尿道下裂患儿包皮组织中FGFR2表达检测及意义

目的 检测成纤维细胞生长因子受体2(fibroblast growth factor receptor 2,FGFR2)蛋白和mRNA在包皮环切组和尿道下裂组包皮组织中的表达情况,初步探究FGFR2蛋白表达、基因功能和尿道下裂的关系.方法 采用Western blotting(免疫印迹)方法和RT-PCR方法;检测两组中FGFR2表达情况;结果包皮环切组和尿道下裂组FGFR2蛋白相对定量值分别为0.39±0.12和0.19±0.09(P＜0.05),尿道下裂组较包皮环切组降低.尿道下裂轻、中、重度三组间FGFR2蛋白相对定量值分别为0.27±0.08、0.16±0.04、0.09±0.03;中度和重度之间比较无统计学差异(P＞0.05).包皮环切组和尿道下裂组FGFR2 mRNA表达量分别为1.30±0.069和1.22±0.052,包皮环切组高于尿道下裂组(P＜0.05).尿道下裂轻、中、重度三组间FGFR2 mRNA定量值分别为1.23±0).054、1.24±0.034、1.16±0.020;轻度和中度之间之间无统计学差异(P＞0.05),重度尿道下裂FGFR2mRNA表达量低于其他两组.结论 尿道下裂患儿包皮组织中FGFR2蛋白和mRNA较包皮环切组表达下调.提示FGFR2和尿道下裂关系密切,进一步研究FGFR2蛋白和基因功能有助于明确FGFR2在尿道下裂发病机制中的重要地位,有可能揭示尿道下裂的发病机制.     

尿道下裂患儿包皮组织Shh基因及其受体Ptch1的表达

目的观察尿道下裂患儿包皮组织中Shh基因及其受体Ptch1的表达,探讨其与尿道下裂发病机制间的关系。方法选取2009年6月-2011年7月在本科首次接受手术治疗、除外合并其他疾病(如隐睾)的尿道下裂患儿30例,选择同期单纯包皮环切患儿30例为对照。术中收集尿道下裂患儿的阴茎背侧包皮,液氮速冻后存于-80℃冰箱,采用反转录-PCR技术检测2组患儿包皮组织Shh基因及其受体Ptch1表达水平。应用SPSS 13.0软件进行统计学分析。结果尿道下裂组患儿阴茎背测包皮组织ShhmRNA及Ptch1 mRNA的表达水平较对照组低,且差异均有统计学意义(Pa<0.05),但轻度尿道下裂、中度尿道下裂及重度尿道下裂3个亚组间Shh mRNA及Ptch1 mRNA表达的差异均无统计学意义(Pa>0.05)。结论尿道下裂患儿阴茎背测包皮组织Shh基因及其受体Ptch1表达下降,可能引起尿道生殖褶部位间充质细胞与上皮细胞转化障碍,尿道板腔化受阻,进而导致尿道下裂发生发展。     

