TGF-β1、TGF-βr Ⅲ在邻苯二甲酸二丁酯诱导尿道下裂新生雄性仔鼠生殖结节中的表达及意义

目的 通过验证大鼠孕期污染邻苯二甲酸二丁酯(DBP)所致尿道下裂雄性仔鼠生殖结节中TGF-β1、TGF-βr Ⅲ的表达异常,探讨其在DBP生殖毒性过程中的作用机制.方法 孕鼠20只,随机分二组,在怀孕(GD)14～18 d期间,每天分别灌胃给予大豆油、DBP750 mg/kg,出生第一天(PND1),统计仔鼠出生数,尿道下裂发生率.PND7,雄性仔鼠称重,测量雄性仔鼠肛门至生殖器距离(AGD),拍摄尿道下裂大体图片,取尿道下裂生殖结节进行病理学切片检查,用实时定量逆转录聚合酶链反应(Real-time quantitative PCR)方法 检测生殖结节中的TGF-β1、TGF-βr Ⅲ的mRNA表达水平.结果DBP染毒后导致新生仔鼠与正常组相比数量明显减少,雄性仔鼠尿道下裂的发生率为:43.75%.PND7时,雄性仔鼠体重明显下降,AGD明显缩短.大体图片和病理切片显示典型尿道下裂改变.同时与对照组相比,DBP导致尿道下裂雄性仔鼠生殖结节中TGF-β1、TGF-βr Ⅲ的tuRNA表达水平明显下降.结论 DBP对母体有着明显的毒性作用,DBP干预了TGF-β1、TGF-βr Ⅲ基因的表达,导致子代的生殖结节发育异常,引起尿道下裂.     

邻苯二甲酸二丁酯对雄性大鼠生殖器官的影响

目的 探讨邻苯二甲酸二丁酯对雄性仔鼠生殖器官的毒性作用及机制.方法 取8周龄SD大鼠,雌性32只,雄性16只,随机分为对照组和染毒组（邻苯二甲酸二丁酯组,即DBP组）.于怀孕第13～21天,对照组按1 mL/只灌服花生油,DBP组按500mg/kg灌服邻苯二甲酸二丁酯.分娩后即对新生鼠点数,观察新生鼠外生殖器,称重和测量肛生殖距离,判断有无尿道下裂畸形.仔鼠出生后第4、15、25及90天,抽取外周静脉血,检测睾酮水平;解剖、记录性腺位置及发育情况,取出性腺,称重,常规病理学检查.结果 新生雄性仔鼠出生后DBP组平均体重明显低于对照组,P＜0.05;DBP组16只孕鼠共产雄性仔鼠91只,隐睾17只,尿道下裂7只,对照组未发现隐睾及尿道下裂雄性仔鼠;出生后第90天,对照组体重、睾丸、附睾及前列腺精囊重量重于DBP组;出生后第15、25、90天血睾酮水平,对照组高于DBP组,P＜0.05.结论 邻苯二甲酸二丁酯对雄性大鼠生殖器官的发育有明显毒性作用,可能是通过影响睾酮的合成、分泌等发挥作用.     

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。     

邻苯二甲酸二丁酯影响雄性大鼠性器官发育的作用机制研究

目的通过检测睾酮合成过程中3个关键酶的活性,探讨邻苯二甲酸二丁酯影响雄性仔鼠生殖器官发育的作用机制.方法取8周龄Sprague-Dawley(SD)大鼠,随机分为对照组和染毒组(邻苯二甲酸二丁酯组,即DBP组).于怀孕第13-21天,对照组按照1mL/只灌服花生油,DBP组按照500mg/kg灌服邻苯二甲酸二丁酯(DBP).分娩后观察有无尿道下裂畸形.于仔鼠出生后第4、15、25及90天,抽取外周静脉血,检测睾酮水平;解剖、记录性腺位置及发育情况,取出性腺,采用免疫组织化学方法检测3β-HSD、P450c17及P450scc的表达.结果 DBP组产雄性仔鼠88只,其中隐睾17只,尿道下裂7只,对照组未发现隐睾及尿道下裂雄性仔鼠;对照组出生后第15、25及90天血浆睾酮水平明显高于DBP组同期水平,P＜0.05.DBP组间质细胞3β-HSD、P450c17及P450scc的表达明显低于对照组,P＜0.05.结论 DBP通过降低睾酮合成过程中关键酶的活性,减少睾酮的合成,进而影响雄性仔鼠生殖器官的发育.     

