邻苯二甲酸二乙基己酯对新生小鼠睾丸及Leydig细胞影响的实验研究

目的:探讨邻苯二甲酸二乙基己酯(DEHP)对新生小鼠睾丸及Leyd ig细胞形态结构及功能的影响。方法:DEHP分别以低、中、高3组剂量[100、200、500 mg/(kg.d)]灌胃作用于怀孕12 d到产后3 d(GD12~PND3)的KM母鼠,观察DEHP对新生雄性仔鼠体重、睾丸重量、Leyd ig细胞形态结构和3β-羟基类固醇脱氢酶(3-βHSD)活性、酶反应面积的影响。结果:DEHP作用于母鼠后,其雄性子代幼鼠体重和睾丸重量减轻,睾丸Leyd ig细胞形态、超微结构发生改变;高剂量组Leyd ig细胞数量明显增多;低、中剂量组睾酮合成关键酶3β-HSD酶活性下降,酶反应面积减小,但高剂量组在仔鼠出生后15 d时酶活性降低[(吸光度值(0.154±0.011)vs空白对照组(0.222±0.013),P<0.01],而酶反应面积增大[(6 303.0±745.6)μm2vs空白对照组(5 091.4±214.4)μm2,P<0.01)]。结论:DEHP能影响新生雄性小鼠体重、睾丸重量、Leyd ig细胞的形态结构和3β-HSD活性,具有抗雄激素效应。     

DEHP对宫内仔鼠睾酮水平、IGF-1和 StAR mRNA表达的影响

目的 探讨不同剂量的邻苯二甲酸二(乙基)己酯(DEHP)对宫内期仔鼠睾酮水平及胰岛素样生长因子1(IGF-1)、类固醇激素合成急性调节蛋白(StAR)基因表达的影响.方法 雌性S-D大鼠24只,随机均分为4组:正常组、低剂量组、中剂量组和高剂量组.后三组给予不同剂量的DEHP灌胃,剂量分别为(10mg/kg·d-1)、(100mg/kg·d-1)、和(750mg/kg·d-1),时间自雌鼠怀孕后第一天开始至仔鼠出生后第一天止.正常组给予等剂量的玉米油.测量各组雄仔出生时肛门生殖器距离(AGD)和血清睾酮(T)水平;并用光镜和电镜观察仔鼠睾丸Leydig细胞形态学改变;Real-timePCR检测睾丸组织中IGF-1和StARmRNA表达水平.结果 与正常组比较,高剂量组AGD明显缩短,中剂量组AGD呈下降趋势;低剂量组T水平升高、中高剂量组T水平降低;低、中、高剂量组StARmRNA表达下降;低剂量组IGF-1mRNA表达升高,中、高剂量组表达下降.光镜下见随着剂量的升高,睾丸Leydig细胞聚集越明显,高剂量组Leydig细胞呈瘤样聚集;电镜下见低剂量组Leydig细胞线粒体和滑面内质网增多,中、高剂量组均减少,并见脂滴增加.结论 DEHP可以影响富内仔鼠睾酮的合成,不同剂量具有不同的效应,其机制可能与DEHP影响IGF-1和StARmRNA表达有关.     

邻苯二甲酸二(2-乙基)己酯对小鼠胎鼠睾丸及睾丸引带的影响

目的:研究邻苯二甲酸二(2-乙基)己酯(DEHP)对小鼠胎鼠睾丸与睾丸引带形态结构及功能的影响,探讨隐睾发生的可能机制。方法:30只健康KM孕鼠随机均分为3组:空白对照组、玉米油对照组、DEHP组。DEHP组以剂量500mg/(kg.d)的DEHP灌胃作用于怀孕12～19d(GD12～GD19)的KM母鼠,观察DEHP对每胎胎鼠数、雌雄性胎鼠比例、雄性胎鼠体重、睾丸重量、睾丸引带形态结构、位置、睾丸到膀胱颈之间的相对距离(TBD)、颅侧悬韧带残留情况、引带内雄激素受体(AR)、雌激素受体(ER)、肌动蛋白、增殖细胞核抗原(PCNA)表达水平的影响。结果:DEHP对每胎胎鼠数、雌雄性胎鼠比例、雄性胎鼠体重无明显影响;DEHP可影响胎鼠睾丸重量及TBD;DEHP组睾丸均有一定程度的下降不全,睾丸引带形态结构无明显差异;光镜下见DEHP组睾丸生精小管、支持细胞存在明显的发育障碍,睾丸Leydig细胞明显增生;DEHP组睾丸引带AR阳性表达率降低(P<0.01)。结论:DEHP可通过抗雄激素效应导致睾丸引带功能障碍,使睾丸下降发生异常而诱导小鼠隐睾;同时造成睾丸Ser-toli细胞、睾丸Leydig细胞和生精细胞的发育障碍和功能改变。     

