CIP4对转化生长因子β1诱导的人肾小管上皮细胞-间充质转分化的影响

目的 观察骨架调节蛋白CIP4（Cdc42 interacting protein 4）对转化生长因子β1（TGF-β1）诱导的人肾小管上皮细胞-间充质转分化（EMT）的影响,并探讨其产生的机制。 方法 10 ?滋g/L TGF-β1刺激72 h诱导人近端肾小管上皮细胞（HK-2细胞）向间充质转分化。Western印迹法检测各组细胞内E-cadherin和α-SMA蛋白的表达。倒置显微镜观察细胞形态的变化。根据GenBank人CIP4的完全cDNA序列,设计1条特异性干扰CIP4表达的RNA片段（CIP4-siRNA）和含野生型CIP4的重组真核表达质粒（pcDNA3.1-hCIP4）,利用lipofactamine 2000将其转染HK-2细胞。Western 印迹法检测对照组、TGF-β1刺激组、CIP4-siRNA转染组、pcDNA3.1-CIP4转染组细胞内CIP4、E-cadherin和α-SMA蛋白的表达,共聚焦显微镜观察 E-cadherin和α-SMA蛋白的分布改变;用PI3K-Akt特异性抑制剂渥曼青霉素（wortmannin） 1 μmol/L干预TGF-β1刺激的HK-2细胞48 h,Western 印迹法检测对照组和干预组CIP4表达的变化。 结果 TGF-β1干预后HK-2细胞E-cadherin蛋白表达显著减少（P ＜ 0.05）,α-SMA蛋白表达显著增多（P ＜ 0.05）,细胞形态由典型的上皮细胞向肌成纤维细胞转变,表明TGF-β1诱导肾小管上皮细胞EMT模型成功。CIP4-siRNA抑制TGF-β1诱导的HK-2细胞表达CIP4后,E-cadherin蛋白表达显著增多（P ＜ 0.05）,α-SMA蛋白表达显著减少（P ＜ 0.05）,部分逆转了上述TGF-β1诱导的肾小管上皮细胞EMT。pcDNA3.1-hCIP4转染使CIP4高表达后,HK-2细胞E-cadherin蛋白表达显著减少（P ＜ 0.05）,α-SMA蛋白表达显著增多（P ＜ 0.05）,诱导了肾小管上皮细胞EMT。用渥曼青霉素干预TGF-β1刺激的HK-2细胞48 h,CIP4可能蛋白表达显著减少（P ＜ 0.05）。 结论 TGF-β1通过PI3K-Akt途径上调CIP4表达,CIP4可能进一步参与TGF-β1诱导的肾小管上皮细胞EMT过程。     

虫草多糖对转化生长因子β1诱导人近端肾小管上皮细胞间充质转分化的影响

目的 探讨虫草多糖（Cp）对转化生长因子β1（TGF-β1）诱导人近端肾小管上皮细胞间充质转分化的影响。 方法 用四甲基偶氮唑盐（MTT）比色法检测不同浓度的Cp(0、0.01、0.1、1、5、10 g/L)对人近端肾小管上皮细胞(HK-2) 增殖的作用。用不同浓度的 TGF-β1（0、0.1、0.5、1、5、10 μg/L）作用HK-2细胞48 h及5 μg/L的TGF-β1作用不同时间（0、12、24、48和72 ｈ），以实时定量PCR和Western印迹方法检测α平滑肌肌动蛋白（α-SMA）、钙黏蛋白（E-cadherin）、纤连蛋白（FN）的表达。用递增浓度的Cp(1、5、10 g/L)预处理HK-2细胞24 h后，观察其对TGF-β1效应的干预作用。 结果 Cp以剂量依赖方式促进HK-2细胞增殖（P < 0.05）。TGF-β1无论在转录水平还是蛋白水平皆能诱导HK-2细胞 α-SMA、FN表达上调，而下调E-cadherin表达。分别用1、5、10 g/L Cp预干预24 h后，同单独5 μg/L TGF-β1刺激组相比，上述因子表达被不同程度逆转。在转录水平，α-SMA mRNA表达被抑制率分别为37.98%、68.08%、84.36%；FN mRNA表达被抑制率分别为46.97%、63.82%、81.85%；E-cadherin mRNA表达分别增加0.67倍、2.69倍、5.43倍（均P < 0.05）。在蛋白水平，α-SMA蛋白表达被抑制率分别为33.40%、47.75%、68.50%；FN蛋白表达抑制率分别为16.26%、65.92%、80.30%；E-cadherin蛋白表达分别增加1.33倍、3.19倍、4.29倍（均P < 0.05）。Cp能明显拮抗 TGF-β1 诱导的HK-2细胞的表型改变。 结论 Cp能有效阻止TGF-β1诱导的人近端肾小管上皮细胞发生转分化。     

