不同价态染砷大鼠肝脏MRP2表达水平与砷甲基化产物关联性

目的 分析亚砷酸钠(iAs3+)和砷酸氢钠(iAs5+)染毒后,对大鼠多药耐药相关蛋白2(MRP2)表达的影响,及其与肝脏砷及甲基化产物的关联性,寻找不同价态砷体内代谢转运间的差异,为进一步阐明砷毒作用机制提供依据.方法 70只Wistar大鼠分成7组,第1组为正常对照组,给予饮用去离子水;第2～4组为染毒iAs3+高、中、低剂量组,分别给予20.0、6.7、2.2 mg/kg体质量亚砷酸钠溶液;第5～7组为染毒iAs5+高、中、低剂量组,分别给予20.0、6.7、2.2 mg/kg体质量砷酸氢钠溶液.各组大鼠于染毒90 d后处死,取肝脏组织,用蛋白印迹法(Western-blotting)测定MRP2表达的变化.采用高效液相-氢化物发生原子荧光光谱分析法(HPLC-HGAFS)分析各组肝脏中砷及甲基化产物含量,并运用Pearson相关法分析MRP2表达与砷及甲基化产物的相关性.结果 对照组、iAs3+和iAs5+高、中、低剂量组MRP2蛋白表达分别为1.21±0.13、1.85±0.09、1.65±0.19、1.61±0.18、1.69±0.04、1.42±0.19、1.27±0.10,iAs3+的高、中、低剂量组和iAs5+高、中剂量组MRP2蛋白表达均高于对照组(P均＜0.05);相同受试物比较,iAs3+和iA83+的高剂量组MRP2蛋白表达均高于低剂量组(P均＜ 0.05);不同受试物同一剂量组比较,iAs3+高、中、低剂量组的MRP2蛋白表达均强于iAs5+高、中、低剂量组(P均＜0.05).同时,iAs3+染毒组肝脏中MRP2蛋白表达量与iAs3+和砷甲基化代谢产物MMA和DMA的含量均呈正相关关系(r值分别为0.575、0.678、0.395,P均＜0.05);iAs5+染毒组肝脏中MRP2表达量与MMA和DMA的含量均呈正相关关系(r值分别为0.593、0.643,P均＜0.05),与iAs3+的含量无相关关系(P＞0.05).结论 随着砷染毒剂量的增加,MRP2表达逐渐上调,从而致使肝细胞膜上MRP2表达代偿性改变.MRP2外排通路可能在iAs3+代谢径路中发挥重要作用.肝脏MRP2与不同价态砷甲基化产物的负荷或水平密切相关.     

锌拮抗砷致大鼠血脂谱异常的研究

目的明确锌拮抗砷致大鼠血脂谱异常的可行性。方法亚慢性毒性染毒试验。取初成年雄性大鼠48只随机分成6组,经口灌胃染毒6周,染毒期间观察大鼠的一般状况,染毒结束后眼眶采血,分离血清。分别采用酶学、免疫透射比浊法、免疫组化进行血脂、载脂蛋白亚类、受体的测定。结果最高剂量染砷组大鼠加锌后血清中TG、TC、LDL-C、apoB升高;HDL-C、apoA-1下降,且高剂量加锌组大鼠血清中TG、LDL-C、HDL-C与最高剂量染砷组相比差异有统计学意义(P<0.05),与对照组相比差异无统计学意义。结论锌对砷致大鼠血脂谱异常具有拮抗作用;为进一步研究及现场使用锌防治砷中毒提供科学依据。     

