pH2AX蛋白在单次热应激致小鼠睾丸生殖功能可逆性中的表达变化

目的:探讨pH2AX在单次热应激致小鼠睾丸生殖功能可逆性中的表达变化。方法:C57雄性小鼠24只,随机按1、7、14 d分为对照组(n=6)和热应激组(n=6)。热应激组和对照组小鼠下腹部分别置于43℃和25℃水浴中干预15 min,于处理后1、7、14 d取睾丸组织。TUNEL检测睾丸细胞凋亡情况,免疫组化染色检测pH2AX蛋白定位及表达,Western印迹检测pH2AX蛋白表达水平。结果:TUNEL结果显示,在热应激后的第1天生精小管中的凋亡细胞数量最多,第7、14天细胞凋亡基本消失。免疫组化显示,pH2AX蛋白在睾丸生精小管基底膜处细胞核表达。Western印迹检测显示,与对照组相比,热应激后的第1天,pH2AX蛋白表达明显增加(0.47±0.02 vs 1.61±0.04,P0.01);第7天(0.85±0.03)与热应激处理第1天相比pH2AX蛋白表达显著减少(P0.01);第14天(1.72±0.02)与热应激处理第1、7天及对照组相比pH2AX蛋白表达显著增加(P均0.01)。结论:小鼠睾丸热应激处理后pH2AX蛋白表达量在睾丸中出现动态变化,这种变化与热应激后睾丸生精细胞的增殖分裂相关。     

过氧化物还原酶4在睾丸热应激时的保护作用

目的:以过氧化物还原酶4(PRDX4)基因全敲除(PRDX4~(-/y))小鼠为模型,研究PRDX4在睾丸热应激时的保护作用。方法:采用CRISPR/Cas 9技术对C57BL/6小鼠进行PRDX4基因全敲除,以野生型小鼠为对照,各24只。生长至性成熟(9周),PRDX4~(-/y)和野生型组各12只小鼠睾丸43℃水浴热应激15 min,1次/d,连续3 d;每组以25℃水浴作为热应激处理的对照。处理前、处理后1 d和处理恢复5周后,各组均分别处死4只小鼠,取双侧睾丸组织,采用HE染色观察组织学变化、TUNEL法检测细胞凋亡率、Western印迹检测PRDX4表达水平、免疫组化染色观察氧化应激因子(HNE和8-OHdG)表达水平。结果:与野生型小鼠比较,PRDX4基因全敲除小鼠的睾丸组织学、细胞凋亡和氧化应激因子表达差异不显著(P>0.05)。43℃水浴热应激处理后PRDX4~(-/y)小鼠[(38.65±2.57)%]和野生型小鼠[(13.21±1.434)%]生精细胞凋亡均显著升高(P<0.01),PRDX4~(-/y)小鼠生精细胞凋亡率增加2倍,5周后生精功能恢复较野生型差。热应激处理后,PRDX4~(-/y)小鼠睾丸8-OHdG(38.25±1.19)较野生型小鼠(24.30±1.65)表达水平显著增加(P<0.01),而两组HNE的表达水平无显著差异(P>0.05);恢复5周后,PRDX4~(-/y)小鼠生精细胞凋亡仍显著高于野生型小鼠[(3.09±0.16)%vs(1.45±0.11)%,P<0.01)],PRDX4~(-/y)小鼠睾丸生精细胞HNE的表达水平也高于对照组(28.57±0.56 vs 19±1.35,P<0.01),8-OHdG的表达水平也仍较对照组升高,但差异无显著性。结论:PRDX4在睾丸处于应激状态时具有重要的保护性作用,并促进睾丸生精功能恢复。     

小鼠无精子症动物模型的构建

目的:建立一种稳定的小鼠无精子症动物模型。方法:60只清洁级C5BL/6小鼠,分为化疗组(1次性腹腔注射白消安10mg/kg体重、环磷酰胺120mg/kg体重)、激素组(每日皮下注射苯甲酸雌二醇40mg/kg,连续注射15d),每组30只。注射后4、12、20周,观察小鼠睾丸生精小管结构的变化以及终止干预后生精功能恢复的情况。结果:化疗组化疗后第4周,睾丸生精小管内精子及精子细胞消失,20周时仍保持无生精状态;激素组连续注射苯甲酸雌二醇后第4周,生精小管内精子及精子细胞消失,但20周时部分生精小管内可以找到精子细胞和精子。化疗组小鼠睾丸重量明显低于激素组(P<0.05)。结论:经过化疗处理的小鼠无精子症模型稳定,雌二醇皮下注射的无精子症模型形成慢而不稳定。相对于雌激素注射方法,化疗是构建小鼠无精子症动物模型比较可靠的方法。     