邻苯二甲酸二丁酯影响类固醇激素合成急性调节蛋白致雄性胎鼠尿道下裂的机制研究CSCD

目的 研究类固醇激素合成急性调节蛋白（StAR）的表达和活性及睾酮在尿道下裂胎鼠体内发生的改变,探讨邻苯二甲酸二丁酯（DBP）诱导雄性胎鼠发生尿道下裂的机制。方法 将40只孕SD大鼠,用随机数表法分为DBP组和对照组（各20只）。于孕13～19 d上午9点给药,DBP组按照800 mg/kg体重DBP混合玉米油（DBP和玉米油总量为3 ml/kg体重）灌胃,对照组按3 ml/kg体重玉米油灌胃。第19天中午12时麻醉孕鼠后取出胎鼠,观察内外生殖器鉴别出DBP组发生尿道下裂的雄性胎鼠及对照组中的雄性胎鼠。从DBP组中发生尿道下裂的雄性胎鼠中选取40只做为尿道下裂组,再从对照组的雄性胎鼠中选取40只做为正常对照组,拍摄尿道下裂图片,测量肛门生殖结节距离,统计尿道下裂发生率,取各组胎鼠生殖结节进行HE染色。各组胎鼠断头取血用酶联免疫吸附测定法（ELISA）检测睾酮水平并取出各组睾丸采用定量逆转录聚合酶链式反应（RT-PCR）、Western blot、免疫荧光、ELISA检测StAR mRNA及StAR的表达,对StAR相对表达量和睾酮含量进行Pearson相关性分析。Western blot检测磷酸化ERK1/2蛋白（P-ERK1/2）,总ERK1/2蛋白（T-ERK1/2）的表达,并通过两者间的比值分析睾丸中ERK1/2蛋白的磷酸化水平以明确StAR蛋白的活性高低。结果 尿道下裂组胎鼠的尿道开口于生殖结节腹侧,对照组的开口于生殖结节顶端。尿道下裂组肛门生殖结节距离（1.77±0.12） mm明显小于对照组（2.25±0.15） mm,组间比较,差异有统计学意义（P〈0.05）。DBP组胎鼠尿道下裂发生率为43.3%（46/106）。尿道下裂组睾酮含量（1.45±0.62） ng/ml低于对照组（4.48±0.93） ng/ml,组间比较,差异有统计学意义（P〈0.05）。尿道下裂组的StAR mRNA、StAR相对表达量及P-ERK1/T-ERK1、P-ERK2/T-ERK2分别为0.23±0.08、0.33±0.07、0.17±0.03和0.19±0.07,对照组的分别为1.00±0.00、1.44±0.19、0.31±0.08和0.43±0.14,组间差异有统计学意义（P〈0.05）。免疫荧光显示睾丸间质细胞中尿道下裂组StAR蛋白的表达少于对照组。尿道下裂组和对照组的Pearson相关系数分别为0.642和0.851（P均〈0.05）。结论 DBP不仅在转录和翻译层面降低StAR的表达,可能还通过降低ERK1/2蛋白的磷酸化水平减弱StAR蛋白的活性使睾丸中睾酮合成障碍,进而影响了雄性胎鼠生殖器的发育。     

Hippo通路相关分子在先天性膈疝肺发育不良大鼠模型中的表达及意义

目的了解Hippo信号通路主要组成分子在先天性膈疝(congenital diaphragmatic hernia,CDH)模型大鼠胎肺组织中的表达情况,初步探讨该通路与CDH肺发育不良的关系。方法应用除草醚建立CDH大鼠模型,选取模型组中单纯左侧膈疝的胎鼠左肺作为研究对象,通过免疫印迹法和RT-PCR分别检测肺组织中Lats1、Taz和Yap(pYap)表达情况并与对照组胎鼠的左肺组织进行比较。结果 Lats1、Taz和Yap在模型胎鼠和正常胎鼠的支气管、肺上皮细胞及肺动脉壁中都有表达。模型组的Yap蛋白及其mRNA的表达量分别为2.333 3±0.367 5和1.243 3±0.103 3明显高于对照组的1.250 9±0.388 6和0.983 3±0.065 0,两组比较,差异有统计学意义(P<0.05);而Lats1蛋白及其mRNA和Yap蛋白磷酸化水平(pYap)的表达量在两组间的差异均无统计学意义(P>0.05)。Taz的mRNA表达量在模型组和对照组分别为1.216 7±0.033 9和1.013 3±0.183 4,组间比较,差异有统计学意义(P<0...     