敌敌畏对仔代大鼠睾丸Leydig细胞影响的研究

目的探讨敌敌畏诱导大鼠尿道下裂发病的可能机制。方法将21只孕SD（Sprague—Dawley，SD）大鼠随机分为对照组、敌敌畏低剂量组（1mg／kg组、4mg／kg组、8mg／kg组）和敌敌畏高剂量组（16mg／kg组、20mg／kg组和24mg／kg组）。于孕12～17d每日每组分别灌喂玉米油1ml、敌敌畏1mg／kg、4mg/kg、8mg／kg、16mg／kg、20mg／kg和24mg／kg，分娩后观察外生殖器形状，测量新生仔鼠的体重和肛生殖窦距，并采集部分雄性仔鼠的睾丸标本行免疫组化染色和电镜观察，将余下的雄性仔鼠饲养至90d后采集血清标本，用放射免疫方法测量血清睾酮浓度，并取睾丸标本行免疫组化染色。结果①对照组所产的雄性仔鼠中未发现尿道下裂，在敌敌畏高剂量组雄性仔鼠中发现3例尿道下裂；②敌敌畏各组新生雄性仔鼠睾丸的Leydig细胞计数与对照组比较，差异有统计学意义，与敌敌畏有剂量依赖关系；敌敌畏各组成年雄性仔鼠睾丸Leydig细胞计数与对照组比较，差异无统计学意义；③敌敌畏各组成年雄性仔鼠血清睾酬浓度与对照组比较，差异无统计学意义；④敌敌畏组新生大鼠睾丸Leydig细胞的滑面内质网较对照组减少。结论孕大鼠染毒敌敌畏可使新生雄性仔鼠睾丸Leydig细胞数量降低；新生仔鼠睾丸Leydig细胞内滑面内质网减少，可能降低了胚胎期雄激素合成的功能，导致雄性仔鼠尿道下裂的发生。     

邻苯二甲酸二丁酯致雄性仔鼠肛门直肠畸形与相关基因表达的研究

目的 邻苯二甲酸二丁酯(di-n-butyl phthalate,DBP)孕晚期染毒诱导子代雄性大鼠发生肛门直肠畸形(anorectal malformations,ARMs),探讨形态学变化及相关基因的表达.方法 孕鼠20只,随机分为两组,在怀孕天数第12～18天,每日分别给予DBP及大豆油850mg/kg灌胃.出生后(PND)1d统计雄性仔鼠肛门闭锁发生率,测量雄性仔鼠体重和肛门生殖器距离(AGD),观察肛门闭锁雄性仔鼠末端直肠及肛周病理学改变.Real-time PCR法检测雄性仔鼠末端直肠组织中Shh、Gli2、Gli3、Bmp4、Wnt5a、Hoxa13、Hoxd13、Fgf10、Fgfr2基因mRNA的表达水平.结果 与对照组相比,DBP染毒组母鼠孕期体重增量减少,孕期延长,仔鼠产量降低(P＜0.05),PND 1d肛门闭锁雄性仔鼠体重及AGD较对照组明显降低(P＜0.05).染毒组雄性仔鼠肛门闭锁发生率大约为39.5％.肛门闭锁雄性仔鼠PND1末端直肠及肛周组织病理显示典型的ARMs.与对照组相比,肛门闭锁雄性仔鼠PND1末端直肠中Shh、Gli2、Gli3、Bmp4、Wnt5a、Hoxa13、Hoxd13、Fgf10、Fgfr2的mRNA表达水平较对照组也明显降低(P＜0.05).结论 DBP孕晚期染毒能明显诱导雄性仔鼠ARMs发生,并对其生长发育具有不良影响,其末端直肠组织中与肛门直肠分化发育相关的基因Shh、Gli2、Gli3、Bmp4、Wnt5a、Hoxa13、Hoxd13、Fgf10、Fgfr2的mRNA的表达水平降低可能促进ARMs的发生.     