白消安致小鼠精子再生模型的精子发生量化评价

目的:量化评价白消安二次腹腔注射制备的小鼠精子再生模型。方法:54只8～10周龄雄性昆明白小鼠随机分为对照组和两个模型组,每组18只。模型组小鼠分别以10mg/kg和15mg/kg白消安间隔24d二次腹腔注射建立精子再生模型,对照组小鼠以50%二甲基亚砜溶液10ml/kg间隔24d二次腹腔注射。采用Johns-en评分量化给药后3、4和8周时生精上皮精子发生,运用实时定量PCR技术检测这3个时期支持细胞GATA结合蛋白4(GATA-4)mRNA和胶质细胞源性神经营养因子(GDNF)mRNA表达水平。结果:对照组Johnsen评分在各时期无显著差异(P>0.05)。给药后3周和4周,两模型组Johnsen评分均显著低于对照组(P<0.01);给药后4周和8周,15mg/kg组Johnsen评分均低于10mg/kg组(P<0.05);给药后8周,15mg/kg组Johnsen评分仍显著低于对照组(P<0.05),而10mg/kg组和对照组间无显著差异(P>0.05)。各组各时期支持细胞GATA-4mRNA表达均无显著差异(P>0.05)。对照组各时期支持细胞GDNF mRNA表达无显著差异(P>0.05)。两模型组支持细胞GDNF mRNA表达在给药后3周均高于对照组,而在给药后4周均低于对照组(P<0.01);给药后8周,15mg/kg组支持细胞GDNF mRNA表达低于对照组(P<0.01),而10mg/kg组与对照组无显著差异(P>0.05)。在以上3个时期,15mg/kg组GDNF mRNA表达均低于10mg/kg组(P<0.01)。结论:10mg/kg白消安间隔24d二次腹腔注射是建立小鼠精子再生模型的适宜剂量,增大剂量可使支持细胞GDNF mRNA表达不足,导致精子发生不能完全恢复。     

睾丸生精小管干细胞移植受体模型建立与移植技术改进

目的:探讨建立稳定的、适合干细胞睾丸生精小管移植的受体小鼠模型和移植技术改进。方法:将雄性ICR小鼠60只随机分为A、B、C、D4组,每组15只。A、B、C组注射白消安的剂量分别为15、30、40mg/kg体重,D组注射50%二甲亚砜作为对照。注射后常规饲养,观察小鼠死亡情况。于注射后4、8、12周称量各组小鼠的睾丸重量,制作睾丸石蜡切片,观察生精小管的中空率和生精上皮结构变化。自主设计干细胞移植装置,改进干细胞移植技术。该装置通过三通连接装置将口含端、注射器端及穿刺端连接在一起,口含端负责试探性的注射,注射器端负责抽吸细胞悬液和注射。结果:注射后只有C组出现1只小鼠死亡。4周时A、B、C组睾丸重量的变化,与作为对照的D组相比有显著性差异(P<0.05);8周时A组与D组无差异(P>0.05),而B、C组与D组差异仍有显著性(P<0.05);12周时各组与D组均无显著性差异(P>0.05)。4、8周时A组的生精小管中空率<50%,B、C组中空率都在50%以上,而D组无明显改变;12周时A、B、C组无显著性差异(P>0.05),恢复近正常状态。自主设计的三通式注射装置注射成功率高,可达90%以上;细胞浪费少,注射双侧睾丸所需细胞悬液量<50μl;所需时间短,注射一侧睾丸<10min。结论:雄性ICR小鼠腹腔内单次注射30mg/kg体重白消安,无小鼠死亡,且4周后生精小管的中空率高(均>50%),适合作为干细胞移植的模型。改进后的移植装置和方法,提高了移植的效率和成功率。为探讨干细胞向雄性配子发育研究提供了简单易学、适于推广的新技术。     

