雄激素受体敲除后雄性小鼠的泌尿生殖系统表型

目的　了解敲除雄激素受体 (AR)后雄性小鼠的泌尿生殖系统表型。　方法　采用Cre lox技术 ,雌性Flox AR小鼠与雄性ACTB Cre小鼠交配 ,PCR方法检测子代小鼠尾巴的基因型 ,筛选出AR敲除小鼠 5只 ,5只AR未被敲除的小鼠作为对照。比较 2组小鼠的泌尿生殖系统表型 ,测量肛门生殖器距离、睾丸重量、血清睾酮及雌二醇浓度。　结果　AR敲除 (ARKO)组小鼠肛门生殖器距离为 (0 .5± 0 .1)cm ,对照组为 (1.1± 0 .1)cm。ARKO组前列腺、精囊、附睾及球海绵体肌均缺如 ,睾丸显著缩小 ,重量 (0 .0 0 6± 0 .0 0 1) g ;对照组前列腺、精囊、附睾及球海绵体肌发育正常 ,睾丸重量为 (0 .0 86± 0 .0 0 2 ) g。ARKO组血清睾酮浓度 (0 .0 5 6± 0 .0 4 5 )nmol/L ,雌二醇浓度 (1390 .1± 2 94 .3) pmol/L ,对照组睾酮浓度 (0 .84 3± 0 .736 )nmol/L ;雌二醇浓度 (786 .2± 15 0 .8) pmol/L。差异均有统计学意义 (P <0 .0 5 )。　结论　敲除雄激素受体后 ,小鼠前列腺、精囊、附睾及球海绵体肌缺如 ,睾丸缩小 ,雄激素水平降低 ,雌激素水平升高 ,提示AR在雄性小鼠泌尿生殖系统发生及雄激素水平调节中发挥重要作用。     

雄激素受体在睾丸内的分布及与生精调节的关系

睾丸间质细胞合成和分泌的雄激素主要有睾酮（testosterone，T）、双氢睾酮。（dihydrotestosterone，DHT）、脱氢异雄酮（dehydroisoandrosterone，DHIA）和雄烯二酮（androstenedione）。在各种雄激素中，双氢睾酮的生物活性最强，睾酮次之。雄激素的作用广泛，在雄激素的诱导下，含有Y染色体的胚胎向男性化方面分化，促进内生殖器发育，而双氢睾酮则主要刺激外生殖器的发育。     

雄激素受体的研究进展

雄激素对正常雄性发育、分化及功能维持十分必要。雄激素的这些作用是由雄激素受体ＡＲ介导的。雄激素虽然是甾体激素中最早被发现的。但其受体的研究却落后于其它家族成员。这是因为ＡＲ在其靶组织中的含量很低，而且易被蛋白酶降解，这使得ＡＲ的测定及纯化十分困难。此...     

【特刊综述】利用雄激素受体敲除（ARKO）小鼠模型认识人类雄激素缺乏

雄激素的作用机理很复杂。最近,使用转基因修饰小鼠模型进行的雄激素受体研究使我们对雄激素作用机理有了更进一步的理解。Cre-loxP系统能够完成组织和／或细胞特异性的敲除。通过使用Cre—loxP系统已经获得了大量整体性或组织特异性雄激素受体（ARK0）敲除模型。这些ARKO模型的多个雄激素作用位点,包括心血管系统、免疫和造血系统、骨骼、肌肉、脂肪组织、前列腺和脑,均已被检测。本篇综述着重阐述了通过针对这些雄激素作用位点的ARKO小鼠模型而取得的人类雄激素缺乏的研究成果,并且指出充分理解Cre-loxPdx鼠模型的优缺点才能正确认识其表型。     

Zfx在小鼠睾丸生精细胞中的表达

目的:了解Zfx基因在各级生精细胞中的表达情况。方法:本试验采集6、8、17日龄和成年小鼠睾丸,采用组合酶消化法制备单细胞悬液,BSA铺设密度梯度法分离生精细胞,运用荧光实时定量PCR(qRT-PCR)及Western印迹法(WB)方法检测Zfx基因的表达。结果:组合酶消化法制备单细胞悬液、BSA铺设密度梯度法可获得纯度>85%的各级生精细胞;Zfx基因在原始A型精原细胞、A型精原细胞、B型精原细胞中表达水平低,前细线期精母细胞、粗线期精母细胞、圆形精子细胞中表达量高,长形精子、成熟精子不表达。结论:Zfx基因在各级生精细胞中呈现阶段性表达,可能是参与调控生精细胞减数分裂的重要转录因子。     