去甲斑蝥素对TGF-β_1诱导的人近端肾小管细胞上皮间质转分化以及转录因子Snail1表达的影响

目的：探讨去甲斑蝥素对体外人源性肾小管细胞株（HK-2）上皮间质转分化以及转录因子Snail1的影响。方法：常规培养HK-2细胞,分为对照组、TGF-β1组、去甲斑蝥素组（NCTD组）。对照组为无血清DMEM培养,TGF-β1组为TGF-β15ng/ml诱导,NCTD组为不同浓度NCTD（0.5、1.0、2.5μg/ml）与TGF-β1（5ng/ml）共同作用,各组作用时间48h。采用RT-PCR、Western blot分别检测α-SMA、E-cadherin与Snail1表达水平的变化。结果：与对照组相比,TGF-β1组α-SMA mRNA和蛋白表达上调（P〈0.05）,E-cadherin mRNA和蛋白表达下调（P〈0.05）;与TGF-β1组相比,NCTD干预组α-SMA mRNA和蛋白表达下调（P〈0.05）,而E-cadherin mRNA和蛋白表达上调（P〈0.05）,且均呈剂量依赖性。与对照组相比,TGF-β1组Snail1 mRNA和蛋白表达上调（P〈0.05）;与TGF-β1组比较,NCTD干预组Snail1 mRNA和蛋白表达下调（P〈0.05）,具有一定的剂量依赖关系。结论：去甲斑蝥素具有抑制肾小管上皮细胞EMT的作用,该作用可能与下调转录因子Snail1的表达有关。     

红细胞生成素抑制C3a介导的肾小管上皮细胞间充质转分化

目的 观察红细胞生成素(EPO)对补体成分C3片段C3a介导的肾小管上皮细胞间充质转分化(EMT)的影响.方法 将人近端肾小管上皮细胞(HK-2)分为6组:对照组、EPO组、TGF-β组、TGF-β+EPO组、C3a组、EPO+C3a组,分别用RT-PCR、Western印迹和细胞免疫荧光方法检测HK-2细胞α-SMA、E-cadherin、C3的mRNA和蛋白表达.结果 与对照组和EPO组比较,C3a或TGF-β干预HK2细胞后,αt-SMA mRNA和蛋白表达增强(均P＜0.05),E-cadherin mRNA和蛋白表达减少(均P＜ 0.05),补体C3 mRNA和蛋白表达增强(均P＜ 0.05);而同时给予EPO干预后,C3a或TGF-β的上述作用可被明显减弱(均P＜0.05).结论 EPO可抑制C3a介导的肾小管上皮细胞EMT.     

