Toll样受体9在狼疮肾炎患者外周血B淋巴细胞和肾组织中的表达及临床意义

目的 探讨Toll样受体9(TLR9)在狼疮肾炎(LN)患者外周血B淋巴细胞和肾组织的表达及其临床意义.方法 分别采用细胞内流式细胞术和免疫组织化学方法对对照组和LN患者外周血B淋巴细胞及肾组织中TLR9表达情况进行比较,并对TLR9表达与临床指标相关性进行分析.结果 活动期LN患者外周血CD19+B淋巴细胞TLR9的表达率高于非活动期LN患者及对照组(P＜0.01),且其表达率与SLE疾病活动指数(SLEDAI)评分、抗双链DNA(dsDNA)抗体滴度呈正相关(P＜0.01).LN患者肾组织中肾小球、肾小管间质TLR9表达均较健康组织增高(P＜0.01),其中国际肾脏病学会/肾脏病理学会(ISN/RPS)的Ⅳ型LN肾组织TLR9表达水平较Ⅱ、Ⅲ、Ⅴ型升高(P＜0.01),且肾小球TLR9表达水平与肾小球细胞增殖程度、肾脏病理活动指数、尿蛋白定量(24 h)呈正相关(P＜0.01).与估计的肾小球滤过率(eGFR)呈负相关(P＜0.01);肾小管间质TLR9表达水平与肾脏病理活动指数、肾小管问质损害程度、尿蛋白定量(24 h)也呈正相关(P＜0.01),与eGFR呈负相关(P＜0.01).结论 TLR9参与了LN患者B淋巴细胞的异常活化及肾脏病理损伤过程.     

狼疮肾炎患者肾组织中载脂蛋白E的表达及意义

目的 了解狼疮肾炎(LN)肾组织中载脂蛋白E(APOE)的表达,并探讨其与LN肾脏病理改变和肾功能损害的关系.方法 用免疫组织化学方法对40例LN患者.肾组织APOE的表达进行检测,并对其与LN的肾脏病理改变和肾功能损害的相关性进行分析.结果 健康肾脏组织有较弱的ApoE表达;LN肾组织APOE的表达较健康肾组织明显增高[肾小球(31.2±11.0)与(3.0±1.6);肾小管(71.64±8.90)与(0.60±0.06)],并且Ⅳ型LN患者肾组织APOE表达量高于非Ⅳ型LN患者[肾小球(38.4±4.5)与(18.0±4.6);肾小管间质(77.6±2.5)与(60.0±3.2)];Spearman相关分析显示LN肾组织ApoE的表达与LN肾脏病理活动指数和肾功能呈显著相关(P＜0.01).结论 LN患者肾脏组织中ApoE的表达增高,尤以Ⅳ型LN为著,其表达量可反映LN肾脏活动性病变及肾脏损害情况.     

狼疮肾炎肾组织核转录因子-κB单核细胞趋化因子-1与CD68的表达及相关分析

目的 对狼疮肾炎(LN)肾组织进行核转录因子-κB(NF-κB)、单核细胞趋化因子(MCP-1)及巨噬细胞特异性抗体(CD68)检测,探讨其与LN肾脏病理及临床指标的关系.方法 应用免疫组织化学二步法检测49例LN肾组织NF-κB、MCP-1及CD68,原位杂交法检测49例LN肾组织NF-κB,并与肾脏病理及临床指标进行分析.结果 ①LN肾组织中NF-κB、MCP-1及CD68表达均比对照组显著升高(P＜0.01);其中Ⅳ型LN肾组织中NF-κB、MCP-1及CD68的表达比非Ⅳ型LN及对照组明显增加(P＜0.01,P＜0.05).原位杂交与免疫组织化学法检测NF-κB差异无统计学意义(P＞0.05).②LN肾组织中,NF-κB的表达与肾组织活动性指数、尿蛋白定量(24 h)及血清肌酐均高于对照组,且三者之间差异有统计学意义(P＜0.01,P＜0.01,P＜0.05);MCP-1与CD68的表达在肾小球和肾小管中仅与肾组织活动性指数(r=0.447,0.532,P＜0.05)、尿蛋白定量(24 h)(r=0.357,0.368,P＜0.05)呈正相关,而与血肌酐之间差异无统计学意义(P＞0.05).结论 NF-κB通过活化MCP-1进一步诱导巨噬细胞可能是LN肾脏损害的原因之一,NF-κB信号途径有望成为抑制巨噬细胞在肾脏局部浸润和增生的一个新的治疗靶点.     