单次热应激处理对小鼠睾丸生精细胞的影响

目的:探讨单次热应激对小鼠睾丸生精功能可复性研究。方法:C57雄性小鼠36只,随机按1、7、14 d各分为对照组(n=6)和热应激组(n=6)。热应激组和对照组小鼠下腹部分别在43℃和25℃水浴中干预15 min,于处理后1、7、14 d取材。计算睾丸器官指数,HE染色观察睾丸组织结构变化,免疫组化染色检测早幼粒白血病锌指蛋白(PLZF)和联会复合体蛋白3(SCP-3)在睾丸组织中的定位及表达,Western印迹检测PLZF蛋白表达水平。结果:热应激组小鼠睾丸器官指数明显低于对照组(P0.01)。HE染色可见,热应激组与对照组相比,热应激后第1天生精细胞排列松散,散落在生精小管中;第7天生精小管萎缩,排列疏松,生精细胞明显减少;第14天生精小管排列较为紧密,生精细胞数量增加,生精细胞层数增多。与对照组相比第1、7天热应激组SCP-3标记的精母细胞明显减少,第14天精母细胞数量开始恢复。Western印迹检测结果显示,PLZF蛋白在热应激处理后1 d表达量显著降低(0.19±0.02 vs 0.64±0.03,P0.01),第7、14天又迅速增加(0.77±0.02、0.77±0.02),且显著高于对照组(P0.01)。结论:小鼠睾丸热应激处理后可致生精功能发生障碍,随着时间的推移,这种损伤具有可复性。     

麒麟丸对无精子症模型小鼠睾丸生精功能的影响

目的:探讨麒麟丸是否促进无精子症模型小鼠生精功能恢复,揭示其调控睾丸生精功能的机制。方法:取15只4周龄ICR雄性小鼠,随机分成模型组、麒麟丸低剂量组、麒麟丸高剂量组,每组5只。采用白消安建立无精子症小鼠模型。建模后麒麟丸组每天给予不同剂量麒麟丸灌胃给药[低剂量组和高剂量组分别为2 000、8 000 mg/(kg·d)],模型组予正常饮食处理,28 d后颈椎脱臼法处死小鼠。HE染色检测小鼠附睾和睾丸组织显微结构,Western印迹检测小鼠睾丸中各级生精细胞、支持细胞和间质细胞特异性标志物表达情况,免疫荧光染色检测睾丸内这些标志物的定位与表达情况。结果:模型组小鼠睾丸中各级生精细胞数量明显减少,大多数附睾管中未见精子; Johnsen评分(5. 2±0. 5)分。麒麟丸高剂量组小鼠生精小管腔内生精细胞排列紧密、层次分明,附睾管腔中见大量精子,未见非精子细胞成分; Johnsen评分(9. 4±0. 6)分。麒麟丸低剂量组小鼠睾丸中各级生精细胞数量相比较模型组有所提升,但是对比高剂量组仍明显减少,部分附睾管中可见精子; Johnsen评分(7. 6±0. 6)分。Johnsen评分模型组显著低于麒麟丸高、低剂量组(P 0. 01),而且麒麟丸高、低剂量组间也有统计学差异(P 0. 05)。Western印迹结果显示,支持细胞标志物GATA4、WT1、SCF、BMP4的表达水平模型组均显著低于麒麟丸高、低剂量组(P 0. 01),高剂量组显著高于低剂量组(P 0. 05或0. 01);精原干细胞和未分化精原细胞标志物UCHL1、STRA8、NGN3、PLZF 3组小鼠间无显著差异;精母细胞标志物DMC1、SYCP3模型组也均显著低于麒麟丸高、低剂量组(P 0. 05或P 0. 01),高剂量组显著高于低剂量组(P 0. 05或P 0. 01)。免疫荧光染色结果显示,SYCP3的表达与Western印迹结果一致; Ki67荧光信号分布在精原细胞,其荧光信号强度模型组低于麒麟丸高、低剂量组;精子顶体标志物PNA主要分布在生精小管内,模型组小鼠没有出现明显的点状信号,麒麟丸高、低剂量组可见大量的荧光信号。结论:麒麟丸可能是通过改善睾丸中支持细胞的功能,从而促进精原细胞进入减数分裂,增加精子的生成。     

葡萄籽原花青素对小鼠睾丸扭转复位后生精功能的保护作用

目的:探讨葡萄籽原花青素(GSP)对小鼠睾丸扭转复位后生精功能的保护作用。方法:24只健康雄性昆明小白鼠(8周龄,25～27 g)随机分为3组:对照组、扭转组、治疗组,每组8只。扭转组及治疗组建立单侧睾丸扭转复位动物模型,治疗组于扭转复位前30 min腹腔注射GSP(50 mg/kg),术后采用腹腔注射方式连续给药3 d,每天1次,每次50 mg/kg。扭转组方法同治疗组,治疗同体积生理盐水。术后第4天取扭转侧睾丸,检测组织病理学参数和生精细胞凋亡指数(AI),并检测睾丸组织超氧化物歧化酶(SOD)和丙二醛(MDA)含量。对照组行假手术。结果:治疗组与扭转组相比,Johnsen评分上升[(7.38±0.92)分vs(5.00±1.85)分,P<0.05],生精小管直径略增大[(178.75±1.58)μm vs(176.50±1.60)μm,P>0.05],生精细胞层数增加[(5.75±0.71)层vs(3.75±1.03)层,P<0.05],生精细胞凋亡指数AI明显降低[(16.25±1.67)%vs(40.50±1.60)%,P<0.05)],SOD活性明显上升[(52.67±3.57)U/mg prot vs(29.04±4.46)U/mg prot,P<0.05],MDA含量明显下降[(2.91±0.04)nmol/mg prot vs(4.63±0.05)nmol/mg prot,P<0.05]。结论:GSP对小鼠睾丸扭转复位后生精功能损伤有明显的保护作用,其作用机制可能与其能清除氧自由基、抑制脂质过氧化、提高机体抗氧化能力有关。     