不同剂量亚砷酸钠染毒大鼠唾液砷水平及其与血砷、尿砷间关系研究

目的 探讨不同剂量砷暴露大鼠唾液中含砷量变化及其与血砷、尿砷间的关系.方法 将32只雄性SD大鼠按体质量随机分为4组,分别为对照组(生理盐水)及低(0.2 mg/kg)、中(2.0 mg/kg)、高(20.0mg/kg)剂量亚砷酸钠染毒组,每组8只.采用经口灌胃染毒方法,隔日1次,连续2周后收集唾液、血液、尿液及脏器,计算脏器系数,应用原子荧光分光光度计(AFS-230)检测血砷、尿砷,应用电感耦合-等离子体质谱(ICP-MS)检测唾液砷.结果 亚砷酸钠染毒组大鼠体质量增长值均低于对照组,其中高剂量组[(76.13±17.19)g]与对照组[(103.00±12.31)g]比较,差异有统计学意义(P＜0.05).高剂量组大鼠肝脏及肾脏脏器系数[(3.92±0.54)％、(0.96±0.15)％]与对照组[(3.27±0.35)％、(0.76±0.05)％]比较,差异有统计学意义(P＜0.05或＜0.01).低、中、高染毒组大鼠唾液砷[(0.044±0.019)、(0.211±0.071)、(1.128±0.380)mg/L]、血砷[(11.832±1.887)、(45.032±7.216)、(121.839±17.323)mg/L]及尿砷[(0.138±0.085)、(0.874±0.328)、(8.843±1.754)mg/L]与对照组[(0.018±0.014)、(2.267±0.370)、(0.025±0.011)mg/L]比较,差异有统计学意义(P均＜ 0.05),且各染毒组之间比较差异有统计学意义(P均＜0.05).大鼠唾液砷与血砷、尿砷之间存在显著正相关,相关系数分别为0.934、0.960(P均＜0.01).结论 高砷暴露可抑制大鼠体质量增长,并对肝脏及肾脏有较强毒性.砷在唾液中存在良好的剂量-反应关系,且唾液砷与血砷、尿砷间存在明显的正相关关系,提示唾液砷也是一种良好的砷暴露标志物.     

亚砷酸钠和砷酸钠染毒大鼠尿液砷形态分析

目的 分析亚砷酸钠和砷酸钠染毒大鼠尿液中砷形态代谢产物水平,寻找不同价态砷代谢的差异,为进一步探讨砷代谢产物与砷毒性作用机制间的关系提供基础数据.方法 Wistar大鼠70只,体质量80～120 g,按体质量随机分为7组,即对照组、亚砷酸钠高、中、低剂量组和砷酸钠高、中、低剂量组,每组10只.各组大鼠染毒1个月后,收集12 h尿液,采用高效液相色谱-氢化物发生原子荧光光谱法(HPLC-HGAFS)测定尿液中砷形态代谢产物水平,并通过二甲基砷酸(DMA)加标回收率测定来评价结果准确度.结果 亚砷酸钠中剂量组尿液中各种代谢物[3价无机砷(iAs~(3+))、5价无机砷(iAs~(5+))、DMA]水平[(121.66±1.26)、(10.26±2.68)、(204.91±0.56)μg/L]高于高剂量组[(113.20±0.75)、(5.16±1.32)、(147.70±0.77)μg/L,P均<0.05]和低剂量组[(79.35±2.12)、(5.13±2.25)、(56.35±1.23)μg/L,P均<0.05];砷酸钠中剂量组尿液中iAs~(3+)和DMA水平[(315.81±1.69)、(245.12±1.18)μg/L]高于高剂量组[(85.03±0.56)、(110.34±1.04)μg/L,P均<0.05]和低剂量组[(22.97±2.67)、(15.75±2.15)μg/L,P均<0.05].亚砷酸钠高、低剂量组尿液中iAs~(3+)和DMA水平高于砷酸钠组(P均<0.05);而亚砷酸钠中剂量组尿液中iAs~(3+)和DMA水平低于砷酸钠组(P均<0.05).亚砷酸钠高、中、低剂量组DMA的平均加标回收率为94.80%～102.70%,砷酸钠高、中、低剂量组DMA的平均加标回收率为95.33%～108.40%.结论 染砷价态及外暴露剂量不同,大鼠体内砷代谢物的形态和水平就不同;不同价态无机砷在大鼠体内的代谢径路不完全相同.     