狼疮性肾炎患者肾组织白介素-10的表达与巨噬细胞浸润的关系及其意义

目的:研究白介素-10(IL-10)在狼疮性肾炎(LN)患者肾组织中表达及巨噬细胞的浸润与肾脏病理和功能损害的关系.方法:采用微波免疫组织化学双重染色法测定正常肾组织和20例LN肾组织巨噬细胞浸润和IL-10的表达及与狼疮肾组织活动指数、肾脏组织学指标和功能损害的相关关系.结果:①正常肾小球及肾小管间质仅个别巨噬细胞存在,几乎无IL-10表达,而在各型LN,肾小球和肾小管间质见浸润的巨噬细胞及IL-10阳性细胞明显增多,且病变严重的Ⅳ型LN尤为明显.②肾小球KP1 巨噬细胞与肾小球细胞数增多,LN活动指数、慢性指数均呈显著相关,IL-10 细胞数也与这些指标相关.肾小管间质中KP1 巨噬细胞数及IL-10 细胞与小管损害、狼疮肾组织活动指数和慢性指数(特别是慢性指数)显著正相关.③肾小球肾小管间质中KP1 细胞数与24h尿蛋白排泄量、尿NAG排泄量有关,IL-10 细胞也与上述指标有关,而与血清学免疫指标无关.④肾小球KP1 与IL-10 细胞呈高度相关性,r=0.79(P＜0.001),小管间质中KP1 与IL-10 细胞也呈高度相关性:r=0.76(P＜0.001).70%KP1 细胞聚集在肾组织损害严重部位,同时IL-10阳性染色物主要存在于KP1 细胞中.结论:肾脏IL-10表达增加与巨噬细胞浸润在LN病理与功能损害中起重要作用.     

中性粒细胞明胶酶相关脂质运载蛋白单核细胞趋化蛋白-1及肿瘤坏死因子样凋亡的微弱诱导剂在狼疮肾炎中的临床意义

目的 探讨狼疮肾炎患者体液中性粒细胞明胶酶相关脂质运载蛋白(NGAL)、单核细胞趋化蛋白-1(MCP-1)和肿瘤坏死因子样凋亡的微弱诱导剂(TWEAK)水平的临床意义.方法 采用酶联免疫吸附法检测51例系统性红斑狼疮(SLE)患者的血清和尿中NGAL、MCP-1和TWEAK水平,其中狼疮肾炎27例、非狼疮肾炎24例.并采集临床资料.采用t检验、Mann-Whitney U检验、Pearson和Spearman 秩相关分析进行统计学分析.结果 ①血清NGAL水平狼疮肾炎组为(122±70)ng/ml高于非狼疮肾炎组(56±34)ng/ml (P＜0.01).血清NGAL与整体SLE疾病活动指数(SLEDAI)评分、肾外SLEDAI评分、肾SLEDAI评分、尿蛋白定量(24 h)、血肌酐水平呈正相关(r=-0.408、0.431、0.339、0.403和0.585,P均＜0.05),与血清C3呈负相关(r=-0.396,P＜0.01).尿NGAL水平与慢性指数呈正相关(r=0.719,P＜0.01).②血清MCP-1狼疮肾炎组(284±1 13) pg/ml高于非狼疮肾炎组(173±69)pg/ml,P＜0.01;尿MCP-1水平在狼疮肾炎组M=1154、(P75 41 178,P5 341)pg/mg肌酐高于非狼疮肾炎组M=456、(P75 714,P25 114)pg/mg 肌酐(P＜0.01).血清MCP-1水平与整体SLEDAI评分、肾外SLEDAI评分、肾SLEDAI评分、尿蛋白定量(24 h)、血肌酐水平均呈正相关(r=0.340、0.416、0.385、0.574和0.654,P均＜0.05),与血清C3水平呈负相关(r=-0.458,P＜0.01).尿MCP-1水平与全身SLEDAI评分、肾外SLEDAI评分、肾SLEDAI评分、血清抗dsDNA抗体、尿蛋白定量(24 h)、血肌酐水平均呈正相关(rs=0.448、0.429、0.459、0.412、0.375和0.419,P均＜0.05).尿MCP-1水平与慢性指数呈正相关(r=0.689,P＜0.01).③6例狼疮肾炎尿中可检测到TWEAK.结论 尿NGAL和MCP-1水平升高提示肾脏慢性损伤.血NGAL和血、尿MCP-1水平在狼疮肾炎活动时均明显升高,可用于监测狼疮肾炎病情活动.TWEAK临床意义需要进一步研究.     