缺血预处理对睾丸缺血再灌注新西兰大白兔睾酮水平及生精细胞凋亡的影响

目的:基于睾丸扭转建立兔睾丸缺血再灌注损伤模型,探索缺血预处理对睾丸缺血再灌注损伤兔的睾酮水平及生精细胞凋亡的影响。方法:雄性新西兰大白兔15只,随机分为3组:A组(对照组)暴露右侧精索,不作缺血处理并关闭;B组(缺血再灌注组)使用无创血管夹夹闭右侧精索致睾丸缺血60 min,再灌注3 d;C组(预处理组)预先夹闭精索3次(缺血5 min/次+灌注5 min/次),余同B组。造模完成后,采用3%巴比妥钠麻醉兔后采集血液并分离血清,测定血清睾酮含量;随后分离睾丸组织,按照缺血侧及健侧分组后采用10%中性福尔马林固定组织,进行HE染色和TUNEL细胞凋亡检测。结果:术后A、C组体重比术前明显增加[(2.65±0.07)kg vs(2.45±0.07)kg、(3.03±0.11)kg vs(2.92±0.07)kg](P均0.05),B组手术前后体重没有变化[(3.05±0.07)kg vs(3.05±0.07)kg](P0.05)。睾酮含量:手术前后A组没有明显差异[(139.59±9.39)ng/L vs(140.19±9.47)ng/L,P0.05];术后B、C组显著低于术前[(74.12±4.00)ng/L vs(148.06±3.31)ng/L、(94.76±3.13)ng/L vs(133.75±6.48)ng/L]和术后A组(P均0.01)。病理结果显示,与A组及B组健侧相比,B组缺血侧睾丸组织内大部分生精小管结构破坏,生精小管内各级生精细胞结构消失,可见凋亡的生精细胞,间质及管腔内有淡伊红色水肿液渗出,凋亡指数明显升高(P0.01),Johnsen评分显著降低(P0.01);与B组缺血侧相比,C组缺血侧睾丸组织基本恢复正常,凋亡指数明显降低(P0.01),Johnsen评分显著增加(P0.01)。结论:缺血预处理明显缓减了睾丸缺血再灌注损伤后睾酮水平下降,并降低了细胞凋亡,为睾丸缺血再灌注损伤的临床治疗提供了潜在干预措施。     

实验性精索静脉曲张大鼠生精状态评价

目的 通过对大鼠实验性精索静脉曲张(varicocele,VC)模型睾丸生精小管生精上皮结构、性激素水平的分析,探讨精索静脉曲张致不育的机制.方法 40只雄性青春期Wistar大鼠随机分为VC8周组(n=12)、VC12周组(13=12)和相应对照组(分别n=8);左肾静脉部分结扎建立实验性大鼠VC模型.术后8周或12周,分别测量各组大鼠:(1)左侧精索静脉直径、睾丸温度及体质量、睾丸生精小管生精上皮;(2)外周血中促卵泡刺激素(FSH)、黄体生成素(LH)和睾酮(T)的水平.结果 VC8周和VC12周组大鼠左侧精索静脉明显扩张,与对照相比血管直径差异有统计学意义(P<0.01);VC8和VC12周组大鼠左侧睾丸体质量均低于自体右侧睾丸和对照组睾丸,差异有统计学意义(P<0.05);光镜下,VC组大鼠双侧睾丸生精小管生精上皮精子发生阻滞、细胞脱落和细胞层数减少等,VC12周组损伤程度较VC8周组明显加重,左侧较右侧显著;与对照组相比,VC组大鼠外周血FSH、LH升高,T降低,差异均有统计学意义(P<0.01).结论 本研究提示:VC对大鼠双侧睾丸生精小管生精上皮产生明显的损害作用,并导致大鼠血中T水平降低和FSH、LH水平升高.     