邻苯二甲酸二-(2-乙基)己酯致小鼠隐睾睾丸和附睾的组织病理学改变

目的 :探讨邻苯二甲酸二 (2 乙基 )己酯 (DEHP)引起的小鼠隐睾睾丸和附睾的组织病理学改变。　方法 :妊娠KM小鼠 4 0只 ,随机分成 5组 ,分别为正常对照组 8只、玉米油对照组 8只、己烯雌酚 (DES)组 8只、DEHP低剂量组 [DEHP 10 0mg/ (kg·d) ]9只和DEHP高剂量组 [DEHP 5 0 0mg/ (kg·d) ]7只。自妊娠第 12d开始到分娩后 3d ,分别持续经口给予DEHP 10 0mg/ (kg·d)、5 0 0mg/ (kg·d)和DES 10 0 μg/ (kg·d)及玉米油 ,观察仔代雄小鼠的隐睾发生率及隐睾睾丸和附睾的组织病理学改变。　结果 :DEHP 5 0 0mg/ (kg·d)组染毒小鼠的隐睾发生率显著增高 ,睾丸和附睾的体积明显减小、重量减轻 ;睾丸生精上皮发育明显异常 ,精曲小管变薄、萎缩 ,间质细胞异常增生 ,电镜下其隐睾精曲小管上皮和间质细胞均出现明显的超微结构改变。同时附睾管腔中的精子数显著减少甚至缺乏。　结论 :高剂量 [5 0 0mg/ (kg·d) ]DEHP可能具有与DES类似的作用 ,是一种诱发隐睾的重要因子。小鼠在孕期及哺乳期接触DEHP后可引起雄性仔鼠性分化异常 ,诱导隐睾发生、睾丸生精上皮损害和生精过程障碍 ,从而对雄性仔鼠生育力产生不利影响。以上作用存在明确的量 效关系。     

强精片对弱精子症SD大鼠MAPK通路的影响研究

目的:观察强精片对弱精子症SD大鼠精液质量及MAPK通路的影响。方法:选取100只SD大鼠,采用单纯随机抽样方法分为空白对照组、模型组、强精片高剂量组、强精片中剂量组、强精片低剂量组,每组20只。除空白对照组外各组大鼠皆予奥硝唑(ORN)200 mg/(kg·d)灌胃造模,空白对照组大鼠1%羧甲基纤维素钠溶液1ml/100g灌胃,高剂量组、中剂量组、低剂量组均在灌胃ORN的同时,予不同剂量强精片:6 700 mg/(kg·d)、3 300 mg/(kg·d)、1 700 mg/(kg·d),每周给药6 d,1次/d,连续20 d。实验结束后予电镜观察睾丸组织、细胞凋亡情况,采用S-P法测定睾丸波形蛋白表达,Western印迹测定大鼠睾丸ERK1/2表达,ELISA法检测精液中TGF-β1表达量测定。结果:(1)电镜结果显示模型组大鼠睾丸精母细胞,细胞核呈圆形,染色质分布均匀,胞浆内,线粒体的脊断裂或消失、严重肿胀,粗面内质网扩张;与模型组相比,高剂量组、中剂量组、低剂量组大鼠睾丸精母细胞,细胞核呈圆形,染色质分布均匀,胞浆内,线粒体,粗面内质网,核糖体等细胞器结构清晰,均与对照组类似。(2)空白对照组、模型组、高剂量组、中剂量组、低剂量组ERK1/2、波形蛋白相对表达量、睾丸组织细胞凋亡率(%)、精液TGF-β1表达量(ng/ml)分别为(1.00±0.00)、(1.26±0.10)、(1.14±0.08)、(1.18±0.05)、(1.19±0.19),(0.16±0.01)、(0.17±0.01)、(0.16±0.01)、(0.17±0.09)、(0.17±0.00),(9.20±3.07)、(42.20±9.17)、(21.60±5.94)、(33.95±6.39)、(40.85±5.61),(627.67±26.07)、(566.73±68.44)、(621.78±30.80)、(583.93±44.24)、(587.69±59.29)。模型组睾丸组织内ERK1/2、波形蛋白表达量高于对照组(P0.01),模型组大鼠睾丸组织凋亡阳性细胞率较对照组明显升高(P≤0.01),而精液中TGF-β1表达量低于空白对照组(P≤0.05);与模型组相比,高剂量组ERK1/2表达量、波形蛋白表达量均明显降低(P0.01),中剂量组ERK1/2表达量、波形蛋白表达量均降低(P0.05),高剂量组大鼠睾丸组织凋亡阳性细胞率明显降低(P≤0.01),中剂量组大鼠睾丸组织凋亡阳性细胞率降低(P≤0.05),而高剂量组精液中TGF-β1表达量明显高于模型组(P0.05)。结论:强精片可能是通过抗氧化应激改善弱精子症大鼠的精子质量。     