Defensinα1-3在狼疮肾炎患者肾组织中的表达及意义

目的探讨defensin α1-3在不同病理类型狼疮肾炎(LN)肾组织中的表达及意义.方法收集47例LN患者的肾活检的肾组织,包括世界卫生组织(WHO)Ⅱ型7例、Ⅲ型6例、Ⅳ型29例、Ⅴ型5例,以及11例泌尿外科手术切除的远离肿瘤的肾皮质区的肾组织作为正常对照,使用defensin α1-3单克隆抗体进行免疫组织化学染色,定量分析defensin α1-3在各个病理类型LN肾组织及正常肾组织中的表达及分布,并分别与相应的增殖细胞核抗原(PCNA)、肾小球病变活动指数(GAI)和肾间质病变活动指数(TIAI)进行相关性的分析.结果各个病理类型LN肾组织及正常肾组织的肾小球、肾间质中均有defensin α1-3的表达,其中肾小球的defensin α 1-3的表达在Ⅱ、Ⅲ、Ⅳ型LN组明显高于正常对照组,肾间质中defensin α1-3表达在Ⅳ型LN组高于其他类型的各组及正常对照组.Ⅳ型LN组的肾小球区域的defensin α1-3的表达与Ⅳ型LN组的肾小球区域的PCNA阳性细胞数(r=0.785,P＜0.05)、及GAI评分之间(r=0.749,P＜0.05)呈正性线性相关.Ⅳ型LN组的肾间质的defensin α 1-3的表达与其TIAI之间呈正性线性相关(r=0.767,P＜0.01).结论defensin α 1-3可能参与了Ⅳ型LN的发病.     

PPARγ在狼疮肾炎患者肾组织中的表达及其意义

目的观察过氧化物酶体增殖物激活受体γ(PPARγ)在狼疮肾炎(LN)不同病变类型肾组织中的表达及其与肾脏病理改变之间的关系,探讨PPARγ在LN发病机制中的可能作用。方法根据临床及肾活检病理诊断,选择LN不同病变类型患者作为研究对象,其中Ⅱ型(6例),Ⅳ型(8例),Ⅴ型(7例)。以肾脏肿瘤切除术中远离肿瘤部位的正常肾组织(6例)为对照。光镜下观察并计数肾脏病变活动指数、间质病变指数；用免疫组织化学方法对各例肾组织PPARγ的表达进行检测,并对其与肾脏病变的相关性进行分析。结果LN患者肾组织。肾小管、肾小球及间质浸润细胞中PPARγ脚表达较正常对照组均显著上调,其中Ⅳ型LN肾组织肾小管、肾小球及间质浸润细胞PPARγ表达显著高于Ⅱ型、Ⅴ型；肾小球PPARγ染色阳性细胞数目与肾脏病理活动积分及间质病变指数之间呈显著正相关(r=0．94；P＜0．01)。结论PPARγ在LN肾组织肾小管、肾小球及间质浸润细胞高表达对于限制LN肾脏病变发展可能具有重要作用     