射线损伤对睾丸Claudin-11 mRNA表达的影响

目的:Claudin-11为支持细胞紧密连接的组分,在构建血睾屏障及维持生精上皮空间构象中呈现重要作用。本研究拟通过射线局部照射致睾丸氧化应激,观察Claudin-11转录水平变化,探讨氧化应激损伤精子发生的作用机制。方法:48只雄性昆明小鼠随机分配至A、B、C、D 4组,每组12只,A组空白对照,B、C、D 3组分别以2、6、10 Gy剂量60Co-γ射线局部照射实验动物下腹部1次,于4周后处死,测定实验小鼠体重及双睾丸重量;HE染色观察睾丸组织学变化;酶联免疫法检测血清性激素水平;实时荧光PCR监测睾丸组织内抑制素βB及Claudin-11转录水平变化。结果:不同剂量射线致睾丸氧化应激损伤后睾丸重量,A组为(182.9±8.43)mg,B组为(129.4±10.81)mg,C组为(87.5±16.83)mg,D组为(56.1±12.36)mg,呈下降趋势,与A组相比,差异有显著性(P<0.05);睾丸指数A组为(4.28±0.31)mg/g,B组为(3.39±0.57)mg/g,C组为(2.46±0.46)mg/g,D组为(1.63±0.44)mg/g,下降更为明显,与A组相比,差异有显著性(P<0.01);组织学分析示,与A组相比,受射线照射3组生精小管分化指数(TDI)下降明显(P<0.01),生精小管直径减小,生精上皮高度显著降低并层次紊乱。血清FSH,A组为(5.77±1.62)IU/L,B组为(6.74±1.95)IU/L,C组为(8.41±2.44)IU/L,D组为(10.93±3.16)IU/L,逐渐升高,D组较A组升高1.9倍。伴随照射睾丸射线剂量增加,组织内抑制素βB mRNA含量下降,Clau-din11转录水平呈现增高趋势,C、D两组Claudin-11 mRNA水平高于正常对照A组(P均<0.01)。结论:射线局部照射致睾丸氧化应激,组织内抑制素βB mRNA降低、血清FSH升高,支持细胞合成及分泌功能受损;同时,睾丸组织Claudin-11表达量则增加。增加的紧密连接组分同升高的FSH致使血睾屏障重建周期延长,生精上皮内处于减数分裂的精母细胞数量减少,导致不育发生。     

Evaluation of lithogenic elements in urine of healthy newborns

The determination of urinary excretion of lithogenic elements in healthy newborns involves factors ranging from urine collection and data handling to maternal influences, which can cause difficulties in analyzing the results. The objective of this study was to determine normal values of parameters related to lithogenesis, such as calcium, uric acid, citrate, and oxalate, in urine of healthy newborns using isolated samples, focusing on variations according to gender, weight, milk ingestion, and family history of lithiasis. Parameters measured in isolated urine samples from 104 healthy newborns (77 males and 27 females) were corrected by creatinine. The ratios were expressed as milligram/milligram of creatinine: calcium/creatinine of 0.10±0.01 (X±SE), uric acid/ creatinine of 1.10±0.10, citrate/creatinine of 0.56±0.04, and oxalate/creatinine of 0.07±0.01. Differences were observed between males and females, in terms of uric acid (0.80±0.07 vs. 1.10±0.10 mg/mg, P<0.05), citrate (0.05±0.06 vs. 0.17±0.05, P<0.05), sodium (0.17± 0.01 vs. 0.05±0.01, P<0.001), and potassium (0.05± 0.01 vs. 0.20±0.02, P<0.001). Interestingly, the urinary concentration of protector factors such citrate and potassium was higher in females than in males with low sodium excretion. Artificial milk feeding leads to higher calcium (0.10±0.06 vs. 0.06±0.01), uric acid (1.40±0.20 vs. 0.90±0.09, P<0.05), citrate (0.90±0.09 vs. 0.50±0.04, P<0.001), and oxalate (0.17±0.03 vs. 0.05±0.01, P<0.001) excretion when compared with breast feeding. There was higher excretion of calcium and sodium in patients under 3 kg. Children with familial antecedents presented some differences in urinary excretion, with higher uric acid (1.50±0.30 vs. 0.80±0.08, P<0.05) but lower calcium (0.05±0.02 vs. 0.10±0.01, P<0.05) and sodium (0.15±0.02 vs. 0.20±0.02, P<0.05) excretion, respectively. This report provides urinary parameters obtained in healthy newborns and correlates them with factors that could be involved in the genesis of osteopenia, renal stones, and/or nephrocalcinosis. Received: 8 May 2000 / Revised: 7 June 2001 / Accepted: 12 June 2001     

Prevention of acute cyclosporin nephrotoxicity by verapamil and atrial natriuretic factor in the rat