邻苯二甲酸二(2-乙己基)酯致小鼠睾丸基因组DNA甲基化水平变化分析

目的:探讨增塑剂邻苯二甲酸二(2-乙己基)酯(DEHP)作用于怀孕小鼠后,其仔代睾丸基因组DNA甲基化水平的改变情况。方法:妊娠KM小鼠随机分为3组:正常对照组、玉米油对照组和DEHP实验组。自妊娠12.5 d到生后第3天分别持续经口予以玉米油和DEHP[500 mg/(kg.d)],于生后第7天取仔代睾丸,应用甲基敏感扩增多态性(MSAP)技术,对仔鼠睾丸组织DNA进行EcoRⅠ/MspⅠ和EcoRⅠ/HpaⅡ两种酶切反应,经ABI 3730DNA自动测序仪电泳及检测后,运用Genescan3.1分析扩增谱带。结果:对CCGG位点,DEHP实验组小鼠仔代睾丸组织DNA甲基化程度明显增高,其甲基化水平为(34.03±3.05)%;而正常对照组和玉米油对照组小鼠仔代睾丸组织基因组DNA甲基化程度分别为(28.37±2.37)%和(28.58±2.45)%。DEHP实验组与正常对照组和玉米油对照组相比,差异具有统计学意义(P<0.05)。结论:DEHP作用孕鼠后可导致仔代睾丸基因组DNA甲基化水平改变,影响基因组表观遗传修饰改变,可能是导致生殖系统损害的重要毒理机制之一。     

邻苯二甲酸单乙基己基酯对原代培养大鼠睾丸Leydig细胞睾酮合成的影响

目的:探讨邻苯二甲酸二乙基己基酯(DEHP)的代谢产物邻苯二甲酸单乙基己基酯(MEHP)对SD大鼠体外培养睾丸间质细胞(Leydigcells)睾酮合成的影响。方法:建立SD大鼠睾丸Leydig细胞体外原代培养模型,MEHP染毒剂量组分为对照(0μmol/L)、62.5、125、250、500、1000μmol/L,通过噻唑蓝(MTT)法观察线粒体活性,放射免疫法测定睾酮浓度,RTPCR法测定Leydig细胞类固醇合成急性调节蛋白(StAR)mRNA表达。结果:MEHP染毒24h后,Leydig细胞线粒体活性在250μmol/L时显著上升,1000μmol/L时显著下降,与对照组比较,差异均有显著性(P<0.01)。基础状态及人绒毛膜促性腺激素(hCG)刺激状态下,Leydig细胞睾酮合成水平均呈上升趋势,与对照组相比,250、500μmol/L剂量组差异均有显著性(P<0.01)。Leydig细胞StARmRNA的表达在62.5、125、250μmol/L时与对照组相比均未见有显著性改变,在500、1000μmol/L时显著下降(P<0.01)。结论:MEHP直接影响原代培养Leydig细胞线粒体活性及睾酮合成,胆固醇跨膜转运的调节因子StAR与MEHP引起睾酮合成上升的原因可能无关。     