苦参碱调控Snail2表达水平抑制转化生长因子β1诱导的人腹膜间皮细胞上皮间充质转分化

目的 研究苦参碱对转化生长因子β1(TGF-β1)诱导腹膜间皮细胞上皮间充质转分化(EMT)后转录因子Snail2的影响.方法 采用TGF-β1刺激人腹膜间皮细胞并同时予不同浓度苦参碱干预处理,实验分为空白对照组、TGF-β1(5 ng/ml)诱导组、TGF-β1+0.4 mg/ml苦参碱干预组、TGF-β1+0.6 mg/ml苦参碱干预组、TGF-β1+0.8 mg/ml苦参碱干预组和TGF-β1+1.0 mg/ml苦参碱干预组.实时荧光定量PCR和Western印迹检测Snail2、上皮标志分子E钙黏蛋白(E-cadherin)和间质标志分子α平滑肌肌动蛋白(α-SMA)、纤维连接蛋白(FN)、胶原Ⅲ(ColⅢ)的表达,Western印迹检测Smad2、Smad3和细胞外调节蛋白激酶1/2(ERK1/2)的蛋白磷酸化水平.结果 TGF-β1(5 ng/ml)刺激能上调人腹膜间皮细胞Snail2、α-SMA、FN和ColⅢmRNA和蛋白的表达水平,上调Smad2、Smad3和ERK1/2的蛋白磷酸化水平,下调E-cadherinmRNA和蛋白的表达水平;苦参碱(0.4、0.6、0.8、1.0 mg/ml)干预处理后能下调Snail2和α-SMA、FN和ColⅢmRNA和蛋白的表达水平以及ERK1/2的蛋白磷酸化水平,上调E-cadherin mRNA和蛋白的表达水平.结论 TGF-β1能诱导人腹膜间皮细胞EMT,苦参碱可能通过ERK1/2信号通路下调Snail2的表达水平来抑制TGF-β1诱导的人腹膜间皮细胞EMT.     

LeftyA真核表达载体的构建及其对转化生长因子-β1诱导人肾小管上皮细胞转分化的影响

目的 构建pcDNA3.1-LeftyA真核表达载体,观察其对转化生长因子-β1(TGF-β1)诱导人肾小管上皮细胞(HK-2)转分化(EMT)的影响.方法 基因克隆技术构建pcDNA3.1-LeftyA真核表达载体,将其瞬时转染HK-2细胞,TGF-β1(10μg/L)刺激后,观察细胞形态变化,检测E-钙黏蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)基因及蛋白的表达.结果 TGF-β1刺激后HK-2细胞内E-cadherin mRNA和蛋白时间依赖性表达下调,α-SMA mRNA和蛋白时间依赖性表达上调,同时E-cadherin表达变化早于α-SMA;LeftyA蛋白可以显著抑制E-cadherin蛋白的下调表达(P＜0.05),其表达比同时间点单纯刺激组高17.6%;同时可逆转α-SMA蛋白的上调表达(P＜0.05),其蛋白表达比单纯刺激组低14.0%.结论 LeftyA蛋白可以抑制TGF-β1所致的EMT.

Abstract：

Objective To construct the eukaryotic expression vector for LeftyA and study the effects of LeftyA on epithelial to mesenchymal transition of human proximal tubular epithelial cells induced by transforming growth factor-β1 ( TGF-β1 ). Methods The pcDNA3. 1-LeftyA was constructed by recombinant DNA technique. After transfection with pcDNA3. 1-LeftyA HK-2 cells were stimulated by TGF-β1( 10 μg/L). The morphological changes, and the expression of E-cadherin, α-SMA and LeftyA mRNA and protein were observed and detected, respectively. Results TGF-β1 could markedly decrease the expression of E-cadherin mRNA and protein in HK-2 cells induced, and dramatically increase the expression of α-SMA mRNA and protein in a time-dependent manner. Forced expression of exogenous LeftyA led to a blockage of TGF-β1 -induced E-cadherin ( 17.6% ) suppression and α-SMA induction ( 14. 0% ). Conclusion Disruption of cell adherence is the beginning stage of EMT, and overexpression of LeftyA can suppress EMT induced by TGF-β1, which suggests a alprostadil role for LeftyA in TGF-β1-induced tubular EMT and renal fibrosis.     

姜黄素对TGF-β1诱导人肾小管上皮细胞转分化及分泌细胞外基质成分的影响

目的：通过研究姜黄素（curcumin,Cur）对转化生长因子-β1（TGF-β1）诱导人肾小管上皮细胞（HK-2）转分化及分泌细胞外基质（ECM）成分的影响,探讨姜黄素在防治肾小管-间质纤维化方面可能的作用机制。方法：应用不同浓度Cur处理经TGF-β1诱导活化的HK-2细胞,通过显微镜观察细胞形态改变,免疫组化技术检测α-平滑肌肌动蛋白（α-SMA）和E-钙黏蛋白（E-cadherin）的表达;应用酶联免疫吸附法（ELISA）检测细胞培养上清Ⅰ型胶原（ColⅠ）、Ⅲ型胶原（ColⅢ）和纤连蛋白（FN）的分泌。结果：（1）正常HK-2细胞表达E-cadherin,不表达α-SMA,经TGF-β1刺激后细胞表型发生转变,E-cadherin的表达明显减弱,α-SMA表达明显增强;（2）不同浓度Cur组与单纯TGF-β1刺激组相比α-SMA的表达有所减弱,而E-cadherin的表达增强;（3）不同浓度Cur抑制了ColⅠ、ColⅢ和FN的分泌,与单纯TGF-β1刺激组相比有统计学差异（P〈0.05）。结论：姜黄素能抑制TGF-β1诱导人肾小管上皮细胞转分化及细胞外基质成分ColⅠ、ColⅢ和FN的合成,在一定程度上具有防治肾小管-间质纤维化的作用。     