乙型肝炎病毒相关性肾炎中Toll样受体4沉积特点及其意义

目的 观察HBV相关性肾炎(HBV-GN)患者肾组织Toll样受体4(TLR4)的表达及分布情况,探讨TLR4与HBV-GN肾组织病变及临床表现的关系.方法 采用免疫组化法检测经肾活检确诊的48例HBV-GN患者及154例非HBV-GN患者的肾组织标本,观察肾组织TLR4的分布特征,明确TLR4与HBsAg的分布关系,结合病史资料,统计并分析其与病理类型,肾小球、肾小管病变程度,肾间质炎性细胞浸润程度及血清HBV等临床指标之间的关系.结果 TLR4以棕红色颗粒状物主要沉积在HBV-GN组患者的肾小管及间质中,且与HBsAg的分布基本一致.HBV-GN组TLR4阳性率及阳性积分均高于非HBV-GN组(P＜0.05).在系膜增生性肾小球肾炎组、局灶节段性肾小球硬化症组TLR4阳性积分略高,但差异无统计学意义(P＞0.05);肾小管病变程度与TLR4的表达显著相关(r =0.748,P＜0.001),且随肾小管萎缩、炎性细胞浸润及间质纤维化的程度加重而升高(r分别为0.577、0.684、0.569,P值均＜0.001),但与肾小球的病变程度无显著相关性(r=0.293,P=0.053).回归分析发现,TLR4表达随血压的升高、GFR的下降、高敏C反应蛋白的增加及24h尿蛋白定量的增多而增加(R2分别为0.869、0.784、0.979、0.615),其余临床指标均无统计学意义.结论 TLR4在HBV-GN肾组织中有异常表达,主要分布于肾小管上皮细胞及间质组织,其分布与HBsAg分布基本一致,表达水平与炎性细胞浸润、肾间质病变及肾功能变化密切相关.故推测,HBV-GN肾组织中的HBV可促进TLR4的异常表达,后者可能参与了HBV-GN的肾脏组织病变的进展.     

慢性移植物抗宿主病狼疮样小鼠肾组织集落刺激因子-1表达及泼尼松的调控作用

目的　观察集落刺激因子 1(CSF 1)在慢性移植物抗宿主病 (GvHD)狼疮样小鼠肾组织中的表达及泼尼松的调控作用。方法　GvHD狼疮样小鼠模型参照有关文献建立。按随机设计原则将模型动物分两组即模型组 (A组 )和泼尼松治疗组 (B组 ) ,另设正常对照组 (C组 ) ,每组均有 8只动物。CSF 1蛋白检测采用免疫组织化学方法 ,mRNA表达采用原位杂交技术检测。结果　①A组肾组织中CSF 1蛋白表达显著高于C组 (P <0 0 5 ) ,肾小球、肾小管 间质中均有表达 ,尤以肾小管 间质表达更为丰富 ,而B组CSF 1在肾小球、肾小管 间质表达均显著减少 (P <0 0 5 )。②A组肾组织中CSF 1mRNA表达显著高于C组 (P <0 0 5 ) ,经泼尼松治疗后CSF 1mRNA受到明显抑制。③A组中CSF 1蛋白和mRNA表达呈正相关关系 (r =0 6 15 ,P <0 0 5 ) ,A组肾组织中CSF 1蛋白表达和 2 4h尿蛋白排泄呈正相关 (r =0 5 78,P <0 0 5 )。结论　慢性GvHD狼疮样小鼠肾组织特别在肾小管 间质中CSF 1蛋白和mRNA表达均显著增高 ,泼尼松在改善肾组织病理改变的同时 ,对CSF 1表达亦起抑制作用 ,提示CSF 1参与狼疮肾炎的发生和发展 ,泼尼松抑制CSF 1过度表达可能是其在狼疮肾炎中发挥治疗作用的机制之一。     