Nephrotexicity is the most common and important side-effectof cyclosporin (CsA) therapy. CsA alters renal haemodynamicswith a reduction in renal blood flow (RBF) and glomerular filtrationrate (GFR) and a significant increase in renal vascular resistances(RVR). The present experimental study investigates whether verapamilor atrial natriuretic factor (ANF) are able to prevent the nephrotoxicityof CsA. All studies were conducted in an in-situ autoperfused rat kidneymodel which allows continuous measurement of renal blood flowwithout dissection of the renal artery. CsA as a 40 mg/kg bolus dose significantly decreased RBF (from2.15±0.1 and 2.19±0.1 before CsA, to 1.29±0.16ml/min/100 g BW, 60 mm after CsA administration) (P<0.05),and GFR (from 0.14±0.1 and 0.13±0.01 before CsA,to 0.08±0.01 ml/min/100 g BW, 60 min after CsA administration)(P<0.05). CsA significantly increased RVR (from 9.5±0.73and 9.8±0.78 before CsA, to 16.7±2.9 mmHgxmin/ml60 min after CsA administration) (P<0.05). Verapamil pretreatment(as continuous intrarenal infusion at the rate of 1.25 µg/kg/min)attenuated the fall in GFR (from 0.16±0.01 and 0.19±0.03ml/min/100 g before CsA to 0.20±0.05 ml/min/100 g BW,60 mm after CsA administration) (NS) and in RBF (from 2.42±0.2and 2.6±0.22 ml/min/100 g before CsA to 1.79±0.17ml/min/100 g BW, 60 min after CsA administration (P<0.05).Pretreatment with ANF (as continuous intrarenal infusion atthe rate of 2.5 µg/kg/min) protected GFR (from 0.11±0.02and 0.18±0.03 ml/min/100 g before CsA, to 0.11±0.03ml/min/100 g BW, 60 min after CsA administration) (NS) and RVR(from 9.53±0.6 and 8.95±0.74 mmHgxmin/ml beforeCsA to 11.93±1.19 minHgxmin/ml, 60 min after CsA administration)(NS)and attenuated the fall in RBF (from 2.17±0.11 and 2.2±0.14ml/min/100g before CsA to 1.56±0.25 ml/min/100 g BW 60mm after CsA administiation)(P<0.05) when compared with initialvalues. These studies suggest that verapamil and ANF can prevent CsA-inducedrenal toxicity. Further studies should evaluate their usefulnessin clinical practice.     

Decreased gallbladder response in leptin-deficient obese mice

Obesity is a major risk factor for gallstone formation, but the pathogenesis of this phenomenon remains unclear. Human data on gallbladder emptying are conflicting, and no animal data exist on the effect of obesity on gallbladder motility. Leptin, a hormone produced by adipocytes, is known to have central effects on neuropeptide Y and cholecystokinin, but the influence of leptin on the biliary effects of these hormones is unknown. Therefore we tested the hypothesis that leptin-deficient C57BL/6J-lep^ob obese mice would have decreased gallbladder responses to excitatory stimuli. Twelve-week-old lean control (C57BL/6J) (n = 22) and C57BL/6J-lep^ob obese (n = 20) female mice were fed a nonlithogenic diet. The mice were fasted overnight and underwent cholecystectomy. Whole gallbladders were placed in 3 ml muscle baths. After optimal length was determined with acetylcholine (10^-5 mol/L, responses to increasing doses of neuropeptide Y (10^-8 to 10^-6 mol/L) and cholecystokinin-8 (10^-10 to 10^-7 mol/L) were measured. Student’s t test and two-way analysis of variance were used where appropriate. Results were expressed as Newtons per cross-sectional area. The lean control mice had significantly greater excitatory responses to acetylcholine than the obese mice (0.37 ± 0.05 vs. 0.16 ± 0.02, P < 0.01). The gallbladder responses were also greater when mice were treated with neuropeptide Y (10^-8 mol/L: 0.00 ± 0.00 vs. 0.00 ± 0.00, NS; 10^-7 mol/L: 0.12 ± 0.02 vs. 0.05 ± 0.01, P < 0.01; 10^-6 mol/L: 0.26 ± 0.08 vs. 0.06 ± 0.01, P < 0.01) and cholecystokinin (10^-10 mol/L: 0.27 ± 0.04 vs. 0.13 ± 0.02, P < 0.01; 10^-9 mol/L: 0.59 ± 0.08 vs. 0.27 ± 0.04, P < 0.01; 10^-8 mol/L: 0.80 ± 0.11 vs. 0.37 ± 0.05, P < 0.01; 10^-7 mol/L: 0.86 ± 0.11 vs. 0.44 ± 0.06, P < 0.01). These data suggest that genetically obese, leptin-deficient mice have decreased responses to acetylcholine, neuropeptide Y, and cholecystokinin. We conclude that decreased gallbladder motility contributes to the increased incidence of gallstones associated with obesity. Presented at the Forty-Second Annual Meeting of The Society for Surgery of the Alimentary Tract, Atlanta, Georgia, May 20–23, 2001 (oral presentation). Supported by grant RO1-DK442 79–07 from the National Institutes of Health.     