高糖通过转化生长因子β信号诱导肾小球内皮细胞向肌成纤维细胞转分化

目的 探讨高糖(HG)能否通过转化生长因子β(TGF-β)途径诱导大鼠肾小球内皮细胞向肌成纤维细胞转分化(EndMT).方法 体外培养大鼠肾小球内皮细胞(GEnC),分为正常对照组(NG,5.5 mmol/L)、高糖组(HG,15、30 mmol/L)、TGF-β抑制剂组(HG+LY36,30 mmol/L葡萄糖+10 μmol/L LY364947)以及高渗对照组(M,5.5 mmol/L葡萄糖+25.5 mmol/L甘露醇)和溶剂对照组(D,5.5 mmol/L葡萄糖+1 ml/LDMSO).采用Western印迹法检测各组细胞内皮细胞标志物claudin5和肌成纤维细胞标志物α-SMA表达变化;实时定量PCR法检测细胞TGF-β1和TGF-β2 mRNA表达改变;免疫荧光法观察细胞形态学变化以及血管内皮细胞标志物VE-cadherin和肌成纤维细胞标志物α-SMA的表达.结果 与NG组比较,HG组claudin5蛋白的表达量随葡萄糖浓度增加而降低(P＜0.05),α-SMA蛋白表达量随葡萄糖浓度增加而升高(P＜0.05),TGF-β1和TGF-β2 mRNA表达均升高(P＜0.05).与HG组比较,TGF-β抑制剂组claudin5蛋白表达量升高(P＜0.05),α-SMA蛋白表达降低(P＜0.05).高渗对照组和溶剂对照组改变差异无统计学意义.激光共聚焦免疫荧光结果显示,高糖处理可引起细胞形态由卵圆形向梭形改变,VE-cadherin表达减少,α-SMA表达增加;TGF-β抑制剂组细胞形态无明显改变.与HG组比较,TGF-β抑制剂组VE-cadherin表达增加,α-SMA表达降低(P＜0.05).结论 高糖诱导大鼠肾小球内皮细胞TGF-β表达增加及内皮细胞-肌成纤维细胞转分化.抑制TGF-β可抑制高糖引起的转分化,提示TGF-β参与了高糖引起的肾小球内皮细胞转分化过程.     

黏着斑激酶在转化生长因子β1诱导的人肾小管上皮细胞转分化中的作用

目的 观察转化生长因子（TGF）β1诱导的正常人近端肾小管上皮细胞（HK-2）转分化（EMT）过程中黏着斑激酶（FAK）的表达及下调FAK的表达后对TGF-β1诱导的HK-2细胞转分化进程的影响。 方法 应用TGF-β1（10 μg/L）刺激HK-2细胞,采用RT-PCR、Western印迹和免疫荧光方法分别检测E钙黏蛋白（E-cadherin）、α平滑肌肌动蛋白（α-SMA）、FAK mRNA和蛋白的表达及磷酸化（p）-FAK（Tyr397）的蛋白表达。应用Lipofectmine2000将FAK siRNA转染HK-2细胞,采用Western印迹观察下调表达FAK对上述指标的影响。 结果 TGF-β1刺激后,HK-2细胞α-SMA蛋白和mRNA水平上调,E-cadherin蛋白和mRNA表达下调。FAK蛋白和mRNA随时间的延长表达逐渐增多,48 h达到高峰。p-FAK（Tyr397）蛋白表达趋势与FAK相同。脂质体转染siRNA后FAK的mRNA和蛋白分别下调了50%和41%,下调表达FAK后可以显著抑制TGF-β1诱导的HK-2细胞α-SMA蛋白的上调表达,逆转 E-cadherin蛋白的下调表达。 结论 在TGF-β1诱导的HK-2细胞转分化进程FAK蛋白表达上调,敲低FAK蛋白表达后可以部分减轻EMT的程度,提示FAK在TGF-β1诱导的肾小管上皮细胞转分化和肾脏纤维化中发挥一定的作用。     