狼疮性肾炎肾组织CD80与CD86的表达及其意义

目的 研究T淋巴细胞活化分子CD80与CD86在狼疮性肾炎患者肾组织中的表达变化及其与临床指标之间的相关性.方法 肾组织标本来源于2004年12月-2008年10月哈尔滨医科大学附属第一医院住院患者,其中女性44例,男性5例,年龄(28±16)岁.用免疫组织化学法检测狼疮性肾炎及微小病变患者肾组织中的CD80与CD86的表达情况,免疫比浊法检测24 h尿蛋白定量,全自动生化分析仪检测血尿素氮、血肌酐及血浆白蛋白等临床指标.结果 CD80在肾小管上皮细胞、肾间质有阳性表达,在肾小球无表达,而CD86在肾小管上皮细胞、肾间质及肾小球均有表达.CD80与CD86在肾小管间质中的表达水平随着狼疮性肾炎患者肾小管间质病变程度的加重而增强(r=0.574,P<0.001;r=0.534,P<0.001),且CD80的表达与CD86的表达成正相关.CD80与CDB6的表达与患者系统性红斑狼疮疾病活动指数积分(r=0.319,P=0.011;r=0.324,P=0.011)、24 h尿蛋白定量(r=0.424,P=0.003;r:0.408,P=0.004)、肌酐清除率(r=-0.535,P=0.000;r=-0.543,P=0.000)及抗dsDNA抗体滴度(r=0.353,P=0.012;r=0.359,P=0.013)具有明显相关性.结论 CD80与CD86在狼疮性肾炎患者肾组织中的表达水平与肾间质病变具有明显相关性,并且与狼疮性肾炎患者肾活检时的肾功能及活动性明显相关.     

妊娠时间与肺结核复发的相关性研究及诊治对策

目的 分析肺结核史患者妊娠时间和肺结核复发间相关性.方法 选取我院收治的有肺结核史的妊娠妇女576例作为研究对象,对其妊娠前肺结核治疗、治愈后妊娠时间、妊娠后复发肺结核等进行分析,总结有肺结核史育龄女性的妊娠时间和肺结核复发之间的关系.结果 肺结核治愈后不同时间段妊娠者的结核复发率比较,差异具有显著性（P＜0.05）,停药后间隔时间越久妊娠,肺结核复发的几率越小.结论 加强孕期痰菌检查,及早发现复发肺结核,提高母婴安全.     

我国骨关节结核诊治的回顾与展望

骨关节结核是危害人们健康的严重感染性疾病,近95％由他处结核病继发而来.罹患骨关节结核疾病后几乎均将致残,严重影响人们的健康、工作和生活.建国以来在党和国家的关心和支持下,骨关节结核的诊治水平取得了长足进步.时至今日,由于多种原因,学科发展和被重视程度受到一定的制约,同整个医疗行业的发展不相适应.回顾过去,展望未来,我们需要重新审视骨关节结核的诊治方法,努力推进骨关节结核诊疗技术的科学发展.     

Effect of phosphorylation of MAPK and Stat3 and expression of c-fos and c-jun proteins on hepatocarcinogenesis and their clinical significance

AIM To study the effect of phosphorylation ofMAPK and Stat3 and the expression of c-fos andc-jun proteins on hepatocellular carcinogenesisand their clinical significance.METHODS SP immunohistochemistry was usedto detect the expression of p42/44~(MAPK), p-Stat3,c-fos and c-jun proteins in 55 hepatocellularcarcinomas (HCC) and their surrounding livertissues.RESULTS The positive rates and expressionlevels of p42/44~(MAPK), p-Stat3, c-fos and c-junproteins in HCCs were significantly higher thanthose in pericarcinomatous liver tissues (PCLT).A positive correlation was observed between theexpression of p42/44~(MAPK) and c-fos proteins, andbetween p-Stat3 and c-jun, but there was nosignificant correlation between P42/44~(MAPK) and p-Stat3 in HCCs and their surrounding livertissues.CONCLUSION The abnormalities of Ras/Raf/MAPK and JAKs/ Stat3 cascade reaction maycontribute to malignant transformation ofhepatocytes. Hepatocytes which are positive forp42/ 44~(MAPK), c-fos or c-jun proteins may bepotential malignant pre-cancerous cells.Activation of MAPK and Stat3 proteins may be anearly event in hepatocellular carcinogenesis.     

