第8周;USP24主要定位于支持细胞和生精细胞的细胞质;与野生型相比,USP24在ARKO小鼠睾丸表达降低;性成熟小鼠睾丸中,USP24定位于成熟精子头部后端及中部。双荧光素酶报告基因实验结果显示,睾酮刺激后USP24启动子的转录活性升高。结论:USP24基因的表达水平的升高与小鼠的性发育相关,且其蛋白在小鼠成熟精子上表达。USP24是受雄激素受体(AR)调控的靶基因。USP24可能参与调控小鼠的精子发生过程。     

趋化素样因子超家族成员5对前列腺癌侵袭行为的影响

目的 探讨趋化素样因子超家族成员5(CMTM5)对前列腺癌细胞的作用及机制.方法 利用划痕实验观察CMTM5对前列腺癌、DU145细胞迁移的影响;蛋白质印迹法检测PI3 K-AKT信号通路相关蛋白的表达;建立18只裸鼠皮下前列腺癌移植瘤模型,实验组肿瘤局部注射CMTM5腺病毒,检测CMTM5过表达对裸鼠前列腺肿瘤生长的影响,应用免疫组织化学方法检测裸鼠肿瘤组织中Ki-67的表达. 结果 过表达CMTM5抑制前列腺癌DU145细胞迁移,减少了PI3K-AKT信号通路中的关键分子pAKT 、NF-kB等蛋白的表达.裸鼠体内实验结果显示,CMTM5组肿瘤体积及质量分别为(573.39±175.24)mm3及(0.55±0.11)g,对照组分别为(1482.50±327.86) mm3及(1.31±0.29)g,2组比较差异均有统计学意义(P＜0.05).CMTM5组肿瘤组织Ki-67表达明显低于对照组. 结论 过表达CMTM5抑制前列腺肿瘤的增殖及迁移能力,其机制与PI3K-AKT信号通路有关.     

趋化素样因子-1致不育小鼠睾丸曲细精管的病理改变

目的　探讨趋化素样因子 1(CKLF 1)致小鼠不育的可能机制。方法　通过以导入CKLFI质粒的不育小鼠为对象 ,观察其睾丸及曲细精管的病理改变 ,探讨小鼠不育的原因。结果导入CKLFI实验鼠不育 ;两组小鼠体重及睾丸重量差异有显著性 (P <0 .0 5 ) ;实验鼠曲精管内各级生精细胞层数少 ,排列紊乱 ,曲细精管壁变薄、皱缩、塌陷 ,细胞排列疏松、脱落。结论　导入CKLFI能使小鼠曲细精管发生病理改变致不育。     

睾丸生精小管界膜和管周肌样细胞调控睾丸功能

精子的发生除了受下丘脑-垂体-性腺轴分泌的激素调节外,还受到睾丸组织本身分泌的一些因子的调控.旁分泌是指某种组织中的一种细胞产生的因子作用于同一组织中的另一种细胞,从而起到调节靶细胞的功能.睾丸内多种细胞分泌的因子是通过旁分泌起作用的,可对精子发生进行更精细的调控.《美国实验生物学会联合会杂志》(FASEB Journal) 2009年12月刊上,Michelle Welsh等研究证实睾酮作用于睾丸中的另一类细胞——管周肌样细胞(peritubularmyoid,PTM)上的受体(AR),调控支持细胞的功能,进而调节精子形成.本文就界膜和管周细胞与旁分泌以及激素对精子的调控进行综述.     

Daxx基因在小鼠睾丸精子发生过程中的表达特征

目的:研究死亡结构域相关蛋白(Daxx)基因在小鼠睾丸精子发生过程中的表达特征,初步探讨其在生精过程中的作用。方法:通过实时荧光定量PCR(q PCR)、Western印迹及免疫荧光等方法检测Daxx在不同周龄野生型小鼠睾丸组织以及成年睾丸支持细胞雄激素受体特异性敲除(SCARKO)和雄激素受体敲除(ARKO)小鼠睾丸中的表达特征。结果:q PCR、Western印迹和免疫荧光结果表明,Daxx基因在出生4周后小鼠睾丸中高表达,且主要定位于细胞核;与野生型小鼠相比,SCARKO小鼠睾丸中DAXX的表达差异不显著(0.853±0.058 vs1.000±0.015),但在生精细胞细胞核中呈极性分布;DAXX在ARKO小鼠睾丸表达显著降低(0.299±0.026 vs1.000±0.015,P0.01)。结论 :Daxx基因在小鼠睾丸发育中期时表达最高。ARKO小鼠中DAXX的表达与野生型相比显著降低,睾丸支持细胞中AR基因特异性敲除影响DAXX定位。DAXX可能参与调控小鼠的精子发生过程。     

NYD-SP6基因在无精症患者和不同年龄段人群睾丸中的表达

目的探讨NYD-SP6基因在无精症患者和不同年龄人群中的表达及意义。方法将睾丸标本按胚胎、成年人、老人不同年龄段分成3组：胚胎组（4例）、成人组（4例）及老人组（4例）；将无精症患者按病理类型分成3组，生精细胞减少组（7例）、生精阻滞组（13例）及唯支持细胞综合征组（3例）。应用实时（Real time）逆转录-聚合酶链反应（RT—PCR）方法定量测定这些睾丸组织NYD—SP6基因的表达水平。结果与成人组比较，胚胎和老人组NYD—SP6基因的表达量降低。差异有统计学意义（P〈0．01）；生精阻滞组和唯支持细胞综合征组的表达量降低，差异有统计学意义（P〈0．01）。而生精细胞减少组与成人组比较差异无统计学意义（P〉0．05）。结论NYD-SP6基因的表达具有时相性，它在成人睾丸的高表达及在生精阻滞和唯支持细胞综合征患者睾丸表达显著降低，提示其在精子发生中具有重要意义。     