p,p'-DDE,β-BHC单独及其联合对睾丸Sertoli细胞JNK MAPK信号转导通路机制的研究

目的:研究2,2-双-(对氯苯基)-1,1-二氯乙烯(p,p'-DDE)和β-六氯化苯(β-BHC)单独作用及联合作用对离体培养大鼠睾丸Sertoli细胞c-jun氨基末端激酶(JNK)丝裂原活化蛋白激酶(MAPK)信号转导通路的作用机制。方法:从大鼠睾丸组织中分离Sertoli细胞进行离体原代培养2d,设溶剂(DMSO)为对照组,分别以终浓度为10、30、50μmol/L的p,p'-DDE,β-BHC及二者联合染毒Sertoli细胞24h,采用二步RT-PCR法检测Sertoli细胞JNK、c-junmRNA的表达水平。结果:①染毒24h后,对照组及10、30、50μmol/Lp,p'-DDE组Sertoli细胞JNKmRNA灰度比值分别为0.068±0.001、0.164±0.002、0.207±0.006、0.499±0.017;c-junmRNA灰度比值分别为0.122±0.002、0.157±0.006、0.218±0.007、0.289±0.004,随着p,p'-DDE剂量的增加,JNK、c-junmRNA的表达水平均逐渐升高,且10、30、50μmol/L组与对照组差异存在显著性(P<0.05)。β-BHC及联合染毒各组随着染毒剂量的增大,Sertoli细胞JNK、c-junmRNA表达水平也显著增加,且呈剂量依赖性关系(P<0.05)。②p,p'-DDE、β-BHC二者联合作用与单独作用相比,10μmol/L的p,p'-DDE,β-BHC及二者联合染毒组,JNKmRNA灰度比值分别为0.164±0.002、0.149±0.003、0.178±0.004;c-junmRNA灰度比值分别为0.157±0.006、0.131±0.004、0.172±0.002,联合染毒组JNK、c-junmRNA灰度比值均显著高于两者单独作用且存在显著性差异(JNK:P<0.05;c-jun:P<0.01);30、50μmol/L染毒剂量组JNK、c-junmRNA表达水平联合染毒组也显著高于单独染毒组(JNK:P<0.05,c-jun:P<0.01),而各DMSO对照组之间mRNA表达水平无显著性差异(P>0.05)。结论:p,p'-DDE、β-BHC及二者联合作用于睾丸Sertoli细胞后,JNK及其下游的c-jun基因转录水平均明显升高,且联合作用比单独作用更加明显。     

DEHP体外对大鼠睾丸间质细胞凋亡Caspase通路的影响

目的:通过对原代培养的大鼠睾丸间质细胞进行邻苯二甲酸二乙基己酯(di-2-ethylhexyl phthalate,DEHP)染毒,探讨DEHP体外对大鼠睾丸间质细胞凋亡通路Caspase-3和Caspase-9表达以及细胞凋亡的影响。方法:体外分离和原代培养大鼠睾丸间质细胞,用不同浓度的DEHP(10、50、100 nmol/L)染毒24 h。荧光定量PCR法检测间质细胞Caspase-3和Caspase-9 mRNA表达,Western印迹检测间质细胞Caspase-3和Caspase-9蛋白表达,流式细胞术检测间质细胞凋亡率。结果:与对照组相比,DEHP各组睾丸间质细胞Caspase-3 mRNA(1.69±0.38 vs3.82±0.39、6.91±0.40、15.47±0.40)和蛋白(0.18±0.09 vs 0.32±0.10、0.61±0.08、0.89±0.09)表达显著增加(P均<0.05),Caspase-9 mRNA(2.24±0.41 vs 5.16±0.43、9.61±0.45、19.22±0.43)和蛋白(0.26±0.07 vs0.40±0.08、0.68±0.09、0.96±0.08)表达也显著增加(P均<0.05),细胞凋亡率(4.36±1.11 vs 7.52±1.09、12.72±1.10、24.59±1.11)也显著增加(P均<0.05),呈浓度依赖关系。结论:DEHP可通过激活细胞凋亡Caspase通路而诱导大鼠睾丸间质细胞凋亡,进而影响间质细胞功能。     

SD大鼠糖尿病创面与正常创面中TGF-β1、TGF-β3的表达差异

目的:通过观察大鼠正常创面与糖尿病模型创面肉芽组织中TGF-β1、TGF-β3不同时段表达水平的差异,探求糖尿病创面“难愈”的可能机制。方法:采用SD大鼠实验性正常创面及糖尿病创面背部全层皮肤缺损模型,分别于不同时段(3天、7天、11天)取材,EnVision二步法免疫组化测定,并行图像扫描定量分析TGF-β1、TGF-β3水平变化。结果:创伤后3天、7天和11天,正常创面模型组TGF-β1表达分别为(25.56±6.86)、(24.79±5.62)和(28.57±6.39),TGF-β3表达分别为(39.69±5.46)、(23.20±2.45)、(13.69±3.63)。而糖尿病溃疡模型组TGF-β1表达分别为(7.10±3.74)、(9.47±1.72)和(7.87±2.53),TGF-β3表达分别为(14.99±1.87)、(18.72±5.01)和(8.68±3.39)。糖尿病性创面肉芽组织中各时段(3天、7天和11天)TGF-β1及TGF-β3表达水平均低于正常创面组(P<0.05)。结论:SD大鼠糖尿病创面TGF-β1及TGF-β3的低水平表达,可能是创面愈合迟缓的原因之一。     