雄激素/雄激素受体对小鼠附睾Caveolin-1表达调控机制的研究

目的:探讨雄激素和雄激素受体(AR)对小鼠附睾Caveolin-1表达的调控机制。方法:通过搜索前期染色体免疫共沉淀-测序(ChIP-seq)实验得到的小鼠附睾AR结合位点的数据库,寻找Caveolin-1基因相关联的AR结合位点。分别取正常小鼠、睾丸阉割小鼠以及睾丸阉割后补充外源雄激素的小鼠的附睾组织,一方面,抽提总RNA,利用逆转录PCR以及逆转录荧光定量PCR检测Caveolin-1基因的mRNA表达水平;另一方面,利用AR抗体进行染色体免疫共沉淀(ChIP),采用ChIP-PCR以及ChIP-qPCR的方法,检测Caveolin-1基因相关联的AR结合位点在体内与AR的结合情况。结果:从小鼠附睾AR结合位点的数据库中找到两个Caveolin-1相关联的AR结合位点,均位于第二内含子区域。睾丸阉割后,Caveolin-1基因的表达显著升高(P<0.05),表达量是对照组的(1.8±0.17)倍;两个AR结合位点的富集倍数分别由正常状态下的(13.5±1.47)倍和(10.5±1.03)倍降至(1.05±0.17)倍和(1.4±0.14)倍(P<0.01)。补充雄激素后,Caveolin-1基因的表达又降至正常水平(P<0.05),为正常对照组的(1.03±0.06)倍。两个AR结合位点的富集倍数则分别升至(16.4±2.6)倍和(10.0±0.92)倍(P<0.01)。结论:Caveolin-1在小鼠附睾内是AR的一个直接靶基因,其表达受雄激素的负调控。该研究为理解雄激素/AR在小鼠附睾内的调控网络提供了新的视角。     

血清抑制素B与睾丸生精功能

目的　探讨血清抑制素B与睾丸生精功能的关系。方法　将男性不育症病人分为原发性无精子症、严重少精子症和梗阻性无精子症 3组 ,另设正常对照组 ,分别测定其抑制素B、FSH及精子密度。结果　上述 4组的抑制素B浓度分别为 4 7.36± 32 .85 ,2 0 0 .92± 86 .4 4 ,2 82 .4 0± 98.2 1,2 36 .79± 10 7.70。结论　血清抑制素B可直接反映睾丸的生精功能状态 ,因而是判断男性生育力更有效的指标     

睾丸外组织雄激素受体的分布及检测

雄激素受体 (AR)是介导雄激素在靶细胞中发挥作用的关键大分子 ,主要存在于男性睾丸组织中。本文综述了AR及其mRNA在泌尿生殖系统的前列腺、附睾、阴茎皮肤等睾丸外组织 ,头皮、海马旁回、脂肪等非泌尿生殖系统组织及在胃癌、喉癌等睾丸外肿瘤组织中的分布情况 ,并对AR的检测方法进行了复习 ,以进一步研究循环血中雄激素对人体各系统的作用     

雄激素受体在雄激素非依赖性前列腺癌的表达

目的探讨雄激素受体(AR)在国人雄激素非依赖性前列腺癌(AIPC)的表达及其临床意义。方法采用免疫组化两步法,检测AR在15例AIPC和20例雄激素依赖性前列腺癌(ADPC)组织标本的表达水平。结果1例AIPC无AR表达,其余14例AIPC及全部ADPC均有AR高表达,AIPC染色的光密值为0.31±0.04较ADPC0.28±0.03升高。结论大多数国人AIPC均高表达AR,AR的异常表达可能在前列腺癌的进展中发挥重要的作用。     

Comparison of p63 and p73 expression in benign and malignant salivary gland lesions