尿道下裂相关miRNA差异表达分析的初步探讨

目的通过筛选尿道下裂、术前经HCG治疗的尿道下裂以及正常包皮组织中miRNA差异表达,探究其与尿道下裂的发生以及术前HCG治疗影响预后的可能途径,为探究尿道下裂病因提供新思路。方法选取9例尿道下裂(其中3例术前进行HCG治疗)以及3例包茎患儿(对照组)为研究对象,分别取包皮组织抽提总RNA,利用芯片技术筛选各组差异性表达的miRNAs,并用RT-PCR技术对筛选结果进行验证。结果芯片筛选结果:与对照组比较,尿道下裂患儿的包皮组织里差异性表达的miRNAs中上调的19个,下调的15个。与术前行HCG治疗组的患儿相比,术前未行HCG治疗的尿道下裂患儿包皮组织里差异性表达的miRNA中上调的19个,下调的25个; RT-PCR验证结果:在差异性表达的miRNA中选择10个miRNA进行验证,其中5个miRNA与芯片结果相符,分别是let-7b-3p、miR-145-5p、miR-376a-3p、miR-377-3p和miR-1-3p。其中let-7b-3p、miR-145-5p、miR-376a-3p、miR-377-3p在尿道下裂组中低表达,在对照组和术前HCG治疗的尿道下裂组中高表达; miR-1-3p在术前HCG治疗的尿道下裂患儿中较未用药组中高表达。结论首次建立尿道下裂患儿miRNAs的基因表达谱,并筛选出在术前行HCG治疗尿道下裂、术前未行HCG治疗尿道下裂与包茎之间存在差异表达的miRNAs。根据筛选出的差异性表达的miRNAs,推测其与尿道下裂的关系,为今后疾病的预防以及提高治疗效果提供新的线索。     

FKBP52与尿道下裂相关性的研究

目的 研究雄激素伴侣蛋白FKBP52和先天性尿道下裂之间的相关性。方法 用免疫组织化学SP法半定量检测45例不同严重程度的尿道下裂患儿尿道板和包皮中FKBP52的表达,以正常男性包皮FKBP52的表达为对照。结果 尿道下裂组的包皮中FKBP52的(36/45,80％)表达较对照组(28/30,93.3％)包皮明显减少,尿道板中FKBP52表达(28/45,62.2％)比尿道下裂包皮(80％)的表达减少差异,均存在统计学意义;轻中度尿道下裂组包皮和尿道板中FKBP52的表达分别为83.3％(20/24)和75％(18/24),重度尿道下裂组分别为76.2％和47.6％(10/21),差异均无明显统计学意义。结论 FKBP52与尿道下裂发病密切相关,与尿道下裂严重程度无明显相关。     

HoxA13基因在尿道下裂胎鼠阴茎中的表达及意义

目的研究尿道下裂胎鼠阴茎中HoxA13核酸及其蛋白的表达,探讨其与尿道下裂发生的相关性。方法选用清洁级C57BL／6小鼠,用邻苯二甲酸二（2-乙基）己脂（DEHP）诱导并建立先天性尿道下裂模型;交配怀孕后,孕鼠随机分为玉米油对照组、实验组（DEHP500mg·kg^-1·d^-1）;各组持续灌胃;于怀孕第19天取出仔鼠,测量各组雄鼠体重、AGD;统计各组胎鼠尿道下裂的发生率,并观测阴茎的组织病理学变化;应用常规RT-PCR及免疫组织化学方法检测诱导出的尿道下裂胎鼠及正常对照胎鼠阴茎组织中HoxA13的mRNA及蛋白表达水平。结果对照组、实验组的AGD值分别为（0．208±0．01）cm、（0．181±0．12）cm,P〈0．01;尿道下裂发生率分别为0％、75．7％,P〈0．01。实验组胎鼠尿道板及包皮于阴茎腹侧隆起融合落后,该组尿道与腹侧皮肤间的皮下组织明显减少。DEHP500mg·kg^-1·d^-1诱导的尿道下裂胎鼠阴茎组织中HoxA13mRNA及蛋白表达较对照组明显降低。结论DEHP能影响胎鼠的生长发育及体重,并导致雄鼠外生殖器发育异常;HoxA13mRNA及蛋白的表达在DEHP诱导的尿道下裂胎鼠阴茎中均较正常对照组降低,提示HoxAl3基因异常表达可能是尿道下裂的发生机制之一。     