氟、砷及其联合染毒对大鼠海马和大脑皮质组织Bcl-2和Bax蛋白表达的影响

目的 探讨氟、砷及其联合染毒对大鼠海马和大脑皮质组织Bcl-2和Bax凋亡蛋白表达的影响.方法 将初断乳SPF级雄性SD大鼠按体质量随机分为对照组、氟处理组、砷处理组和氟砷联合组,每组10只.氟处理组大鼠饮用120 mg/L氟化钠(NaF)水溶液,砷处理组大鼠饮用70 mg/L亚砷酸钠(NaAsO2)水溶液,氟砷联合组大鼠饮用含120mg/L NaF和70 mg/L NaAsO2的水溶液,对照组大鼠饮用蒸馏水.3个月后采用免疫组织化学和Western blot法检测大鼠海马和大脑皮质组织Bcl-2和Bax凋亡蛋白的表达.结果 ①免疫组织化学法检测结果:大鼠海马和大脑皮质组织细胞质中Bcl-2和Bax凋亡蛋白呈现为棕黄色颗粒物;与对照组和氟、砷处理组比较,氟砷联合组大鼠海马和大脑皮质Bcl-2阳性神经元细胞数较少;与对照组比较,氟、砷处理组和氟砷联合组大鼠海马和大脑皮质Bax阳性神经元细胞数较多,尤其以氟砷联合组的阳性细胞数最多.②Western blot法检测结果:对照组、氟处理组、砷处理组、氟砷联合组大鼠海马组织Bcl-2、Bax蛋白表达分别为0.84±0.22、0.76±0.10、0.75±0.24、0.28±0.05和0.44±0.19、0.81±0.14、1.22±0.45、1.45±0.26,其中氟砷联合组Bcl-2表达明显低于其他3组(P均＜0.05),而氟、砷处理组和氟砷联合组Bax表达均明显高于对照组(P均＜ 0.05),且砷处理组和氟砷联合组Bax表达高于氟处理组;上述4组大鼠大脑皮质Bcl-2、Bax蛋白表达分别为0.51±0.18、0.50±0.12、0.49±0.19、0.33±0.19和0.39±0.18、0.79±0.30、0.79±0.35、0.80±0.18,其中氟、砷处理组和氟砷联合组大鼠Bax蛋白表达明显高于对照组(P均＜0.05).氟、砷处理组和氟砷联合组大鼠海马和大脑皮质组织Bcl-2/Bax表达比值(0.96±0.28、0.72±0.45、0.33±0.05和0.69±0.37、0.57±0.10、0.37±0.18)均明显低于对照组(1.91±1.32、1.44±0.29,P均＜0.05),尤以氟砷联合组最明显.结论 在一定染毒剂量下,氟和砷均促进大鼠海马及大脑皮质组织Bax蛋白的表达,降低海马及大脑皮质组织的Bcl-2/Bax表达比值.氟和砷对大鼠海马组织Bcl-2蛋白表达和大脑皮质组织Bcl-2/Bax蛋白表达比值的影响存在协同作用.     

维生素C、锌制剂拮抗慢性砷中毒的实验研究

目的观察补充锌制剂、维生素C对染砷小鼠肝脏组织超氧化物岐化酶(SOD)水平、丙二醛(MDA)含量的影响,比较这两种物质针对砷染毒小鼠的抗氧化作用的强弱.方法昆明种小鼠饮用含砷水同时通过灌胃给予不同种类的抗氧化剂,8周后,测定小鼠肝脏SOD活性及MDA含量.结果干预8周后,测定小鼠肝脏组织的SOD活性及MDA的含量,可见维生素C组的小鼠肝SOD活性上升、MDA含量下降达到正常对照组水平(P<0.05),锌制剂组仅小鼠肝SOD活性上升,达到正常对照组水平(P<0.05)而MDA含量与染毒对照组差异无统计学意义(P>0.05).结论提示补充一定剂量的锌制剂、维生素C对染砷小鼠的脂质过氧化有拮抗作用,维生素C对慢性砷中毒的防治效果要优于锌制剂.     