BACKGROUND: The p63 and p73 genes are members of the p53 family and play an important role in stem cell identity and cellular differentiation and are expressed in basal and myoepithelial cells. In this study, we examined the expression of p63 and p73 in 50 various benign salivary gland lesions and 45 malignant salivary gland tumors. METHODS: The 95 salivary gland tumors were selected from the archives of the Department of Pathology and Laboratory Medicine at the Hospital of the University of Pennsylvania. Sectioned formalin-fixed paraffin-embedded tissue cut at 3 mum was immunostained with antibodies that recognize all isozymes of p63 and p73 and evaluated with respect to percentage of positive cells and localization. RESULTS: In benign lesions, p63 and p73 nuclear reactivity was seen in 46 (92%) of 50 and 47 (94%) of 50 cases, respectively. In malignant tumors, p63 and p73 were seen in 34 (76%) of 45 and 40 (89%) of 45 cases, respectively. A significant difference between p63 and p73 positivity was only seen in adenoid cystic carcinomas (p = .006). Also, p73 was found in tumors with minimal basal/myoepithelial differentiation. CONCLUSIONS: Hence, p63 and p73 expression is retained in both benign and malignant salivary gland tumors with basaloid or myoepithelial differentiation. Hence, p63 seems to be a more specific marker of myoepithelial differentiation than p73.     

皮肤扩张过程中p63和细胞角蛋白的表达特征及意义

目的:初步观察皮肤扩张术对表皮细胞增殖分化的影响并探讨其机制。方法:用iWstar大鼠制作扩张动物模型,将72个扩张器埋于36只wistar鼠背部浅筋膜下,利用表皮干细胞特异表达p63和角蛋白19及短暂扩充细胞表达角蛋白14的特点,采用免疫组织化学Powervision M二步法检测扩张过程中表皮干细胞和短暂扩充细胞的分布与数量差异。结果:扩张皮肤表皮层增厚,表皮干细胞和短暂扩充细胞的数量明显增多,在棘层和颗粒层可见表皮干细胞和短暂扩充细胞的阳性表达。结论:皮肤扩张术能诱导表皮干细胞增殖分化,表皮干细胞在扩张过程中对机械应力和创伤修复的响应机制可能是其主要的生物学基础。     

基因物质p63、β1整合素对正常人不同部位皮肤表皮干细胞的表达和意义

目的　探讨基因物质 p63、β1整合素对正常人不同部位皮肤表皮干细胞的表达情况及意义。方法　在 5例正常人尸体上各取 2 1个不同部位的全层皮肤组织 ,采用免疫组织化学SP法 ,用基因物质 p63、β1整合素作为第一抗体标记表皮干细胞 ,并通过图像分析 ,研究表皮干细胞在正常人不同部位皮肤中的表达情况。结果　 (1)头皮 p63的PU值 [阳性单位 (% ) ] (4 2 .17± 3 .10 )最大 ,与面皮、腋部、上臂内侧、前臂内侧、腰背部、手背、足背及足底差异有显著性 (P <0 .0 5 ) ;足背p63的PU值(2 1.19± 6.89)最小 ,与身体其他部位差异均有显著性 (P <0 .0 5 )。头皮CD2 9的PU值 (2 9.13± 4.99)最大 ,与身体其他部位差异均有显著性 (P <0 .0 5 ) ;足底CD2 9的PU值 (11.89±1.5 2 )最小 ,阴囊与面皮、腰背、上臂内外侧、手掌、手背、腹部及足底差异有显著性 (P <0 .0 5 )。(2 )正常人不同部位多毛组与汗毛组 p63、CD2 9的PU值差异有显著性(P <0 .0 0 0 1)。(3 )躯干组与四肢组p63、CD2 9的PU值差异无显著性 (P >0 .0 5 )。(4 )屈侧与伸侧p63、CD2 9的PU值差异无显著性 (P >0 .0 5 )。结论　 (1) p63、CD2 9对正常人不同部位皮肤表皮干细胞的表达不同 ,头皮、阴阜、阴囊皮肤组织中的阳性表达较高 ,而其他部位皮肤组织中     