环境内分泌干扰因子DEHP与隐睾发病相关性的实验研究

目的　探讨邻苯二甲酸二异辛酯 (DEHP)诱导KM小鼠隐睾发生的作用及致病机制。方法　以己烯雌酚 (Diethylstilbestrol,DES)为阳性对照 ,孕鼠在妊娠期第 12d至分娩后第 3d每日灌胃给药DEHP ,分为低剂量组、高剂量组 ,观察雄性仔鼠隐睾发生情况及睾丸形态、组织学改变 ;应用免疫组织化学方法检测睾丸雌激素受体和雄激素受体的表达水平 ,应用放射免疫测定法测定仔鼠血清睾酮、雌二醇、卵泡刺激素和黄体生成素水平。结果　随给药剂量的增大 ,孕鼠未出现明显毒性反应 ,而雄性仔鼠隐睾发生率明显增高 ;隐睾鼠的睾丸、附睾重量及睾丸体积较非隐睾鼠明显降低 ,光镜和电镜下隐睾曲细精管上皮和间质细胞均出现明显异常和超微结构改变 ;同时雄性仔鼠血清睾酮、黄体生成素水平降低 ,卵泡刺激素水平增高 ,雌激素受体表达水平降低。结论　在雄性小鼠性分化的关键时期 ,DEHP可导致其隐睾发生 ,其发生率与剂量存在一定的相关性 ,其作用机制尚需进一步研究。     

含CpG基序的寡聚脱氧核糖核苷酸辅助乙肝疫苗对孕鼠和仔鼠的免疫效果

目的　观察不同剂量CpG 182 6联合乙肝疫苗注射孕鼠后 ,对孕鼠本身及其仔鼠的特异性免疫效果。方法　分别用不同剂量 (10、2 0、4 0 μg/只 )CpG 182 6作为乙肝疫苗佐剂辅助免疫孕鼠 ,ELISA方法检测孕鼠和仔鼠血清乙肝表面抗体 (HBsAb)水平 ,并观察仔鼠存活数量和生长发育指标 (体质量、身长、胎脑和胎仔体质量系数 )变化。结果　给孕鼠注射CpG 182 6 乙肝疫苗或单纯乙肝疫苗后 ,孕鼠血清HBsAb水平总是高于仔鼠水平 (P <0 .0 1) ,但孕鼠与仔鼠血清HBsAb水平间无相关性存在 (r =0 .379　P >0 .0 5 ) ;CpG 182 6 乙肝疫苗免疫孕鼠后 ,2 0 μg/只剂量组孕鼠和仔鼠血清HBsAb含量均高于 10、4 0 μg/只剂量组 ,单纯乙肝疫苗组及空白对照组 (P <0 .0 5 ) ,但仔鼠存活数量及体质量、身长、胎脑和胎仔体质量系数等生长发育指标间均无显著差异 (P均>0 .0 5 )。结论　CpG 182 6 (2 0 μg/只 )联合乙肝疫苗免疫孕鼠时 ,既能显著提高孕鼠及其仔鼠血清特异性免疫抗体水平 ,又不对仔鼠生长发育造成不良影响 ,是一种在妊娠期即可刺激免疫系统不成熟个体免疫活性的理想免疫佐剂。     