EphB2在先天性肛门直肠畸形直肠末端表达的研究

目的 研究酪氨酸蛋白激酶受体--EphB2在先天性肛门直肠畸形直肠末端组织中的mRNA表达和蛋白表达的水平,探讨EphB2与先天性肛门直肠畸形发生的关系.方法 RT-PCR方法和Western蛋白印迹方法,检测31例不同类型先天性肛门直肠畸形直肠后壁末端、5例后天瘘及8例正常直肠后壁末端EphB2在蛋白水平和mRNA水平的表达情况,应用单因素方差分析比较正常组和畸形组、不同类型的畸形组之间EphB2表达水平的差异.结果 ①先天性肛门直肠畸形末端肠壁EphB2基因mRNA相对表达量明显低于正常对照组(0.99±0.11,P=0.00).不同类型的畸形组之间,高位组(0.43±0.07)和中位组(0.47±0.10)之间差异无统计学意义(P=0.33),但二者明显低于低位组(0.64±0.06,P=0.00).后天瘘组(0.97±0.09)与正常对照组比较差异没有统计学意义(P=0.63);②Western蛋白印迹结果显示,先天性肛门直肠畸形高位组(0.21±0.05)、中位组(0.24±0.04)和低位组蛋白表达水平均明显低于后天瘘组(0.51±0.05)和正常对照组(0.52±0.03,P=0.00).不同类型的畸形之间,高位组和中位组之间无明显差异(P=0.11),但二者明显低于低位组(P＜0.01).后天瘘组与正常对照组比较差异没有统计学意义(P=0.67).结论 EphB2在先天性肛门直肠畸形直肠末端低表达.EphB2的表达和肛门直肠畸形的发生有关,EphB2在先天性肛门直肠畸形的发生中可能具有重要作用.     

信号转导子与转录激活子3和缺氧诱导因子-1α在神经母细胞瘤中的表达及其意义

目的检测信号转导子与转录激活子3(STAT3)与缺氧诱导因子-1α(HIF-1α)基因在神经母细胞瘤(NB)中的表达,探讨STAT3 mRNA及HIF-1αmRNA的表达与NB的发生、发展及预后的关系。方法收集30例行手术切除的NB患儿的瘤体组织(研究组)及瘤旁组织(对照组)。其中预后不良型(UFH)20例,预后良好型(FH)10例。应用反转录PCR法,检测STAT3 mRNA、HIF-1αmRNA在瘤体组织及相应的瘤旁组织中的表达情况。结果 STAT3 mRNA水平在对照组、FH组和UFH组的表达分别为0.114±0.002、0.267±0.073、0.529±0.325,3组间比较差异有统计学意义(F=22.00,P=0.001);HIF-1αmRNA水平在对照组、FH组和UFH组的表达分别为0.519±0.251、0.673±0.124、0.779±0.145,3组间比较差异有统计学意义(F=111.82,P=0.001)。且FH组和UFH组STAT3 mRNA和HIF-1αmRNA表达水平分别与对照组比较差异均有统计学意义(Pa<0.05),FH组与UFH组比较差异亦有统计学意义(Pa<0.05)。研究组STAT3 mRNA、HIF-1αmRNA相对表达水平分别为0.530±0.051、0.489±0.032,二者表达呈正相关(r=0.497,P<0.05)。结论 STAT3与HIF-1α在NB中过量表达,NB患者预后较差,STAT3与HIF-1α可相互作用促进肿瘤的发生发展。PCR技术可检测NB中STAT3 mRNA及HIF-1αmRNA表达水平,判断其生物学行为,用于指导临床。     

尿道下裂患儿阴茎包皮内板组织成纤维细胞生长特点分析

目的 体外观察尿道下裂患儿阴茎包皮内板组织成纤维细胞生长特点,探讨成纤维细胞生长发育异常与尿道下裂发生的关系.方法 组织块法原代培养尿道下裂患儿的阴茎包皮内板组织成纤维细胞以及细胞增殖试验(CCK8法)检测其增值情况和流式细胞术分析其细胞周期.结果 在细胞原代培养实验中近端型尿道下裂组的成纤维细胞移出组织块的时间(9.5±1.0)d明显长于中远端型组(4.8±0.8)d和对照组(4.0±0.6)d(P值均＜0.05),同时细胞生长融合成束所需时间也较中远端型组和对照组明显延长(P值均＜0.001);在细胞增殖实验及细胞周期分析中显示近端型组尿道下裂受检的成纤维细胞同样表现为细胞增殖明显低于中远端型组和对照组[(17.1±2.2)d比(10.5±1.4)d比(9.0±1.1)(P值均＜0.001)],中远端型低于对照组(P＜0.05).结论 尿道下裂患儿生殖器成纤维细胞生长和增殖能力下降,可能是尿道下裂,尤其是近端型尿道下裂发病的重要因素之一,提示成纤维细胞功能越差,尿道下裂临床表现的程度可能越严重.     