膀胱癌组织中Ki-67、p63的表达与肿瘤复发的关系

目的:探讨浅表性膀胱癌组织学形态以及Ki67、p63表达与肿瘤复发的关系。方法:将3年来随访的60例浅表性膀胱癌分为复发组和无复发组。免疫组化法检测Ki-67和p63的表达并比较复发组与无复发组的差异。结果:在平均28个月的随访中,40例复发,复发1~4次,Ki-67和p63阳性率在两组之间有明显差别,复发组均明显高于无复发组(P<0.05)。在不同病理分级TCC中p63和Ki-67表达有明显差异,病理分级高的明显高于分级低的(P<0.01)。结论:Ki-67、p63阳性表达尤其是联合阳性表达对判断膀胱癌复发具有重要意义。     

p63 gene analysis in Mexican patients with syndromic and non-syndromic ectrodactyly.

Ectrodactyly is a congenital limb malformation that involves a central reduction defect of the hands and/or feet which is frequently associated with other phenotypic abnormalities. The condition appears to be genetically heterogeneous and recently it has been demonstrated that mutations in the p63 gene, a homologue of the tumor suppressor gene p53, are the cause of at least four autosomal dominant genetic syndromes which feature ectrodactyly: ectrodactyly, ectodermal dysplasia, and facial clefting (EEC), split hand/split foot malformation (SHFM), limb-mammary syndrome (LMS), and acro-dermato-ungual-lacrimal-tooth syndrome (ADULT). In this study, genetic analysis of the p63 gene in a group of 13 patients with ectrodactyly (syndromic and isolated) was performed. Four patients with syndromic ectrodactyly had p63 heterozygous point mutations that affect the DNA binding domain of the protein. One of these subjects exhibited the typical features of EEC syndrome as well as ankyloblepharon being, to our knowledge, the first case combining these traits. This finding supports the view of a clinical overlap in this group of autosomal dominant syndromes caused by p63 mutations and demonstrates that there are exceptions in the previously established p63 genotype-phenotype correlation.     

ΔNp63蛋白在膀胱移行上皮癌中的表达及其临床意义

目的 :探讨 p5 3基因家族新成员截短型p6 3(△Np6 3)在膀胱癌组织中的表达及其意义。 方法 :采用免疫组织化学SP法检测 4 0例膀胱移行上皮癌 (TCC)、6例膀胱内翻性乳头状瘤和 8例正常膀胱移行上皮中△Np6 3的表达 ,并分析△Np6 3表达与膀胱癌病理类型、临床分期的关系。 结果 :正常膀胱移行上皮、膀胱内翻性乳头状瘤、TCC中△Np6 3的阳性表达率分别为 37.5 % (3/ 8)、6 6 .7% (4/ 6 )、10 0 % (40 / 4 0 ) ,组间差异有统计学意义 (P <0 .0 1)。TCCG3 级与G2 级△Np6 3的强阳性、中度阳性表达率显著高于G1级 (P <0 .0 1)。Ta～T1期以△Np6 3弱阳性为主 (6 6 .7% ) ,随TCC浸润程度的增加 ,△Np6 3染色强度逐渐增强。T2 期△Np6 3强阳性表达率为 35 .3% ,T3 ～T4期增至 6 3.6 %。结论 :△Np6 3在TCC中高表达 ,与TCC病理分级、临床分期密切相关 ;△Np6 3可能参与TCC的发生、发展 ,是评估TCC预后的潜在因素之一。     

AMACR和p63表达在前列腺癌临床诊断中的价值

目的 探讨前列腺癌组织中α-甲基酰基辅酶A消旋酶（AMACR）和p63表达及其在临床诊断中的价值。方法 采用免疫组织化学方法检测39例前列腺癌和24例朗性前列腺增生组织标本中AMACR和p63的表达，分析其与前列腺癌组织分化程度、临床分期的关系。结果 AMACR和p63在前列腺癌中阳性表达率分别为85％（33／39）和10％（4／39），在前列腺增生组织中阳性表达率分别为21％（5／24）和88％（21／24），比较差异均有统计学意义（P〈0．01）。AMACR高表达和p63低表达与前列腺癌分化程度、临床病理分期无相关性（P〉0．05）。结论 AMACR高表达和p63低表达可能是前列腺癌形成的早期事件，AMACR和p63可能作为前列腺癌的早期诊断指标和治疗新靶点。     

p63 Immunohistochemistry Differentiates Salivary Gland Oncocytoma and Oncocytic Carcinoma from Metastatic Renal Cell Carcinoma