花姜酮对燃煤型砷中毒大鼠肝组织Bax、Bcl-2蛋白表达及肝纤维化的影响

目的探讨花姜酮对燃煤型砷中毒大鼠肝组织Bax、Bcl-2蛋白表达及肝纤维化的影响。方法将18只健康清洁级断乳Wistar大鼠(体质量80～100 g)随机均分为三组,对照组大鼠喂标准饲料90 d后处死;造模治疗组大鼠喂含砷100 mg/kg的饲料,且由股静脉穿刺给予10 mg/kg花姜酮溶液,1次/周,持续90 d后处死;单纯造模组大鼠同样喂含砷100 mg/kg的饲料,且由股静脉穿刺给予等量的生理盐水,1次/周,持续90 d后处死。比较三组大鼠毛发与尿液中砷含量、血清肝功能指标,并采用Western blot法检测肝组织Bax、Bcl-2、α-SMA、TGFβ-1蛋白表达水平。结果三组大鼠的毛发与尿液中砷含量、血清AST与ALT水平、肝组织Bax、Bcl-2、α-SMA、TGFβ-1蛋白表达水平比较,差异均有统计学意义(P0.05)。对照组的毛发与尿液中砷含量、血清AST与ALT水平、肝组织Bax、α-SMA、TGFβ-1的相对表达量显著低于单纯造模组与造模治疗组,肝组织Bcl-2的相对表达量显著高于单纯造模组与造模治疗组;造模治疗组的毛发与尿液中砷含量、血清AST与ALT水平、肝组织Bax、α-SMA、TGFβ-1的相对表达量显著低于单纯造模组,肝组织Bcl-2的相对表达量显著高于单纯造模组。结论花姜酮能显著缓解燃煤型砷中毒大鼠的肝损伤和肝纤维化,具有一定抗砷中毒和护肝功能,可能与其通过下调肝组织Bax表达、上调Bcl-2表达发挥抗凋亡作用有关。     

PPARγ、NF-κB的表达与砷暴露致大鼠肝纤维化的相关性

目的:初步探讨过氧化物酶体增殖物激活受体γ(PPARγ)、核转录因子B(NF-κB)的表达与水砷暴露致大鼠肝纤维化的相关性.方法:110只SD大鼠随机分成对照组(自来水)、模型组(浓度100mg/L亚砷酸钠溶液)、自然恢复组(浓度100mg/L亚砷酸钠溶液+自来水).对照组和模型组分别于第l、2、3、4月末各处死10只,自然恢复组先给予砷溶液,分别在第l、2、3月末取出10只改给予1mo自来水饮用后处死.肝组织病理学检查以观察肝纤维化的动态变化,实时荧光定量RT-PCR法和Western blot法检测PPARγ、NF-κB的mRNA及蛋白表达水平.结果:(1)病理结果:HE染和Masson染色可见,随砷暴露时间的延长,肝细胞变性、坏死增多,汇管区炎症细胞浸润加重,纤维组织增生增多,肝纤维化趋势明显.砷暴露1mo脱离自然恢复1mo后较同月模型组肝细胞变性、坏死及炎细胞浸润程度明显减轻,胶原生成减少.砷暴露2、3mo脱离自然恢复1mo后较同月模型组病理结果差异不明显;(2)mRNA水平:模型组PPARγ mRNA含量逐渐降低,与对照组比较差异均有统计学意义(174.99±41.48,114.55±21.30,6...     

亚慢性砷暴露小鼠血和肝中砷形态及谷胱甘肽水平检测分析

目的 探讨亚慢性饮水砷暴露小鼠血和肝中砷形态的分布,评价砷对血和肝中还原型谷胱甘肽(GSH)水平的影响.方法 小鼠以自由饮水方式饮用含砷量为0(对照)、25、50、100mg/L的水溶液,连续染砷6周.采用氢化物发生-超低温捕集-原子吸收分光光度法,检测小鼠血和肝中无机砷(iAs)、一甲基砷酸(MMA)和二甲基砷酸(DMA)的水平,计算甲基化率.采用二硫双硝基苯甲酸(DTNB)法测定全血和肝组织中GSH的水平.结果 小鼠血和肝中iAs、MMA和DMA水平随染砷剂量的增加而升高.各染砷组小鼠血和肝中的砷一甲基化率(PMI)和二甲基化率(SMI)与对照组比较差异均有统计学意义(P＜0.05),其中50 mg/L染砷组小鼠肝中SMI[(50.45±2.94)%]明显高于25 mg/L组[(41.68±7.09)%]和100 mg/L组[(41.19±8.87)%],组间比较差异有统计学意义(P＜0.05).各染砷组小鼠血和肝中砷形态的构成比(iAs:MMA:DMA)分别为2:3:5和4:3:3.有机砷(MMA+DMA)的水平分别约占总砷的80%和60%.各染砷组小鼠血和肝中GSH水平随染砷剂量的增加而下降,与对照组比较差异均有统计学意义(P＜0.05),但各染砷组之间的小鼠血和肝中GSH水平比较差异均无统计学意义(P＞0.05).结论 当砷暴露水平达到一定程度后,肝脏对iAs的甲基化代谢能力可达到饱和.血中砷形态变化不同于肝组织,提示机体内的其他组织器官也可能具有砷甲基化代谢能力.血和肝中GSH水平是反映砷毒性的较好指标.     