Activating Transcription Factor 3: An Estrogen Responsive Gene in the Aetiology of Hypospadias

PurposeHypospadias is a common malformation in boys and the complex aetiology remains elusive. Initially mainly androgen, but also estrogen imbalances are likely involved in the aetiology of hypospadias. ATF3, a hormone responsive gene, has been reported to be up-regulated during sexual development, as well as in hypospadic genital skin. We investigated ATF3 as a candidate gene for hypospadias.Material and MethodsGenotyping of eight tagged SNPs was performed in 330 boys with hypospadias and in 380 healthy controls. In addition, screening for mutations in ATF3 was conducted in 102 boys with hypospadias. ATF3 expression in the foreskin of boys with hypospadias and in healthy controls, as well as in the human foetal genitalia, was analyzed by immunohistochemistry.ResultsThree common SNPs, spanning a region of about 16 kb in intron 1 of ATF3, are associated with hypospadias. Sequence analysis revealed the gene variant c536A > G (pArg90Gly) in one patient; and c817C > T in the 3'UTR in two other patients. These variants were not present in the 380 controls. ATF3 expression was observed in the developing male urethra, in the developing vagina and in the rectal region in both sexes; and in the pre-pubertal genital tissue.ConclusionsSequence variants in the ATF3 gene influence the risk to hypospadias. Its responsiveness to estrogen may underlie this risk effect. In addition, other ATF3 dependent biological aspects, such as cell survival and cell death, response to stress stimuli or the control of epithelial-mesenchymal interactions, may be of importance.     

尿道下裂患儿包皮组织中表皮生长因子

目的　探讨表皮生长因子（EGF）及其表皮生长因子受体（EGFR）在尿道下裂发生中的作用。方法　应用免疫组织化学染色方法对15例尿道下裂患儿包皮组织和尿道板中EGF、EGFR表达进行检测。结果　15例尿道下裂患儿包皮组织中EGF、EGFR阳性细胞指数分别为195±4、153±8,均低于正常儿童为212±4，225±6（P<0.01），尿道板中EGF、EGFR阳性细胞指数分别为153±8、118±7,亦明显少于其包皮组织（P<0.01）。结论　EGF、EGFR在包皮组织中的不足表达和不均衡分布可能与尿道下裂的发生有关。     

Quantitative Measurement of the Androgen Receptor in Prepuces of Boys with and Without Hypospadias

PurposeIdentifiable causes of hypospadias, a midline fusion defect of the male ventral urethra, are still infrequently well-known. The aim of this study was to quantify the androgen receptor mRNA and androgen receptor protein in prepuces of boys with and without hypospadias.Material and MethodsForty prepuce specimens of circumcised boys, aged between 12 and 14 months, with (n=20) and without hypospadias (n=20), were investigated. Immediately after surgery all probes were fixed in formaldehyde and small parts were deep frozen in liquid nitrogen at -80° C. The total RNA of the specimens was isolated and cDNA was written. With the aid of real time PCR the amount of present androgen receptor mRNA was measured. For quantification of present androgen receptor protein, Western Blot (Bradford method) and immunohistochemistry for androgen receptor using standardized automated procedures (Discovery XT, Ventana) were performed. Statistical analyses using the the Kolmogorov-Smirnov and Mann-Whitney U-test were done.ResultsOur observations show that the androgen receptor mRNA is significantly increased in the prepuce of hypospadiac boys compared to specimens of boys with phimosis.(p<0,01) similarly the amount of androgen receptor protein is increased compared to healthy boys. (p<0.01) Our results provide evidence that the rise of androgen receptor mRNA and protein seems to be an indirect expression of a decreased androgen receptor DNA binding capability possibly indicating further missing polypeptide encoding.ConclusionsOur results provide evidence that the different expression of androgen receptor mRNA indicates the extent of a defect androgen receptor signalling in boys with hypospadias.     