左旋精氨酸对低出生体重仔鼠PI3K和PKB的影响

目的：生命早期应用左旋精氨酸（L-Arg）干预低出生体重仔鼠,检测其肝脏磷脂酰肌醇-3-激酶（PI3K）和蛋白激酶B(PKB)的表达,探讨L-Arg对改善胰岛素抵抗的影响。方法：以孕期低蛋白饮食法建立低出生体重仔鼠模型,18只孕鼠随机分为对照组、模型组和干预组,每组6只。对照组孕期给予21%正常蛋白饲料建立正常出生体重仔鼠模型,模型组及干预组孕期给予10%低蛋白饲料建立低出生体重仔鼠模型,3组孕鼠所生仔鼠分别纳入仔鼠对照组、模型组、干预组。生后21 d 仔鼠的哺乳期内,3组孕鼠均给予21%正常蛋白饲料喂养,对照组及模型组给予正常饮用水喂养,干预组给予富含L-Arg（200 mg/kg.d）的饮用水喂养。断乳后,3组仔鼠均给予21%正常蛋白饲料及正常饮用水喂养。于仔鼠生后1 周、3 周、8 周时,分别留取3组仔鼠的肝脏标本,用Western blot法检测肝脏PI3K和PKB的蛋白表达。结果：1 周时干预组仔鼠肝脏PI3K的蛋白表达高于模型组（P=0.045）,且与正常组相比差异无统计学意义（P=0.503）。8 周时干预组仔鼠肝脏PKB蛋白表达明显高于模型组（P=0.039）,且与正常组比较差异无统计学意义（P>0.05）。结论：生命早期补充L-Arg可促进蛋白质的合成,增加肝脏PI3K及PKB的蛋白表达,促进胰岛素信号传导,改善胰岛素抵抗。     

早期环境干预对脑损伤仔鼠脑神经丝蛋白表达的影响

目的 观察早期环境干预对宫内感染致脑损伤仔鼠脑组织神经丝蛋白(NFP)的表达及对其神经行为的影响.方法 孕第17天的孕鼠腹腔注射脂多糖(LPS)450 μg/(kg·d),连续2 d,建立感染性脑损伤仔鼠模型(LPS组);对照组腹腔注射同等剂量的9 g/L盐水.孕22 d前分娩的仔鼠为早产鼠,二组均去除早产鼠.仔鼠出生后,立即取孕鼠子宫胎盘行HE染色观察宫内感染情况;仔鼠出生24 h,随机选取对照组和LPS组仔鼠脑组织,常规HE染色,观察脑白质损伤情况.LPS组足月产仔鼠随机分为干预组与非干预组,干预组于仔鼠出生8 d起进行早期抚触和丰富环境刺激;对照组和非干预组常规饲养.仔鼠出生21 d采用悬吊试验进行神经行为学检测;应用免疫组织化学方法 对各组仔鼠脑组织NFP阳性染色进行分析.结果 LPS组胎盘病理检测可见腹腔注射LPS造成的宫内感染;1 d仔鼠脑白质结构稀疏.对照组悬吊试验得分最高,干预组次之,非干预组最低,两两比较差异均有统计学意义(Pa<0.01).NFP的阳性表达在对照组最高,干预组其次,非干预组最低;两两比较差异均有统计学意义(Pa<0.01).结论 早期环境干预可促进宫内感染致脑损伤的恢复,NFP表达增加是其可能的机制之一.     