氟对大鼠成骨细胞骨保护蛋白表达的影响

目的 观察氟对体外培养大鼠乳鼠成骨细胞骨保护蛋白(osteoprotegerin,OPG)mRNA和蛋白表达的影响.方法 体外培养Wistar大鼠乳鼠成骨细胞,按染氟剂量分为0(对照)、1、2、4 mg/L组.分别在染氟48、72 h后提取RNA,采用反转录聚合酶链反应(RT-PCR)法检测OPG mRNA表达;采用酶连免疫吸附实验(ELISA)法检测培养液上清中的OPG蛋白表达.结果 大鼠乳鼠成骨细胞在体外染氟培养48、72 h,OPG mRNA表达组间比较差异均有统计学意义(F值分别为333.48、808.34,P<0.05).在染氟48 h,1、2、4 mg/L组OPGmRNA表达(0.810±0.043、0.819±0.031、0.870±0.044)与对照组(0.800±0.040)比较,差异均有统计学意义(P<0.05);在染氟72 h,1、2、4 mg/L组(0.933±0.047、1.031±0.051、1.240±0.062)高于对照组(0.805±0.020),差异均有统计学意义(P<0.05);OPGmRNA表达,72 h均高于48 h,染氟剂量和染氟时间有交互作用(F=204.16,P<0.05).在染氟培养48、72 h,OPG蛋白表达组间比较差异均有统计学意义(F值分别为7.49、41.31,P<0.05);组间两两比较,仅4 mg/L组(0.228±0.014、0.277±0.048)与对照组(0.205±0.012、0.229±0.010)比较,差异有统计学意义(P<0.05);OPG蛋白表达虽然72 h均高于48 h,但染氟剂量和染氟时间无交互作用(F=1.21,P>0.05).结论 氟可促进成骨细胞OPG mRNA和蛋白表达,这种作用与染氟的剂量以及作用时间有关.     

一氧化氮在砷中毒中作用的研究

目的探讨砷对机体ＮＯ的影响及ＮＯ在砷中毒发病中的作用。方法采用动物实验和人群调查方法。结果慢性染砷实验中,小鼠血清、肾脏ＮＯ２－／ＮＯ３－含量中、高剂量组显著降低,肝脏、心脏ＮＯ２－／ＮＯ３－含量各剂量组显著降低,且呈剂量—效应关系。全血ＧＳＨ含量低剂量组显著升高,中、高剂量组显著降低,肝脏、心脏ＧＳＨ含量各剂量组均显著下降,肾脏ＧＳＨ含量中、高剂量组显著降低。红细胞、心脏ＳＯＤ活性中、高剂量组显著下降,肝脏ＳＯＤ活性低剂量组显著升高,中、高剂量组显著下降,肾脏各剂量组未见显著差异。人群调查,砷病区病人血中ＮＯ２－／ＮＯ３－、ＧＳＨ含量显著低于非病区健康人,且二者呈正相关,红细胞ＳＯＤ活性未见显著差异。结论砷可导致ＮＯ含量的下降。砷可导致ＧＳＨ含量和ＳＯＤ活性的变化,进而影响ＮＯ合成和代谢     