带蒂包皮岛状皮瓣在尿道下裂再次手术中的应用

目的 总结带蒂包皮岛状皮瓣在尿道下裂再次手术中应用的适应证、手术技巧及其并发症.方法　2005年9月-2009年12月本院应用带蒂包皮岛状皮瓣再次手术治疗尿道下裂再次手术患儿35例,年龄3～15岁.其中22例为第2次手术,9例为第3次手术,4例为第4次手术,所有病例尿道局部瘢痕增生明显或尿道板已被破坏.手术方式分为2种:应用加盖岛状皮瓣法(Onlay术式)24例,应用岛状皮瓣管状尿道成形法(Duckett术式)11例.带蒂包皮岛状皮瓣长度为1～4 cm,新尿道采用F8或F10号的硅胶气囊尿管作为支架管,术后10～14 d拔除.结果　35例中27例(77.1%)一次手术成功,阴茎伸直良好,外形美观,排尿正常,成功率为77.1%.术后并发症:尿瘘6例,5例已行尿瘘修补,痊愈,1例等待修补;尿道狭窄2例,1例先行尿道狭窄处尿道造瘘,然后再次行尿瘘修补,痊愈,另1例已行尿道造瘘等待修补.结论　带蒂包皮岛状皮瓣可应用于尿道下裂再次手术,术后阴茎外形美观,但需注意根据手术适应证选择,而且手术操作较复杂,有一定的并发症.     

Lichen sclerosus et atrophicus in children with phimosis and hypospadias

 This prospective study was designed to evaluate the incidence of lichen sclerosus et atrophicus (LSA) in a pediatric population with hypospadias and phimosis in order to discuss the indications for circumcision and utilization of preputial skin for urethral surgery. All 115 boys, 55 with congenital phimosis, 45 with acquired phimosis, 13 with hypospadias, and 2 with recurrent chronic balanitis, underwent full-thickness biopsies of the foreskin that were examined by a single pathologist. Of the patients with acquired phimosis, 88% showed inflammatory features in the foreskin; 60% had LSA. Of the patients with congenital phimosis, 82% showed inflammatory disease in the prepuce; 30% had LSA. Of the patients operated upon for hypospadias, 61% showed histologic findings of chronic inflammation of the foreskin and LSA was evident in 15%. The high incidence of LSA in the prepuce of patients with phimosis suggests that circumcision should be performed to correct this disease. The frequent presence of chronic inflammation is a possible cause of stenosis when the foreskin is used to perform a urethroplasty in patients with hypospadias. Accepted: 30 April 2001     

Surgical technique for antegrade dissection of the preputial vascular pedicle during hypospadias repair

ObjectiveBoth layers of the prepuce can be used as vascularized flaps in hypospadias repair. The aim of this communication is to describe an antegrade approach to the harvesting of the vascular pedicle.Surgical techniqueThe vascular pedicle is isolated at the level of the penopubic junction where it is most robust. Dissection then proceeds distally towards the prepuce fanning laterally.ResultsOf 306 boys presenting for hypospadias repair in 2002–2008, this approach was utilized in 235 (77%). The vascularized flap was used as 1) a classic single or double faced onlay flap in 95, 2) a vascularized layer to cover a tubularized incised plate urethroplasty in 68, 3) a tube-onlay in 39, 4) a tubularized urethroplasty in 23, 5) for ventral skin coverage alone in 7, and 6) for a planned first stage repair in 3. Age at repair averaged 12 months (range 2–160) and follow up 36 months (range 2–68).ConclusionsAntegrade dissection of the vascular pedicle is a simple, expeditious and reliable way of isolating the prepuce on its blood supply. The versatility of the resulting flaps allows the repair to be adapted to the individual anatomic conditions as opposed to one type of repair being used for all variants.