Plasticizer di(2‐ethylhexyl)phthalate interferes with osteoblastogenesis and adipogenesis in a mouse model

Plasticizer di(2‐ethylhexyl)phthalate (DEHP) can leach from medical devices such as blood storage bags and the tubing. Recently, epidemiological studies showed that phthalate metabolites levels in the urine are associated with low bone mineral density (BMD) in older women. The detailed effect and mechanism of DEHP on osteoblastogenesis and adipogenesis, and bone loss remain to be clarified. Here, we investigated the effect and mechanism of DEHP and its active metabolite mono(2‐ethylhexyl)phthalate (MEHP) on osteoblastogenesis and adipogenesis. The in vitro study showed that osteoblast differentiation of bone marrow stromal cells (BMSCs) was significantly and dose‐dependently decreased by DEHP and MEHP (10–100 µM) without cytotoxicity to BMSCs. The mRNA expressions of alkaline phosphatase, Runx2, osteocalcin (OCN), Wnt1, and β‐catenin were significantly decreased in DEHP‐ and MEHP‐treated BMSCs during differentiation. MEHP, but not DEHP, significantly increased the adipocyte differentiation of BMSCs and PPARγ mRNA expression. Both DEHP and MEHP significantly increased the ratios of phosphorylated β‐catenin/β‐catenin and inhibited osteoblastogenesis, which could be reversed by Wnt activator lithium chloride and PPARγ inhibitor T0070907. Moreover, exposure of mice to DEHP (1, 10, and 100 mg/kg) for 8 weeks altered BMD and microstructure. In BMSCs isolated from DEHP‐treated mice, osteoblastogenesis and Runx2, Wnt1, and β‐catenin expression were decreased, but adipogenesis and PPARγ expression were increased. These findings suggest that DEHP and its metabolite MEHP exposure may inhibit osteoblastogenesis and promote adipogenesis of BMSCs through the Wnt/β‐catenin‐regulated and thus triggering bone loss. PPARγ signaling may play an important role in MEHP‐ and DEHP‐induced suppression of osteogenesis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1124–1134, 2018.

     

Effects of exposure of pre-pubertal boars to di(2-ethylhexyl) phthalate on their frozen-thawed sperm viability post-puberty

Late effects of pre-pubertal oral exposure to di(2-ethylhexyl) phthalate (DEHP), a plastic softener used in, for example, polyvinyl chloride-products, on semen quality in young boars have not been clear-cut. The aim of this study was to determine whether stress imposed on spermatozoa would reveal such effects. Semen was collected from post-pubertal boars (8-9 months of age), which had been exposed to 300 mg kg(-1) body weight of DEHP per os three times a week from 3 to 7 weeks of age and from control siblings given placebo (water). The semen was cryopreserved and examined for plasma membrane integrity post-thaw using the short hypo-osmotic swelling test and flow cytometry (propidium iodide /SYBR-14). Sperm motility was assessed by computer-assisted sperm analysis. No significant difference in plasma membrane integrity could be found between the groups. The DEHP-exposed group had a significantly lower percentage of linearly motile spermatozoa at 30 min (P < 0.05) and 120 min (P < 0.001) after thawing, and a larger amplitude of lateral displacement of the head 120 min after thawing (P < 0.05), compared with controls. In summary, spermatozoa from boars pre-pubertally exposed to low doses of DEHP, showed kinematic deviations post-thaw that could be related to DEHP exposure.     