锌对砷中毒小鼠肝脏Fas和FasL表达影响的实验研究

目的 探讨砷对小鼠肝脏Fas、FasL表达的影响及锌的拈抗作用.方法 选用健康昆明种小鼠60只,按体质量分成5组:阴性对照组(不给砷也不给锌),单纯给砷组(55 mg/L NaAsO2溶液),砷+低锌组、砷+中锌组、砷+高锌组均给55 mg/L NaAsO2溶液,并分别给20、40、80 mg/L ZnSO4溶液.经口连续灌胃(按体质量灌胃量为0.02 ml/g.1次/d)6周,待实验结束后处死小鼠,采用免疫组化法测定小鼠肝脏巾凋亡因子Fas、FasL表达.结果 干预组随着给锌量的增加,肝脏Fas、FasL的表达率逐渐降低.低、中锌+砷组的Fas的表达率[83.33%(10/12)、50%(6/12)]、FasL表达率[66.67%(8/12)、41.67%(5/12)]与阴性对照组Fas[8.33%(1/12)]、FasL[0.00%(0/12)]表达率比较,差异有统计学意义(P均<0.05);中、高锌+砷组的Fas的表达率[50%(6/12)、25%(3/12)]与阳性对照组[8.33%(1/12)]比较,差异有统计学意义(P均<0.01),中、高锌+砷组的FasL的表达率[41.67%(5/12)、16.67%(2/12)]与阳性对照组[91.67%(11/12)]比较,差异有统计学意义(P均<0.05).结论 砷可使小鼠肝脏Fas、FasL的表达率增高,促进肝脏的凋亡,锌可拮抗砷对细胞凋亡的作用.

Abstract：

Objective To investigate the effects of arsenic on liver Fas/FasL expression in mice and to observe the antagonize effect of zinc. Methods Sixty health Kunming mice were divided into five groups according to their body mass: negative control group(no arsenic and no zinc), arsenic group(55 mg/L NaAsO2 solution), low dose zinc intervention group(55 mg/L NaAsO2 solution and 20 mg/L ZnSO4 solution), middle dose zinc intervention group (55 mg/L NaAsO2 solution and 40 mg/L ZnSO4 solution), and high dose zinc intervention group (55 mg/LNaAsO2 solution and 80 mg/L ZnSO4 solution). Everyday the solution was given by oral gavage at a dose of 0.02 ml/g body weight for six weeks. The expression of Fas/FasL in mice liver was examined by immunohistochemistry.Results With the amount of zinc increasing, the expression of both Fas and FasL in mice liver decreased gradually. The expression rates of Fas[83.33%(10/12), 50%(6/12)] and FasL[66.67%(8/12), 41.67%(5/12)]in low dose zinc intervention group and middle dose zinc intervention group, respectively, were different from the expression rate of Fas[8.33%(1/12)] and FasL[0.00%(0/12)] in the negative control group(all P < 0.05). The Fas expression rate of middle dose zinc intervention group[50%(6/12)] and the high dose zinc intervention group [25%(3/12)] was compared with the arsenic group[8.33%(1/12)], and the difference was statistically significant (all P < 0.01 ). The FasL expression rate of the middle dose zinc intervention group [41.67% (5/12 )] and the high dose zinc intervention group[16.67%(2/12)] was compared with positive control group[91.67%(11/12)], and the difference was statistically significant (all P < 0.05). Conclusions Arsenic can increase the expression of Fas and FasL in mice liver, and promote apoptosis in liver, zinc may antagonize the effect of arsenic.     

多药耐药相关蛋白1与人肝细胞耐砷性关系的研究

目的探讨多药耐药相关蛋白1（MRP1）与人肝细胞（L-02）耐砷性之间的关系。方法采用人肝细胞L-02细胞株,噻唑兰法（MTT）进行24h急性NaAsO2毒性试验,选择细胞生存率在90％～95％时的NaAsO2浓度为诱导浓度,同时设1组不加砷诱导的L-02细胞作为同步对照。置于细胞培养箱中37℃,5％CO2常规培养6周,定期换液传代,每周用MTT法计算半数致死浓度（LC50）及细胞的生存率;6周后,将加砷诱导的L-02肝细胞与同步对照细胞在培养基中培养至细胞80％融合,然后进行24h急性砷毒性试验（NaAsO2终浓度为0、2．5、5．0、10μmol／L）;采用real-time PCR检测细胞内MRP1mRNA的表达情况;MTT法检测细胞的存活率;石墨炉原子吸收光谱法检测各组细胞内总砷浓度。结果实验组细胞MRP1mRNA的表达明显高于对照组（P〈0．05）;24h急性砷毒性试验中,实验组不同砷浓度下细胞生存率和LC50明显高于对照组（均P〈0．001）;实验组细胞内总砷浓度明显较同步对照组低（P〈0．001）。结论L-02肝细胞具有可诱导的对砷的耐受性,其耐砷性与MRP1高表达有关。     