维生素E拮抗邻苯二甲酸二(2-乙基)己酯尿道发育毒性的实验研究

目的 探寻维生素E(Vitamin E,VitE)对邻苯二甲酸二(2-乙基)己酯[Di(2-ethylhexy1)phthlate,DEHP]所致大鼠尿道发育毒性的拮抗作用及其可能机制.方法 GD12(gestation day12)SD孕鼠随机分为4组,每组20只:玉米油对照组、DEHP组(500 mg·kg-1·d-1)、DEHP(500mg·kg-1·d-1)+VitE(200mg·kg-1·d-1)组和VitE组(200mg·kg-1·d-1).各组分别于母鼠孕期12～19d(GD12～19)持续经口灌注给药.各组分别留取10只孕鼠让其正常分娩,出生第一天,即对新生大鼠计数,并在解剖显微镜下测量雄性新生鼠的肛门生殖器距离(anal genital distance,AGD)并称体重;雄性仔鼠70日龄时逐个检查尿道下裂的发生情况.余孕鼠在GD19d行破宫产取仔代鼠,应用逆转录-聚合酶链反应(RT-PCR)的方法检测胎鼠阴茎TGF-β1,TGF-βR3 mRNA的表达水平.DNA末端原位标记染色法(TUNEL法)检测胎鼠阴茎尿道上皮细胞凋亡情况.结果 各组TGF-β1mRNA表达水平分别为:正常组为0.63±0.07、DEHP组为0.96±0.12、DEHP+VitE组为0.65±0.07、VitE组为0.62±0.06,DEHP组表达明显较其他各组增高(P＜0.05).各组TGF-βR3mRNA表达水平分别为:正常组为0.47±0.10、DEHP组为0.75±0.10、DEHP+VitE组为0.49±0.09、VitE组为0.46±0.09,DEHP组表达明显较其他各组增高(P＜0.05).各组胎鼠阴茎凋亡指数分别为:正常组为(30±2.0)%、DEHP组为(8.8土1.1)%、DEHP+VitE组为(28.9±1.6)%、VitE组为(29.6±2.0)%,DEHP组凋亡细胞数较其他各组相比明显减少,差异有统计学意义(P＜0.01).导致大鼠尿道下裂的发生.VitE可降低DEHP上调的胎鼠阴茎TGF-β1,TGF-βR3 mRNA表达水平,恢复胎鼠阴茎尿道上皮细胞的凋亡水平.结论 VitE对DEHP所致尿道发育毒性具有拮抗作用,其机制可能与调控TGF-βs及胎鼠阴茎尿道上皮细胞的凋亡有关.

Abstract：

Objective To study the therapeutic effects of Vitamin E (VitE) on urethral development toxicity induced by di(2-ethylhexyl) phthalate (DEHP). Methods From gestational day 12 to gestational day 19 (GD 12-19) , the timed-pregnant Sprague-Dawley (SD) rats were fed with the eiwith 20 in each group: DEHP group, DEHP + VitE group, and VitE group. The control rats were fed with corn oil. Ten pregnant rats of each group delivered full-term infant rats. The male infant rats were counted, and the anal genital distance (AGD) and body weight were measured. Hypospadias morbidity was also counted on male rats after 17 postnatal days. In the rest 10 pregnant rats of each group, fetuses were harvested on GD19. Semi-quantitive RT-PCR was used to measure mRNA expression of TGF-β1 and TGF-βR3. TUNEL staining was used to measure cell apoptosis in the fetus' genital tubercles. Results Hypospsdias was observed in male rat after 17 postnatal days. The TGF-β1 mRNA of the control group, DEHP group, DEHP + VitE group, and VitE group is 0. 63 ± 0. 07,0. 96±0. 12, 0. 65 ±0. 07, and 0. 62± 0. 06, respectively. The TGF-βR3 mRNA of the control group,DEHP group, DEHP + VitE group, and VitE group was 0. 47 ± 0. 10, 0. 75 ± 0. 10, 0. 49 ± 0. 09,and 0. 46 ± 0. 09, respectively. The TGF-β1 mRNA and TGF-βR3 mRNA was up-regulated in the fetus of DEHP group (P＜0. 05). Cell apoptosis rate in fetus' genital tubercles on GD19 of the control group, DEHP group, DEHP + VitE, and VitE group was 30% ± 2. 0%, 8. 8% ± 1.1%, 28. 9% ±1.6%, and 29. 6% ± 2. 0%, respectively. Cell apoptosis rate was significantly reduced in the fetus of DEHP group (P＜0. 01). In the GD19 fetus treated with VitE, hypospadias morbidity, the TGF-β1 and TGF-βR3 mRNA expressions were significantly decreased. And cell apoptosis rate was significantly increased. Conclusions Vitamin E ameliorates the urethral development toxicity induced by DEHP via regulating TGFβs expression and cell apoptosis in rat fetus.     