Glucocorticoid diminishes vascular endothelial growth factor and exacerbates proteinuria in rats with mesangial proliferative glomerulonephritis

Glucocorticoids are widely prescribed for renal diseases. It is believed that glucocorticoids attenuate immune-mediated renal diseases by suppressing the cell-mediated immune system. However, there is evidence that glucocorticoids influence the expression of such growth factors as vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), and connective tissue growth factor (CTGF), which are known to influence the development or progression of renal diseases. Therefore, we undertook this study to determine whether glucocorticoids regulate proteinuria or extracellular matrix (ECM) production by altering these growth factors. Mesangial proliferative glomerulonephritis was induced in rats by intravenous injection of monoclonal antibody (OX-7), and dexamethasone (20 mg/kg) was administered intraperitoneally from the third to seventh disease day. Glomerular expression of VEGF, TGF-β1, and CTGF, the amount of urinary protein, and glomerular ECM were measured on the seventh disease day. The nephritic group showed proteinuria and greater VEGF, TGF-β1, and ECM production. Dexamethasone aggravated proteinuria (protein, 0.4 ± 0.1 mg/mg creatinine in the NC group, 6.3 ± 2.0 mg/mg creatinine in the DC group, and 21.1 ± 1.9 mg/mg creatinine in the D-Dex group; P < 0.05) and diminished VEGF release (22 ± 3 pg/mg total protein in the NC group, 292 ± 26 pg/mg total protein in the DC group, and 198 ± 23 pg/mg total protein in the D-Dex group; P < 0.05). Expression of TGF-β1, CTGF, and ECM was not altered significantly by dexamethasone treatment. We found that glucocorticoid diminishes VEGF release and at the same time exacerbates proteinuria in rats with this type of glomerulonephritis. © 2002 by the National Kidney Foundation, Inc.     

Urinary phthalate metabolites and semen quality: a review of a potential biomarker of susceptibility

Phthalates are a class of chemicals with widespread general population exposure. Some phthalates are reproductive and developmental toxicants in laboratory animals. Advances in the field of phthalate research in humans are dependent on the development and implementation of biomarkers to assess exposure and outcome, as well as potential markers that may be indicative of increased susceptibility. Recently, we incorporated a novel biomarker of potential 'susceptibility' into our study on the relationship of phthalates with semen quality and sperm DNA damage among men recruited from an infertility clinic. We measured urinary concentrations of three di(2-ethylhexyl) phthalate (DEHP) metabolites, mono(2-ethylhexyl) phthalate (MEHP) and two oxidative metabolites, mono-(2-ethyl-5-hydroxylhexyl) phthalate (MEHHP) and mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP). We calculated the percent of DEHP excreted as the hydrolytic monoester (i.e., MEHP). We referred to this as %MEHP and considered it a phenotypic marker of the proportion of DEHP excreted in the urine as MEHP. In our sperm DNA study, we found novel results for the DEHP metabolites. Although MEHP was positively correlated with the oxidative metabolites, the association of sperm DNA damage with MEHP, as compared to MEHHP and MEOHP, were in opposite directions. We hypothesized that MEHP is the bioactive toxicant and further metabolism to MEHHP/MEOHP may lower internal burden of MEHP and thus be protective from sperm DNA damage. An alternative explanation may include that the relative percentage of DEHP excreted as MEHP was a surrogate for the function of phase I enzymes. Men with high %MEHP may have higher levels of sperm DNA damage because of poor metabolism (detoxification) of other genotoxic chemicals. Our hypothesis that %MEHP may represent a phenotypic marker of metabolism is novel but requires further exploration to confirm.     

A study on the effect of phthalate esters and their metabolites on idiopathic infertile males

Phthalate plasticisers in medical, cosmetic and consumer products might pose serious health implications in humans including infertility. We sought to investigate the correlation, if any, between the phthalates and their metabolites and sperm quality parameters, and male infertility. Phthalate esters (15) and their metabolites (5) were estimated in the blood serum and urine samples from the age-matched 152 infertile and 75 fertile males using gas chromatography (GC) and high-performance liquid chromatography (HPLC). Finally, the data were analysed to correlate phthalate exposure and semen quality parameters in the infertility group. The estimated levels of DEHP, DBP, DIBP, BEHIP, BPBG, DPP, DIOP, DIHP, DMP, DINP, BIOP, DMOP and DICHP were significantly higher in the infertile males compared to the fertile males (p < .05 or p < .01). However, these were not found to be associated with the semen quality parameters (sperm count, motility and sperm morphology). Similarly, HPLC data revealed that the associations between semen parameters (sperm count, sperm motility and sperm morphology) and phthalate metabolite (MEHP and MBP) concentrations in urine samples from the infertile males were mostly unremarkable or statistically nonsignificant. Conclusively, environmental exposure to phthalates and their impacts on male infertility were statistically insignificant in our study groups.     