Upper Extremity Hemodynamic Changes after Radial Artery Harvest for Coronary Artery Bypass Grafting

t -test. There were no significant changes between preoperative and postoperative pressures in the brachial, radial, and ulnar arteries, and thumb, index, long, ring, or little fingers. Pressure changes in the thumb and index finger approached but did not achieve a statistical difference. Peak systolic velocity (PSV), end diastolic velocity (EDV), and resistive index (RI) in the distal ulnar artery changed significantly between preoperative and postoperative measurements. PSV changed from 0.50 ± 0.05 m/sec to 0.67 ± 0.04 m/sec (p= 0.02); EDV changed from 0.03 ± 0.03 m/sec to −0.10 ± 0.05 m/sec (p= 0.05); and RI changed from 0.97 ± 0.05 to 1.13 ± 0.05 (p= 0.02). Palmar arch evaluations revealed significant changes at rest and with ulnar compression between preoperative and postoperative measurements: (1) at rest EDV changed from 0.03 ± 0.02 m/sec to −0.05 ± 0.02 m/sec (p < 0.01); (2) at rest RI changed from 0.96 ± 0.05 to 1.12 ± 0.05 (p= 0.01); (3) with ulnar compression the PSV changed from 0.23 ± 0.05 m/sec to 0.005 ± 0.01 m/sec (p < 0.01); and (4) with ulnar compression the RI changed from 0.82 ± 0.11 to 0.27 ± 0.12 (p < 0.01). Eight patients had a variety of complaints at the follow-up visit, the majority being numbness and tingling. No patients reported symptoms of claudication or rest pain at the follow-up visit. The data suggest that while statistically significant changes in velocity and arterial resistance do occur, patients seem to tolerate radial artery harvesting without clinical consequences. The ideal method of preoperative evaluation remains to be determined.     

蛋白激酶C-alpha对小鼠尿浓缩功能的调节机制初探

目的通过观察蛋白激酶C(PKC)-alpha基因敲除鼠血清加压素(AVP)水平及髓内水通道蛋白2(AQP-2)分布、表达和转运状态的变化,初步探讨PKC-alpha在小鼠尿浓缩功能中的调节机制。方法使用代谢箱收集PKC-alpha基因敲除鼠及SV129野生鼠24 h尿液并取血,采用渗透计检测尿渗透浓度。ELISA法检测正常饮食下24 h尿尿素排泄量。放射性免疫法(RIA)检测血清AVP水平。免疫荧光及半定量免疫印迹技术检测小鼠内髓AQP-2的分布和表达情况。分别使用V2受体拮抗剂SR141263及不同浓度去氨基精加压素(DdAVP)腹腔内注射,检测3 h内尿渗透浓度、尿量以观察两组小鼠AQP-2转运状态。结果正常饮食下PKC- alpha基因敲除鼠24 h尿尿素排泄量[(3．25±0．18)mmol／24 h比(3．83±0．42)mmol／24 h,P= 0．24]以及血清AVP水平[(4．64±0．43)pmol／L比[(5．03±0．44)pmol／L,P=0．55]与野生鼠相比,差异均无统计学意义。两组小鼠内髓AQP-2的分布及表达相似(P=0．48)。不同浓度DdAVP腹腔注射后两组小鼠尿量变化曲线完全一致。SR141263腹腔注射后PKC-alpha基因敲除鼠及野生鼠尿渗透浓度改变之间差异也无统计学意义[(0．20±0．02)mmol／L比(0．20±0．04) mmol／L,P=0．97]。结论PKC-alpha参与调节的小鼠尿浓缩功能与血清AVP水平、髓内AQP-2状态无关。     