Overweight and Obesity and Progression of ADPKD

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Replication and Inhibitors of Enteroviruses and Parechoviruses

The Enterovirus (EV) and Parechovirus genera of the picornavirus family include many important human pathogens, including poliovirus, rhinovirus, EV-A71, EV-D68, and human parechoviruses (HPeV). They cause a wide variety of diseases, ranging from a simple common cold to life-threatening diseases such as encephalitis and myocarditis. At the moment, no antiviral therapy is available against these viruses and it is not feasible to develop vaccines against all EVs and HPeVs due to the great number of serotypes. Therefore, a lot of effort is being invested in the development of antiviral drugs. Both viral proteins and host proteins essential for virus replication can be used as targets for virus inhibitors. As such, a good understanding of the complex process of virus replication is pivotal in the design of antiviral strategies goes hand in hand with a good understanding of the complex process of virus replication. In this review, we will give an overview of the current state of knowledge of EV and HPeV replication and how this can be inhibited by small-molecule inhibitors.     

Effect of phosphorylation of MAPK and Stat3 and expression of c-fas and c-jun proteins on hepatocarcinogenesis and their clinical significance

AIM To study the effect of phosphorylation ofMAPK and Stat3 and the expression of c-fos andc-jun proteins on hepatocellular carcinogenesisand their clinical significance.METHODS SP immunohistochemistry was usedto detect the expression of p42/44MAPK, p-Stat3,c-fos and c-jun proteins in 55 hepatocellularcarcinomas (HCC) and their surrounding livertissues.RESULTS The positive rates and expressionlevels of p42/44MAPK, p-Stat3, c-fos and c-junproteins in HCCs were significantly higher thanthose in pericarcinomatous liver tissues (PCLT).A positive correlation was observed between theexpression of p42/44MAPK and c-fos proteins, andbetween p-Stat3 and c-jun, but there was nosignificant correlation between p42/44MAPK and p-Stat3 in HCCs and their surrounding livertissues.CONCLUSION The abnormalities of Ras/Rat/MAPK and JAKs/ Stat3 cascade reaction maycontribute to malignant transformation ofhepatocytes. Hepatocytes which are positive forp42/ 44MAPK, c-fos or c-jun proteins may bepotential malignant pre-cancerous cells.Activation of MAPK and Stat3 proteins may be anearly event in hepatocellular carcinogenesis.     

心电图、心电向量图与冠状动脉造影结果对比分析

目的:通过分析心电图(Electrocardiogram,ECG)和心电向量图(Vectorcardiogram,VCG)的改变与冠脉造影（CAG）结果进行对比,探讨ECG、VCG在冠状动脉病变中的诊断价值。方法: 选择2008年1月~2009年12月临床拟诊断为冠心病患者108例,行常规ECG、VCG检查,并于1周内进行CAG,对检查结果依据各自的诊断标准进行判定,以CAG为标准诊断法,利用四格表法,计算相关评价真实性的指标并进行比较。结果: ①VCG检测的灵敏度、特异度、准确度显著高于ECG(P<0.05,P<0．01)。②ECG、VCG阳性率与冠脉病变支数组间比较:在单支病变、双支病变中,VCG阳性率明显高于ECG(P<0.05),左主干或三支病变无统计学意义;组内比较:ECG组左主干或三支病变组较单支病变、双支病变阳性率高(P<0.05,P<0.01);VCG组左主干或三支病变组较单支病变阳性率高(P<0.05);与双支病变阳性率比较无统计学意义;③ECG、VCG阳性率与冠脉病变程度组间比较:冠脉病变狭窄50%～69%的VCG阳性率明显高于ECG (P<0.05),其他两组阳性率比较无统计学意义;组内比较:ECG组冠脉病变狭窄≥90%较50%～69%、70%～89%的阳性率高(P<0.05,P<0.01); VCG组狭窄≥90%较50%～69%阳性率高（P<0.01),其他无统计学意义。结论: VCG对冠心病检测价值显著高于ECG。     

Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence

Here we report the structural characterization of the product formed from the reaction between hydroethidine (HE) and superoxide (O(2)(.-)). By using mass spectral and NMR techniques, the chemical structure of this product was determined as 2-hydroxyethidium (2-OH-E(+)). By using an authentic standard, we developed an HPLC approach to detect and quantitate the reaction product of HE and O(2)(.-) formed in bovine aortic endothelial cells after treatment with menadione or antimycin A to induce intracellular reactive oxygen species. Concomitantly, we used a spin trap, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), to detect and identify the structure of reactive oxygen species formed. BMPO trapped the O(2)(.-) that formed extracellularly and was detected as the BMPO-OH adduct during use of the EPR technique. BMPO, being cell-permeable, inhibited the intracellular formation of 2-OH-E(+). However, the intracellular BMPO spin adduct was not detected. The definitive characterization of the reaction product of O(2)(.-) with HE described here forms the basis of an unambiguous assay for intracellular detection and quantitation of O(2)(.-). Analysis of the fluorescence characteristics of ethidium (E(+)) and 2-OH-E(+) strongly suggests that the currently available fluorescence methodology is not suitable for quantitating intracellular O(2)(.-). We conclude that the HPLC/fluorescence assay using HE as a probe is more suitable [corrected] for detecting intracellular O(2)(.-).     

Effects of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice

Non-invasive techniques to monitor stress hormones in small animals like mice offer several advantages and are highly demanded in laboratory as well as in field research. Since knowledge about the species-specific metabolism and excretion of glucocorticoids is essential to develop such a technique, we conducted radiometabolism studies in mice (Mus musculus f. domesticus, strain C57BL/6J). Each mouse was injected intraperitoneally with 740 kBq of 3H-labelled corticosterone and all voided urine and fecal samples were collected for five days. In a first experiment 16 animals (eight of each sex) received the injection at 9 a.m., while eight mice (four of each sex) were injected at 9 p.m. in a second experiment. In both experiments radioactive metabolites were recovered predominantly in the feces, although males excreted significantly higher proportions via the feces (about 73%) than females (about 53%). Peak radioactivity in the urine was detected within about 2h after injection, while in the feces peak concentrations were observed later (depending on the time of injection: about 10h postinjection in experiment 1 and about 4h postinjection in experiment 2, thus proving an effect of the time of day). The number and relative abundance of fecal [3H]corticosterone metabolites was determined by high performance liquid chromatography (HPLC). The HPLC separations revealed that corticosterone was extensively metabolized mainly to more polar substances. Regarding the types of metabolites formed, significant differences were found between males and females, but not between the experiments. Additionally, the immunoreactivity of these metabolites was assessed by screening the HPLC fractions with four enzyme immunoassays (EIA). However, only a newly established EIA for 5alpha-pregnane-3beta,11beta,21-triol-20-one (measuring corticosterone metabolites with a 5alpha-3beta,11beta-diol structure) detected several peaks of radioactive metabolites with high intensity in both sexes, while the other EIAs showed only minor immunoreactivity. Thus, our study for the first time provides substantial information about metabolism and excretion of corticosterone in urine and feces of mice and is the first demonstrating a significant impact of the animals' sex and the time of day. Based on these data it should be possible to monitor adrenocortical activity non-invasively in this species by measuring fecal corticosterone metabolites with the newly developed EIA. Since mice are extensively used in research world-wide, this could open new perspectives in various fields from ecology to behavioral endocrinology.     

大鼠骨髓间充质干细胞的分离培养和外源基因的导入

目的探讨绿色荧光蛋白基因转染骨髓间质干细胞的可行性。方法采用F icoll-PaqueTMP lus淋巴细胞分离液,根据细胞密度梯度原理,分离大鼠骨髓间充质干细胞(rM SC s)并进行体外原代培养和传代扩增,倒置相差显微镜观察细胞生长情况,免疫细胞化学法对其初步鉴定。流式细胞仪分析转染效率。结果原代和传代培养的细胞呈现梭形外观,具有较强的生长增殖能力;细胞均一表达CD44、CD54、CD106、CD29抗原。电穿孔法转染rM SC s转染率为32.8%±3%。结论采用比重为1.077 g/L的F icoll-PaqueTMP lus能分离获得大鼠骨髓间充质干细胞,经原代培养和传代培养能够迅速扩增。电穿孔法具有较高的介导外源基因表达于rM SC s的效率。