邻苯二甲酸二酯与特发性少弱精子症关系的探讨

目的:通过检测精浆中邻苯二甲酸二酯(DEHP)的含量,探讨DEHP与男性特发性少弱精子症之间的关系。方法:收集潍坊地区3家医院特发性少弱精子症所致不育患者100例,分为A组:常年接触蔬菜大棚塑料薄膜者50例;B组:常年食用塑料包装盒饭者50例;另设立C组(对照):为精液正常之志愿者50例。采用计算机辅助精子分析仪检测精子浓度和活率,采用反相液相高效色谱法测定精浆中DEHP的浓度。结果:1A、B、C3组中DEHP含量分别为(0.72±0.48)、(0.71±0.49)、(0.21±0.18)mg/L,C组与A、B组比较,差异均有统计学意义(P均0.05)。2精浆中DEHP浓度与精子活率呈负相关(A组:r=-0.354,B组r=-0.348,P0.05)。结论:常年接触某些塑料制品的男性不育患者,精浆中DEHP浓度高于正常人群,精浆内DEHP过量蓄积可能是不育的重要因素之一。     

Uraemic pruritus and exposure to di(2-ethylhexyl)phthalate (DEHP) in haemodialysis patients

Uraemic pruritus is a frequent and disabling symptom in patientson dialysis. The pathogenesis of uraemic pruritus is neverthelessstill obscure. We investigated whether di (2-ethylhexyl ) phthalate(DEHP), the most commonly used plasticizer in polyvinyichloride(PVC) haemodialysis tubings, is a possible pathogenetic factorin uraemic pruritus. Serum concentrations of DEHP and its majorderivatives mono-(2-ethylhexyl ) phthalate (MEHP), 2-ethylhexanol(2-EH) and phthalic acid (PA) were determined in uraemic patientsbefore and after a haemodialysis session and compared with theoccurrence and intensity of pruritus in these patients. Twenty-onepatients on regular haemodialysis for at least 6 months wereexamined. The severity of uraemic pruritus was assessed usinga standard questionnaire (pruritus score). The quantitativeanalysis of DEHP and its derivatives was carried out by GC/selectedion monitoring mass spectrometry. Fourteen out of 21 patients(66%) complained about uraemic pruritus to a variable degree.The post-dialysis serum concentrations of DEHP, MEHP and 2-EHwere significantly higher than the corresponding pre-dialysisvalues, whereas the post-dialysis concentrations of PA (0.122±0.078µg/ml) were significantly lower than pre-dialysis levels(0.194±0.101 µg/ml, P=0.00068). Neither pre- norpost-dialysis serum concentrations of DEHP, MEHP, PA or 2-EHwere correlated with the severity of uraemic pruritus. Additionally,serum concentrations of DEHP and its metabolites did not differsignificantly in patients with and without pruritus. These findingssuggest that patients on haemodialysis are regularly exposedto considerable amounts of DEHP and metabolites. Phthalic acid,one of the presumed end products of DEHP metabolism, might beeliminated at least in part by haemodialysis. The expositionto DEHP and metabolites during haemodialysis, as assessed bymeasuring serum concentrations, bears no immediate realtionto the occurence or intensity of uraemic pruritus.     

结缔组织生长因子介导转化生长因子β1促进瘢痕成纤维细胞转分化的研究

目的 了解结缔组织生长因子(CTGF)介导TGF-β1发挥促人增生性瘢痕Fb(HSFb)转分化的作用.方法 体外培养人HSFb,取5份细胞标本分别加入不同浓度TGF-β1(0、2.5、5.0、7.5、10.0 ng/mL),作用48 h后待测.余下标本分为:空白对照组;CTGF刺激组,培养液中加入终浓度10.0 ng/mL重组人CTGF;TGF-β1刺激组,培养液中加入终浓度10.0 ng/mL重组人TGF-β1;CTGF反义寡脱氧核苷酸(ASODN)转染组,细胞转染CTGF ASODN后,加入培养液;CTGF ASODN转染+TGF-β1刺激组,细胞转染CTGF ASODN后2 h,加人含终浓度10.0 ng/mL重组人TGF-β1的培养液.蛋白质印迹法分析不同浓度TGF-β1刺激对细胞CTGF表达的影响,比较各组α-平滑肌肌动蛋白(α-SMA)表达变化;流式细胞仪检测α-SMA阳性细胞百分率.结果 TGF-β1浓度为10.0 ng/mL时,CTGF的表达明显高于未受刺激的细胞(P<0.05).CTGF刺激组与TGF-β1刺激组α-SMA表达明显高于空白对照组(P<0.01).CTGF ASODN转染组以及CTGF ASODN转染+TGF-β1刺激组α-SMA表达与空白对照组接近(P>0.05).上述各组细胞α-SMA阳性细胞百分率依次为(10.8±2.8)%、(29.1±4.0)%、(28.7±4.8)%、(10.7±2.3)%、(14.3±2.9)%,统计学分析结果类似于α-SMA表达.结论 CTGF是TGF-β1发挥促人HSFb转分化的重要下游效应分子之一.     