雄激素对HIBD新生大鼠脑保护作用的量效关系及副作用研究

目的 探讨雄激素对HIBD新生大鼠脑保护作用及量效关系,初步揭示雄激素的远期副作用.方法 3日龄SD大鼠72只随机分为:假手术组(n=8)、HIBD对照组(n=32)、雄激素组(n=32).雄激素组和HIBD对照组各分为4个剂量组,每个剂量组8只.3日龄时雄激素组每个剂量组每只大鼠分别腹腔注射30 mg/kg、60 mg/kg、120 mg/kg和240 mg/kg的丙酸睾酮,HIBD对照组每个剂量组每只大鼠腹腔注射相应毫升数的花生油,每日1次,连续3 d.7日龄时雄激素组和HIBD对照组制作HIBD模型.所有动物饲养至成年后进行水迷宫实验以测定空间学习记忆能力;成年雌鼠每日做阴道涂片,明确每只大鼠的完整发情周期,对于处于发情期的雌鼠及时与雄鼠合笼,次日晨雌鼠做阴道细胞涂片检查,并观察有无阴栓,以查到精子或观察到阴栓作为受孕成功标志,记录各组雌鼠的妊娠率以及妊娠胚胎数目,进行统计学处理,比较组间差异.计算成年雄鼠睾丸系数和附睾系数.结果 水迷宫实验中,HIBD对照组较假手术组平均潜伏期(27.71±3.19)s和首次穿越平台时间(5.34±0.83)s明显延长,HIBD对照组较假手术组穿越目标象限次数(18.88±1.89)明显减少,均有统计学意义(P均＜0.01,P=0.0005＝.雄激素组中各相应剂量组平均潜伏期(34.89±3.68,33.71±3.38,33.84±3.45,35.43±2.43)和首次穿越平台时间(5.39±1.51,6.28±2.07,5.09±1.61,5.85±0.87)较HIBD对照组明显缩短,穿越目标象限次数(12.75±2.05,14.88±3.36,14.88±2.36,14.38±1.60)较HIBD对照组明显增加,均有统计学意义(P均＜0.01,P=0.0001＝.雄激素组各剂量组以上指标相比均无统计学意义(P均＞0.05,P=0.159).雄激素各剂量组雄性大鼠睾丸系数(0.89 ±0.07,0.92±0.08,0.88±0.11,0.87±0.09)和附睾系数(0.25±0.02,0.24±0.05,0.26±0.04,0.23 ±0.05)与假手术组和HIBD组相比,差异均无统计学意义(P＞均0.05,P=3.207),雄激素组各剂量组间相比无统计学差异(P＞0.05,P=6.663);雄激素各剂量组雌性大鼠的怀孕率(均100%)及妊娠胚胎数(14.52±3.34,14.69±2.28,14.98±2.67,15.38±3.07)与假手术组和HIBD组相比,差异均无统计学意义(P＞0.05,P=5.085).结论 HIBD对大鼠的空间学习记忆能力有影响,可降低其空间学习记忆能力.雄激素干预后可明显降低HIBD对大鼠学习记忆能力的影响.雄激素的早期干预对成年后大鼠的生殖能力无影响.