虫草素通过下调TGF-β抑制高糖诱导的大鼠肾小管上皮细胞转分化

目的：探讨虫草素对高糖诱导的大鼠肾小管上皮细胞转分化的影响。方法：体外培养大鼠近端肾小管上皮细胞株（NRK52E细胞株）,分为正常对照组（葡萄糖5.5 mmol/L,NG组）、高糖组（葡萄糖30 mmol/L,HG组）、高糖＋虫草素组（葡萄糖30 mmol/L＋虫草素10μg/ml,HG＋C组）。分别于刺激12 h,24 h,48 h后收集细胞。应用定量RT-PCR测定NRK52E TGF-β,E-cadherin,α-SMA mRNA的表达;Western印迹方法检测TGF-β、E-cadherin、α-SMA蛋白的表达。结果：高糖刺激后NRK52E细胞的TGF-β和α-SMA mRNA及蛋白表达明显高于正常糖组（P〈0.01）,而虫草素组TGF-β和α-SMA mRNA及蛋白表达显著低于高糖组（P〈0.05）;高糖诱导的NRK52E细胞E-cadherin mRNA及蛋白水平明显降低（P〈0.01）;而虫草素组NRK52E细胞E-cadherin mRNA及蛋白水平显著高于高糖组（P〈0.05）。结论：虫草素可以明显抑制高糖诱导的大鼠肾小管上皮细胞转分化,其机制可能是通过下调TGF-β实现。     

Expression of toxin-related human mono-ADP-ribosyltransferase 3 in human testes

目的:研究单腺苷二磷酸-核糖基转移酶3(ART3)的 mRNA 在人类睾丸中是否表达其相应的蛋白,如果有表达是否细胞特异性表达。方法:用逆转录聚合酶链反应(RT-PCR)确定人类睾丸和精子中 ART3的mRNA 水平。用磷脂酶 C 处理转染了 ART3的 HEK-293-T 细胞,并通过它检测 ART3的糖基化磷酸肌醇链。用荧光激活细胞分类(FACS)分析和免疫组织学方法分别检测 ART3蛋白在成熟精子和睾丸中的表达。结果:ART3蛋白在睾丸和精母细胞中有表达,而在精原细胞、精子细胞和精子中未检测到,FACS 分析进一步确认精子中无 ART3蛋白。在精原细胞瘤和睾丸间细胞中也未检测到 ART3蛋白。结论:本研究首次发现ART3蛋白在睾丸中表达,尤其是在精母细胞中,这表明 ART3执行一个特定的功能,此功能只在精子发生的某个特殊阶段发挥作用。     