扶正解毒祛瘀方联合奥沙利铂对人结直肠癌裸鼠移植瘤的影响

目的：探讨扶正解毒祛瘀方（简称“扶正方”）联合奥沙利铂（oxaliplatin，L-OHP）对人结直肠癌 HT-29 裸鼠移植瘤的影响。方法：构建裸鼠移植性结直肠癌 HT-29 肿瘤模型，将其随机分为空白组、扶正方组、L-OHP 组及扶正方 + L-OHP 组，每组 10 只。空白组给予生理盐水灌胃，扶正方（50 g/kg）为灌胃给药；L-OHP（10 mg/kg）为腹腔注射给药（ip）1 次。扶正方 +L-OHP 组为 L-OHP 腹腔注射给药（ip）1 次，扶正方灌胃（ig）给药，每日 1 次，连续 21 d。观察扶正方对皮下移植瘤的作用及与 L-OHP 合用的效果；并用 RT-PCR 及 Western blotting 法检测细胞周期相关因子细胞周期素D1（cyclin D1）、细胞周期蛋白依赖激酶 4（CDK4）、增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）和细胞周期蛋白依赖性激酶抑制剂 P21（P21）的表达，比较各组的变化。结果：扶正方组、L-OHP 组、扶正方 +L-OHP 组的抑瘤率分别为 9.36%、29.69%、44.54%，均能抑制结肠癌移植瘤的生长。免疫器官脾的脏器指数空白组（0.41±0.04）、合用组（0.45±0.11），而扶正方组为（0.47±0.05），均高于 L-OHP 组（0.40±0.11）；且扶正方组显著高于空白组（P<0.01）。 PCR 与 Western blotting 结果均显示，与空白组比较，L-OHP 组、扶正方 +L-OHP 组 cyclin D1、CDK4、PCNA 的表达下调（P<0.05，P<0.01），P21 的表达上调（P<0.01）；与 L-OHP 组比较，扶正方 +L-OHP 组 PCNA（P<0.01）表达下调，P21的表达上调（P<0.01）。结论：扶正方与 L-OHP 单独和联合应用均对结肠癌小鼠移植瘤的生长有抑制作用，且两药合用不仅能增强化疗效果，同时在一定程度上抑制了 L-OHP 的毒性，其机制可能与影响周期相关因子的表达有关。     

Effect of heavy exercise on mineral metabolism and calcium regulating hormone in humans

Summary The relationship between acid base status and mineral metabolism after heavy exercise has been examined in 12 healthy subjects. Following burst exercise (duration 60–130 sec) to the point of exhaustion, blood pH had decreased (7.42±0.01 vs. 7.18±0.02,P<0.001) and plasma ionized calcium had increased (1.09±0.01 vs. 1.22±0.02 mmol/liter,P<0.001). Log ionized calcium concentration showed a significant negative correlation with pH (r=−0.90). Although plasma total calcium increased after exercise (2.47±0.05 vs. 2.67±0.04 mmol/liter,P<0.001), this change was not seen if the observed values were corrected for the accompanying increase in plasma protein concentration, suggesting that hemoconcentration accounted for these increments. Significant increases were also seen in plasma inorganic phosphate concentration, though not in plasma magnesium. Radioimmunoassay of parathyroid hormone using two different region-specific assays, one directed at the mid-region/carboxy-terminal and the other at the amino-terminal portion of the molecule, and of calcitonin, showed no change during exercise-induced hypercalcemia. The results do not suggest significant skeletal buffering of this type of acidosis and indicate that the changes in ionized calcium associated with short bursts of intense exercise are directly related to acidosis and that those in total calcium are a consequence of hemoconcentration.     

The effects of muscle-building exercise on bone mineral density of the radius,spine, and hip in young men

Summary We previously demonstrated that muscle-building exercise is associated with increases in serum Gla-protein, serum 1,25(OH)₂D, and urinary cyclic AMP. These studies were interpreted to mean that this form of exercise increases bone formation and modifies the vitamin D-endocrine system to provide more calcium for bone. The present investigation was carried out in normal young adult white men to determine the effects of exercise on bone mineral density at weight-bearing and nonweight-bearing sites. Twelve men who had regularly engaged in muscle-building exercises (use of weights, exercise machines, or both) for at least 1 year and 50 age-matched controls (aged 19–40 years) were studied. The body weights of the two groups were not different from each other (78±2 vs. 74±1 kg, NS). Bone mineral density (BMD) of the lumbar spine, trochanter, and femoral neck was measured by dual-photon absorptiometry, and BMD of the midradius was measured by single-photon absorptiometry. It was found that muscle-building exercise was associated with increased BMD at the lumbar spine (1.35±0.03 vs. 1.22±0.02 g/cm²,P<0.01), trochanter (0.99±0.04 vs. 0.86±0.02 g/cm²,P<0.01), and femoral neck (1.18 ±0.03 vs. 1.02±0.02 g/cm²,P<0.001) but not at the midradius (0.77±0.02 vs. 0.77±0.01 g/cm², NS). These studies provide additional evidence that muscle-building exercise is associated with increases in BMD at weight-bearing sites but not at nonweight-bearing sites.