Osteotesticular protein tyrosine phosphatase expression in rodent testis

PURPOSE: In the last decade the novel receptor-like protein tyrosine phosphatase was identified and termed osteotesticular tyrosine phosphatase (OST-PTP) due to its restricted expression in bone and testis. OST-PTP expression is regulated during osteoblast differentiation and it shows stage specific expression in the testis. Confined OST-PTP expression in the basal compartment of the seminiferous tubules suggests that this protein may be a good candidate for a rodent germ stem cell marker. To test this hypothesis we determined the exact pattern of OST-PTP expression in the rodent testis. MATERIALS AND METHODS: Adult mouse and rat paraffinized testis sections were subjected to in situ hybridization using riboprobes against the receptor and catalytic domains of the protein OST-PTP. RESULTS: OST-PTP testicular expression in rodents is not confined to the spermatogonia, as previously suggested, but is also present in Sertoli cells in a stage independent pattern. This finding excludes the hypothesis that OST-PTP is a germ stem cell identification marker in rodents. In addition, we report the identification of a testicular OST-PTP isoform lacking part of a catalytic domain that is widely expressed during spermatogenesis in all cell types. CONCLUSIONS: This finding implies tight control over OST-PTP expression in the testis, which in turn suggests an important role for OST-PTP and its isoform in male germ cell differentiation.     

Identification of a novel testis-specific gene and its potential roles in testis development／spermatogenesis

Aim: To identify and characterize a novel gene with potential roles in testis development and spermatogenesis.Methods: A cDNA microarray was constructed from a human testis large insert cDNA library and hybridized with probes of human or mouse adult and fetal testes. Differentially expressed genes were isolated and sequenced. RT-PCR was used to test the tissue distribution of the genes of interest and in situ hybridization was performed to localize the gene expression in the mouse testis. A range of bioinformatical programs including Gene Runner, SMART, NCBI Blast and Emboss CpGPlot were used to characterize the new gene‘s feature. Results: A novel testis-specific gene,NYD-SPS, was differentially expressed in fetal and adult testes. The deduced protein structure of NYD-SP5 was found to contain an IQ motif (a short calmodulin-binding motif containing conserved lie and Gin residues), a Carbamate kinase-like domain, a Zn-dependent exopeptidase domain and a lactate dehydrogenase (LDH) C-terminal-like domain. RT-PCR analysis revealed that NYD-SP5 was predominantly expressed in the testis but not in other 15 tissues examined. In situ hybridization and RT-PCR examinations revealed that the expression of NYD-SP5 was confined in the male germ cell but not present in the somatic cell in the testes. Conclusion: NYD-SP5 is a newly found testisspecific gene with potential roles in testis development and spermatogenesis through a calmodulin-activated enzyme.     

Hormonal regulation and germ cell-specific expression of heat shock protein 60 (hsp60) in the testis of macaque monkeys (Macaca mulatta and M. fascicularis)

Decrease of heat shock protein 60 (hsp60), a mitochondrial chaperonin, in germ cells of men has been shown to be associated with low spermatogenic efficiency. In the present study, we have investigated the hormonal regulation of hsp60 in a pre-clinical primate animal model. Hsp60 production in the testes of the intact cynomolgus monkey ( Macaca fascicularis ) and animals that had been treated with the GnRH antagonist Cetrorelix for 25 days was studied by immunohistochemistry. In addition, testes of untreated adult rhesus monkeys ( Macaca mulatta ) and immature animals either exposed to human chorionic gonadotrophin (hCG), human follicle stimulating hormone (FSH) or hCG and FSH in combination, as well as vehicle-treated controls were analysed. In adult monkeys, specific hsp60 staining was observed in Leydig cells, spermatogonia and early primary spermatocytes. The labelling in Sertoli cells was not stage dependent. The hsp60 staining pattern was unaffected by gonadotrophin releasing hormone (GnRH) antagonist treatment. Western blot analysis confirmed the presence of a single band of 60 kDa in␣testicular homogenates of the cynomolgus monkey. In the testis of immature rhesus monkeys, hsp60 immunoreactivity was visible in gonocytes, spermatogonia and in Sertoli cells, whereas interstitial cells were negative. In the experimental study, hCG alone or in combination with FSH caused a substantial and marked upregulation of the chaperonin in Leydig cells. Human FSH alone did not affect hsp60 expression. We conclude that hCG is an important regulator of Leydig cell hsp60 expression during development, whereas FSH in immature animals and GnRH in adult monkeys is of less importance.     

Developmental expression of ACRV1 in humans and mice

To identify the developmental expression of the ACRV1 gene in humans and mice, testes cDNA samples were collected at different post-natal days (days 4, 9, 18, 35, 54, and 6 months) from Balb/c mice and were hybridised to the mouse whole genome 430 2.0 Array (Affymetrix Inc.) chip. The characteristics of ACRV1 were analysed using various cellular and molecular biotechnologies. The results showed that the expression of mouse ACRV1 was not detected in mouse testes on days 4, 9, and 18 but was present on days 35, 54, and 6 months. Using RT-PCR analysis of mouse ACRV1, we determined that mouse ACRV1 was expressed specifically in the mouse testis, and its expression began at days 35. Western blot analysis demonstrated that human ACRV1 was primarily expressed in human testes, and immunofluorescent and immunohistochemistry staining showed that human ACRV1 protein was predominantly located in round and elongated spermatids in human testes, indicating that ACRV1 may play an important role in mammalian spermatogenesis and may be a target of a contraceptive vaccine.