Metastatic renal cell carcinoma (RCC) can pose diagnostic challenges in the head and neck often resembling benign and malignant oncocytic lesions. Immunohistochemical panels have been reported to help with this differential but are not entirely specific or sensitive. We have noticed that p63 routinely stains salivary gland oncocytomas but not metastatic RCC. Nineteen oncocytomas, 9 cases of oncocytosis, 9 oncocytic carcinomas and 16 head and neck metastatic RCC were studied. Morphologic features evaluated were cytoplasmic character (clear versus oncocytic), Fuhrman nuclear grade, mitotic rate, growth pattern, presence of lumens/blood lakes and stromal characteristics. Tumors were stained with antibodies to p63, renal cell carcinoma marker (RCCm), CD10, and vimentin. Eight benign oncocytic tumors (29%) had clear cell features while 6 metastatic RCC (37%) had oncocytic features. Median Fuhrman nuclear grade was 2 in oncocytoma and oncocytosis and 3 both oncocytic carcinoma and metastatic RCC. Mitotic rates were only significantly different between benign oncocytic tumors and metastatic RCC. All oncocytomas had lumina compared to half of metastatic RCC, all of which also demonstrated blood lakes. Seven benign oncocytic tumors (25%) and 5 oncocytic carcinomas (56%) had RCC-like vascular stroma. All primary salivary gland tumors were positive for p63, predominately in basal cell-type distribution. None of the metastatic RCC was positive. RCCm was entirely specific but lacked sensitivity for metastatic RCC while CD10 and vimentin showed variable sensitivity and specificity. While clinical history and morphology usually are adequate, demonstration of p63 staining can definitively exclude metastatic RCC from the differential diagnosis of similar appearing tumors in salivary glands, namely oncocytoma and oncocytic carcinoma, with 100% specificity and sensitivity. While RCCm, CD10, and vimentin performed adequately, they were significantly less reliable than p63 with both false positives and false negatives. The results of this study were presented at the 96th Annual Meeting of the United States and Canadian Academy of Pathologists in San Diego, CA, March 2007.     

Use of p63 and CD10 in the differential diagnosis of papillary neoplasms of the breast

Papillary neoplasms of the breast represent a complex spectrum ranging from benign to malignant lesions. The myoepithelial cell (MEC) layer is generally continuous in papillomas and increasingly discontinuous to absent in atypical and malignant counterparts. Identification of MECs can be difficult on morphological grounds and currently relies on immunomarkers. We investigated the potential role of p63 and CD10 in 20 papillary lesions and compared them with 1A4 and calponin. In 18 cases, adjacent normal breast tissue was available for study. All four markers were diffusely positive in all samples of normal tissue and benign papillomas indicating similar sensitivity in the identification of MECs. Intense positivity was found in 100% of the cases with 1A4 and CD10, but in only 76% with calponin and in 60.5% with p63 (differences statistically significant, p < 0.05), suggesting that the former two render more reproducible results. The most specific markers were p63 and CD10 which showed cross-reactivity in 0% and in up to 33% of the cases respectively. 1A4 and calponin showed diffuse cross-reactivity in all cases. When assessing benign versus atypical papillomas, the best parameters were diffuse positivity using CD10 or p63, and continuous MEC layer, mainly using CD10. When comparing benign papillomas to carcinomas all parameters were equally useful with 1A4 and CD10. Regardless of the marker, intense positivity was the only parameter that could distinguish atypical papillomas from papillary carcinomas. p63 staining, which renders a nuclear and mostly discontinuous reactivity, was not as useful as the other markers when the parameter continuous MEC layer was evaluated. Although CD10 seems to combine the highest specificity and reproducibility with a good sensitivity, reproducibility of 1A4 is higher. Thus, a minimum panel to assess papillary lesions should include both markers. Although p63 is the most specific, its nuclear and discontinuous pattern may lead to erroneous diagnosis, especially in the differentiation between benign papillomas and atypical/malignant lesions.