Choleretic effect of inchinkoto, a herbal medicine, on livers of patients with biliary obstruction due to bile duct carcinoma

Aim:  To investigate the choleretic effects of inchinkoto (ICKT) on livers of patients with biliary obstruction due to bile duct carcinoma.
Methods:  Twenty-seven patients with bile duct carcinoma who were due to undergo biliary drainage and subsequent major hepatectomy were randomly assigned to preoperative ICKT ( n  = 13) or untreated ( n  = 14) groups. ICKT was administered from the day of admission until one day before surgery. Changes in bile constituents, expression of multidrug resistance-associated protein (MRP) 2, MRP3 and MRP4 in the liver, and the incidence of postoperative complications were included as end-points.
Results:  The biliary concentration of total bilirubin was significantly increased after administration of ICKT (23.7 ± 2.8 mg/dL before ICKT; 34.0 ± 4.0 mg/dL after ICKT, P  < 0.05). The biliary concentration of total bile acids was also significantly increased. Protein levels of MRP2 and MRP3 in the crude plasma membrane fraction of livers of treated patients were significantly higher than those without treatment. MRP2 staining in the livers of patients without ICKT treatment was weak and diffuse around the bile canaliculi, whereas staining in patients with ICKT treatment was strong and restricted to the bile canaliculi.
Conclusion:  ICKT exerts a choleretic effect on the livers of patients with biliary obstruction. This beneficial effect was associated with increased expression of MRP2. ICKT thus has therapeutic potential for treatment for obstructive cholestasis due to bile duct carcinoma.     

The expression levels of plasma membrane transporters in the cholestatic liver of patients undergoing biliary drainage and their association with the impairment of biliary secretory function

OBJECTIVES: Percutaneous transhepatic biliary drainage (PTBD) has been believed to reduce hyperbilirubinemia in patients with obstructive cholestasis and to lessen liver injury through bile acid retention. The efficacy may be closely related to the capability of cholestatic liver to produce and secrete bile, which in turn depends on the expressions and functional activities of plasma membrane transporters in the liver. The aim of the present study was to determine the expression levels of these transporters in the cholestatic liver of patients undergoing PTBD. METHODS: A total of 24 patients who had experienced obstructive cholestasis and had undergone preoperative PTBD were included in the study. Liver biopsy specimens were analyzed to determine the expression levels of the multidrug resistance-associated proteins (MRP) MRP2 and MRP3 and the canalicular bile salt export pump BSEP in the liver. RESULTS: The messenger RNA (mRNA) levels of MRP2, the canalicular bilirubin conjugate export pump, and bile salt export pump (BSEP) were unchanged in liver specimens from the 14 patients well drained by PTBD but were reduced in specimens from the 10 patients poorly drained, compared to the levels of control subjects. Immunostainings of MRP2 and BSEP outlined the canalicular membrane domain but seemed fuzzy to varying degrees in specimens obtained from cholestatic liver, especially in specimens from liver that had been poorly drained, in contrast to the linear and intense localization in the liver of control subjects, correlating with the impaired bilirubin conjugate and bile acid secretion. The mRNA of MRP3, functioning as an inducible export pump for bilirubin conjugate and bile acid, was expressed not only in the cholestatic liver but also in the liver of control subjects, and the mRNA level was increased in specimens from both the cholestatic liver that had been well drained and from the liver that had been poorly drained. Immunostaining of MRP3 was observed in the epithelia of intrahepatic bile ducts in the liver of both control subjects and cholestatic patients, and in the epithelia of proliferated bile ductules and the hepatocytes surrounding the portal tracts in the cholestatic liver. CONCLUSIONS: From the results of the present study, it is concluded that 1) the mRNA and immunohistochemical expression levels of MRP2 and BSEP may be altered in the cholestatic liver of patients undergoing PTBD; 2) both the decreased mRNA levels and the diminished canalicular membrane localization may be associated with the impairment of bile formation and secretion, i.e., the efficacy of PTBD; and 3) upregulated MRP3 in the cholangiocytes and hepatocytes may play a significant role in bile acid transport in the cholestatic hepatobiliary system.     