五子衍宗丸对少弱精子症模型大鼠精子线粒体膜电位及超微结构影响

目的:研究五子衍宗丸对少弱精子症模型大鼠精子线粒体膜电位(MMP)水平及线粒体超微结构的影响。方法:取体重为200～220 g雄性SD大鼠60只,随机分成正常组,模型组,对照组(黄精赞育胶囊组),五子衍宗丸低、中、高剂量组,除正常组外,其他各组大鼠灌服雷公藤多苷[30 mg/(kg.d)],连续8周,制备少弱精子症模型。造模结束后,正常组、模型组给予等容量蒸馏水[10 ml/(kg.d)],对照组给予黄精赞育胶囊溶液[3.01 g/(kg.d)],五子衍宗丸各组分别给予水提液,低剂量组[2.30 g生药/(kg.d)],中剂量组[4.60 g生药/(kg.d)],高剂量组[9.20 g生药/(kg.d)],连续30 d。末次给药后30 min,采用荧光流式细胞技术(JC-1染色)测定精子MMP水平(JC-1+%),并通过透射电镜观察精子线粒体超微结构的改变。结果:①MMP:JC-1+%和强度分别为:正常组70.80±4.92、4 360±945;模型组33.77±6.19、1 4685±496;对照组56.34±10.35、3 277±895;五子衍宗丸低剂量组40.80±10.40、2 016±767;中剂量组59.40±6.51、3 897±643;高剂量组60.71±7.81、3 371±6467。造模各组大鼠精子线粒体JC-1+%及强度均明显下降,与正常组比较差异有显著性意义(P均<0.05);连续给药30 d后,给药各组均能明显提高精子MMP,增加JC-1+%,除低剂量组外,其他用药各组与模型组比较差异有显著性意义(P均<0.05)。②精子线粒体超微结构:雷公藤造模后,精子外膜松散、变性,线粒体肿胀、大小不一、线粒体膜不完整,轴丝结构不清或出现断裂。给予五子衍宗丸30 d后,精子外膜及线粒体膜结构完整,减少线粒体肿胀,轴丝及微管结构基本正常。结论:雷公藤多苷能降低精子MMP水平,破坏线粒体的结构。五子衍宗丸能明显提高少弱精子症模型大鼠精子MMP水平,减轻精子线粒体结构损伤。保护精子线粒体结构与功能的完整是五子衍宗丸治疗少弱精子症的机制之一。     

转化生长因子、核心蛋白聚糖在慢性非细菌性前列腺炎大鼠模型前列腺组织中的检测及意义

目的 研究慢性非细菌性前列腺炎(chronic nonbacterial prostatitis,CNP) SD大鼠前列腺组织中转化生长因子(TGF-β1)和核心蛋白聚糖(DCN)的表达,并初步探讨前列腺组织纤维化的发病机制.方法 将60只6月龄SD大鼠随机分为对照组、30 d和45 d CNP模型组各20只.CNP模型组采用去势+高剂量苯甲酸雌二醇肌注方法建立.采用双抗体夹心法检测SD大鼠前列腺组织中TGF-β1、DCN的含量.结果 ①45 d、30 d CNP模型组大鼠TGF-β1表达水平[(140.78 ±37.94)、(106.28 ±30.63) ng/L]均显著高于对照组[(13.42-±4.24)ng/L,P＜0.05];且45 d CNP模型组高于30 d CNP模型组(P＜0.05);②45 d、30 d模型组DCN表达水平[(947.06±114.28)、(722.96±110.66) ng/L]显著高于对照组[(63.97±15.34) ng/L,P＜0.05];且45 d CNP模型组高于30 d CNP模型组(P＜0.05);③TGF-β1、DCN在CNP大鼠模型前列腺组织中表达呈正相关.结论 TGF-β1、DCN可能参与CNP发病过程,在前列腺纤维化的形成中起着重要的作用.