Abstract：

Objective To explore brain-protective effect of androgen, its dose-effect relationship and long-term adverse reaction. Method Seventy two 3-day-old SD rats were randomized into androgen group( n = 32) , HIBD model group ( n = 32 ) and sham operated group ( n = 8 ). The androgen group and HIBD model group were further randomized into 30 mg/kg group, 60 mg/kg group, 120 mg/kg group and 240 mg/kg group, respectively. In androgen group and HIBD group, every rat was given testosterone or peanut oil, one time a day. Three days later, HIBD model was established by occlusion of the left common carotid artery and inhalation of 8% oxygen plus 92% nitrogen for 2.5 hours. Adult rats' ability of learningand memory was determined by water maze test. Escape latencies were recorded and analyzed by statistics.Vaginal cells of all female rats were examined everyday for identifying their estrous cycle. Female rats were allowed to live with normal adult male rats if the female rats were in estrous period. Vaginal cells were examined everyday until sperm was seen, which was the signal of gestation. Pregnancy rate and the number of embryos were recorded and analyzed by statistics. Acropetal coefficient was calculated. The testes and epididymis were taken from adult male rats, histopathological sections were made, and the structure of testis and epididymis were studied under light microscope. Result In Morris experiment, escape latencies (EL)of HIBD group were much longer than those of sham operation group( 27.71 ± 3.19)s, time of first enter target ( 1st ET) was later than that of sham operation group(5.34 ±0.83)s, times of target cross (TC) was less than that of sham operation group ( 18.88 ± 1.89 ) ( P ＜ 0.01, P = 0. 0005 ). EL of androgen group (34.89 ± 3.68,33.71 ± 3.38,33.84 ± 3.45, 35.43 ± 2.43 ) were much shorter than that of HIBD group,1st ET (5.39 ± 1.51,6.28 ±2.07,5.09 ± 1.61, 5.85 ±0.87)was earlier than that of HIBD group, TC ( 12.75 ± 2.05,14.88 ± 3.36,14.88 ± 2.36,14.38 ± 1.60) was more than that of HIBD group ( P ＜ 0.01,P=0.0001). Among the four doses groups of androgen group, EL, 1st ET and TC had no statistical significance (P ＞0.05, P =0.159). There were no statistical significance between male rats of androgen group [Testes acropetal coefficient ( 0.89 ± 0.07,0.92 ± 0.08, 0.88 ± 0.11,0.87 ± 0.09 ), epididymis acropetal coefficient ( 0.25 ± 0.02,0.24 ± 0.05,0.26 ± 0.04,0.23 ± 0.05 )], HIBD group and sham operation group( P ＞ 0.05, P = 3.207 ). Among the four doses groups of androgen group had no statistical significance ( P ＞ 0.05, P = 6.663 ). There were no statistical significance between female rats of androgen group( pregnancy rate, 100%; times, 14.52 ± 3.34,14.69 ± 2.28,14.98 ± 2. 67, 15.38 ± 3.07 ), HIBD group and sham operation group in pregnancy rate and times. Conclusion The intellectual ability of rats decreased after HI. Androgen could reduce the effect of HI on intellectual ability. Androgen had no adverse reaction to the reproductive capacity of adult rats.     

苗勒管抑制物质在尿道下裂发病中的作用

目的 探讨苗勒管抑制物质(MIS)在尿道下裂发病中的作用.方法 分别检测54例单纯型尿道下裂及36例无泌尿生殖系统疾病儿童血清苗勒管抑制物质、睾酮及雄烯二酮的水平.结果 尿道下裂组和对照组儿童的血清MIS平均值分别为(122.0±21.7)rig/ml和(80.5±11.4)ng/ml,P＜0.01.对照组儿童血清睾酮为(1.55±0.42)Pg/ml,尿道下裂组儿童为(0.64±0.31)Pg/ml,P＜0.05.对照组儿童血清雄烯二酮的水平明显高于尿道下裂组儿童,分别为(1.41±0.28)ng/ml和(0.66±0.19)ng/ml,P＜0.01.尿道下裂儿童较高的MIS水平和较低的睾酮水平呈较弱的负相关.结论 尿道下裂儿童血清苗勒管抑制物质的水平与睾酮水平呈负相关,苗勒管抑制物质可能通过抑制睾酮的合成进而在尿道下裂的发病中发挥作用.     

Ex-premature infant boys with hypospadias are similar in size to age-matched, ex-premature infant boys without hypospadias

Objective

Studies have postulated that hypospadias, prematurity, and low birth weight are linked by defects in androgen signaling. To determine whether premature, hypospadiac boys are small and remain so, we compared their size at birth and at hypospadias repair to premature boys who underwent post-neonatal circumcision.

Methods

We identified premature boys admitted to Texas Children’s Hospital who underwent either hypospadias repair or circumcision after 4 months of age. Age, weight, and height at birth and surgery were recorded.

Results

Fifty-four boys had hypospadias and 34 did not. For hypospadiac boys, the mean birth weight and age, height, and weight at surgery were lower than for boys without hypospadias. More importantly, length-for-age and weight-for-age percentiles were also lower for hypospadiac boys. When subset analysis was performed on boys younger than 2 years at surgery, however, there were no significant differences in height or weight between hypospadiac and non-hypospadiac boys.