慢性砷暴露人骨髓间充质干细胞的生物学特性观察

目的 观察抗砷细胞模型慢性砷暴露人骨髓间充质干细胞(CAsE-hFBMSCs)的生物学特性,探讨长期低剂量砷暴露对人骨髓间充质干细胞(hFBMSCs)的影响.方法 常规条件培养hFBMSCs,48 h急性砷毒性实验MTS法检测不同剂量砷刺激条件下[0(对照)、0.25、0.50、1.00、2.00、4.00、8.00、20.00、40.00、80.00、120.00μmol/L]第2代(P2)hFBMSCs细胞生存率,根据检测结果用1.00μmol/L亚砷酸钠刺激hFBMSCs 14周,建立抗砷细胞模型CAsE-hFBMSCs,作为实验组,同时设立对照组,并采用荧光激活细胞分选术鉴定诱导前后细胞表型,流式细胞术检测第2、9、16代(P2、P9、P16)细胞周期,软琼脂克隆形成实验检测细胞是否发生恶性转化.结果 <10μmol/L时,急性砷刺激促进hFBMSCs增殖,而≥10μmol/L时,急性砷刺激则抑制细胞的生长.14周时实验组半数致死剂量(LC_(50))为(89.42±0.64)μmol/L,对照组为(52.48±0.71)μmol/L,组间比较差异有统计学意义(t=123.89,P<0.05);抗砷细胞模型CAsE-hFBMSCs的细胞表型CD34、CD45表达阴性,CD29、CD90、CD166表达阳性,与对照组比较细胞表型未见明显改变;CAsE-hFBMSCs的细胞周期变化较大,与对照组[(8.44±0.45)%、(9.14±0.14)%、(82.42±0.60)%]比较,P2实验组G2/M期[(17.72±5.47)%]和S期细胞[(25.34±3.36)%]增加,G0/G1期细胞减少[(56.96±8.83)%],P16细胞周期恢复至与对照组相近的水平:软琼脂克隆形成实验中,抗砷细胞CAsE-hFBMSCs未见克隆形成.结论 长期低剂量砷刺激对hFBMSCs生物学特性无明显影响.     

MRP2和NF-κB在阻塞性黄疸大鼠肝细胞中表达变化及意义

目的研究MRP2及NF—κB在阻塞性黄疸肝细胞中表达的变化,进一步阐明二者的关系。方法健康sD大鼠64只,将大鼠随机分为2组：假手术组（SHAM组）、胆总管结扎组（CBDL组）。建立大鼠阻塞性黄疸模型,SHAM组仅游离胆总管,不结扎离断胆总管。CBDL组予以结扎并切断胆总管。分别于术后的第3、7、14、21d各处死8只,用免疫组化法观察上述两组肝组织MRP2、NF—κBp65蛋白表达。结果SHAM组在第3、7、14、21dMRP2高水平表达,而NF—κBp65低水平表达。CBDL组大鼠NF—κBp65水平表达随着时间的延长而显著增高,MRP2低水平表达随着时间的延长而显著下降。结论阻塞性黄疸时,NF—κB的激活可能是引起MRP2表达下降的重要原因,NF—κB对MRP2调控可能起重要作用。     

Developmental expression of canalicular transporter genes in human liver

BACKGROUND/AIMS: BSEP, MRP2, and MDR3 are major hepatic canalicular transporters mediating bile secretion. Their expression in human liver during development has not been reported. METHODS: Human liver samples from fetus at gestational age 14-20 weeks, adult livers and liver samples of infants with biliary atresia were tested for mRNA expression of BSEP, MDR3, MRP2, NTCP, FIC1, and FXR genes by using real-time RT-PCR. Immunohistochemical staining of BSEP, MDR3, and MRP2 were performed on fetal and adult livers. RESULTS: All the genes tested were expressed at mid-gestational age. MDR3 and NTCP showed significant lower levels in fetal livers compared to adults. In patients with biliary atresia, all the genes tested showed higher mean expression levels than adults except for NTCP, but not statistically significant. The immunohistochemical staining of MRP2 in fetal liver was canalicular, BSEP showed both intracellular and canalicular staining, and MDR3 staining was faint, only occasional canalicular pattern could be seen. CONCLUSIONS: The major canalicular transporter genes are expressed at mid-gestational stage during human fetal development, but are different in expression level and targeting pattern, indicating differential regulation and maturation.