Conclusion

Our series suggests that premature, hypospadiac boys are born smaller than age-matched, non-hypospadiac controls. However, there were no age-corrected size differences between hypospadiac and non-hypospadiac boys at surgery. This implies that hypospadiac boys exhibit post-neonatal ‘rebound’ growth. Global growth deficits, if any, do not persist in hypospadiac boys.     

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.     

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.     

二氧化硫对低氧性肺动脉高压大鼠内源性硫化氢体系的调节作用

目的 研究二氧化硫( SO2)对低氧性肺动脉高压大鼠肺动脉内源性硫化氢(H2S)/胱硫醚-γ-裂解酶( CSE)以及H2S/巯基丙酮酸转硫酶(MPST)体系的调节作用.方法 将雄性Wistar大鼠(32只)随机分为对照组、低氧组、低氧+ SO2组和低氧+天冬氨酸异羟肟酸(hydroxamate,HDX)组,每组8只.低氧处理采用常压低氧的方法,氧浓度为10％,每天低氧6h,持续21 d.对照组大鼠在常氧环境中饲养.低氧处理结束后采用右心导管法测定肺动脉平均压,采用硫电极法测定肺组织H2S含量和H2S产率,采用免疫组化法检测各组大鼠肺小动脉内膜及中膜CSE和MPST的蛋白表达.结果 低氧组大鼠肺动脉平均压较对照组高[(33.38 ±6.32) mm Hg vs(16.74±3.81) mm Hg,1 mm Hg=0.133 kPa,P＜0.01];低氧+SO2组大鼠肺动脉平均压较低氧组低[(29.65±2.53)mm Hg vs(33.38±6.32) mm Hg,P＜0.01],低氧+HDX组大鼠肺动脉平均压较低氧组高[(39.44±6.26) mm Hg vs(33.38±6.32) mm Hg,P＜0.01].低氧组大鼠肺组织H2S含量[(2.02±0.43) μmol/g vs (3.11±0.42) μmol/g,P＜0.01]及H2S产率[(19.64±3.48) nmol/(g· min)vs(28.20±5.95) nmol/(g·min),P＜0.05]均较对照组低.给予SO2供体后,低氧+SO2组肺组织H2S含量[(2.73±0.20)μmol/g vs(2.02±0.43)μmol/g,P＜0.01]及H2S产率[(26.24±1.92) nmol/(g· min)vs(19.64±3.48) nmol/(g· min),P＜0.01]均较低氧组升高.当给予内源性SO2生成酶抑制剂HDX后,低氧+HDX组肺组织H2S含量[(1.64±0.23) μmol/g vs (2.02±0.43)μmol/g,P＜0.05]及肺组织H2S产率[(13.94±3.63) nmol/(g·min) vs (19.64±3.48) nmol/(g·min),P＜0.05]均较低氧组低.低氧组大鼠肺小动脉内膜[(0.31±0.02)vs(0.36±0.01),P＜0.01]及中膜[(0.27±0.01)vs (0.30±0.01),P＜0.01]中CSE蛋白表达均较对照组低.低氧+SO2组大鼠肺小动脉内膜CSE蛋白表达较低氧组高[(0.35±0.02) vs (0.31 ±0.02),P＜0.01].低氧+HDX组大鼠肺小动脉内膜CSE蛋白表达较低氧组低[(0.26±0.01) vs (0.31±0.02),P＜0.01].与对照组相比,低氧组大鼠肺小动脉内膜及中膜MPST的蛋白表达差异没有统计学意义.低氧+SO2组大鼠肺小动脉中膜MPST的蛋白表达较低氧组高[(0.32±0.02) vs (0.29±0.01),P＜0.01];而与低氧组相比,低氧+ HDX组大鼠肺小动脉内膜及中膜MPST的蛋白表达差异没有统计学意义.结论 外源性给予SO2供体可上调低氧性肺动脉高压大鼠肺小动脉内膜H2S生成酶CSE及肺小动脉中膜MPST蛋白表达,促进H2S生成增多,从而间接缓解低氧性肺动脉高压的形成.