Modulation of human chondrocyte metabolism by recombinant human interferon

OBJECTIVES: Interferon gamma (IFN gamma) is found to be elevated in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis, suggesting its implication in joint disease pathogenesis. In this study, we investigated the effects of IFN gamma on the production of cytokines (IL-6, IL-8, IL-10), prostaglandin E(2)(PGE(2)), proteoglycans (PG), nitric oxide (NO), interleukin-1 receptor antagonist (IL-1ra) and stromelysin by non-stimulated and IL-1 beta-treated human chondrocytes. The role played by NO in the responses of chondrocytes to IFN gamma was also examined by incubation of chondrocytes with N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase. METHODS: Enzymatically isolated human chondrocytes were cultured for 48 h in the absence or presence of IL-1 beta, IFN gamma or N(G)-monomethyl-L-arginine (L-NMMA) added solely or in combination. The productions of IL-6, IL-8, IL-10, IL-1ra and stromelysin were measured by enzyme amplified sensitivity immunoassays (EASIA). PG and PGE(2)were quantified by specific radioimmunoassays (RIA). Nitrite concentrations in the culture supernatants were determined by a spectrophotometric method based upon the Griess reaction. RESULTS: As expected, IL-1 beta highly stimulated NO, IL-6, IL-8, IL-10, IL-1ra, PGE(2)and stromelysin synthesis, but dramatically decreased PG production. NO, IL-6, IL-1ra and PGE(2)production by non-stimulated chondrocytes was dose-dependently increased by IFN gamma while PG production was inhibited. In the absence of IL-1 beta, IL-10 was undetectable in the culture supernatants. At the doses of 10 and 100 U/ml, IFN gamma markedly inhibited the constitutive and IL-1 beta-stimulated IL-8, IL-10 and stromelysin productions. Interestingly, IFN gamma synergized with IL-1 beta to increase NO, IL-6, IL-1ra and to depress PG production. As previously reported, the inhibition of NO synthesis by the competitive inhibitor L-NMMA led to enhancement of IL-6, IL-8 and PGE(2)production by IL-1 beta treated chondrocytes, but did not significantly modify IL-10, PG and MMP-3 productions. Inhibition of NO synthase significantly inhibited the stimulating effect of IFN gamma on IL-6 and IL-1ra but did not affect the inhibitory effect of IFN gamma on IL-8, PG or stromelysin production. CONCLUSIONS: These findings suggest that IFN gamma and IL-1 synergistically stimulate the production of IL-6, IL-1ra, NO and PGE(2)and inhibit PG synthesis. By contrast, IL-1 beta and IFN gamma have opposite effects on IL-8, IL-10 and stromelysin productions. These effects are not reversed by L-NMMA, suggesting that NO is not the principal mediator involved in responses of chondrocytes to IFN gamma.     

Endothelialization of human collagen surfaces with human adult endothelial cells

Artificial vascular grafts are currently used to restore blood flow to ischemic tissue. Although the long-term patency of large diameter grafts is relatively acceptable, small diameter (less than 4 mm) grafts exhibit poor long-term patency rates. One technique to create a nonthrombogenic surface on artificial prostheses has been to seed with endothelial cells derived from autologous vessels. We have examined the interaction of human adult endothelial cells with the natural collagen surfaces presented by human amnion. Scanning electron microscopic evaluation revealed that human adult endothelial cells adhered rapidly to both the basement surface (collagen types IV and V) and interstitial surface (collagen types I and III) of amnion. However, the adherence of cells was significantly greater on the basement membrane surface. In addition, human adult endothelial cells rapidly formed close cell-to-cell interactions on basement membrane as compared with cells seeded onto the interstitial surface. These results suggest that seeding of endothelial cells onto artificial surfaces will be facilitated if the surface simulates the natural basement membrane to which endothelial cells natively adhere.     

Potent oncolytic activity of human enteroviruses against human prostate cancer

BACKGROUND: Oncolytic virotherapy offers a unique treatment modality for prostate cancer, especially stages that are resistant to current therapies, with the additional benefit of preferentially targeting tumor cells amongst an environment of healthy tissue. Herein, the low pathogenic enteroviruses; Coxsackievirus A21 (CVA21), as well as a bio-selected variant of Coxsackievirus A21 (CVA21-DAFv) and Echovirus 1 (EV1) are evaluated as novel oncolytic agents against human prostate cancer. METHODS: The surface expression of viral receptors required for enterovirus cell attachment/entry, including intercellular adhesion molecule-1 (ICAM-1), decay-accelerating factor (DAF) and integrin alpha(2)beta(1) on a number of human prostate cancer lines was assessed by flow cytometry. Susceptibility to viral oncolysis was determined via in vitro cell lysis assays performed on cell monolayers cultured in micro titer plates. The in vivo oncolytic efficacy of the enteroviruses was assessed using xenograft models in immune compromised SCID-mice following systemic challenge. RESULTS: The majority of prostate cancer lines tested expressed surface ICAM-1 and/or DAF, or alpha(2)beta(1), facilitating significant degrees of oncolysis following in vitro viral challenge. Systemic delivery of each of the three viruses induced reduction of xenograft tumor burdens in vivo, and a therapeutic dose-response was demonstrated for escalating doses of EV1 in the LNCaP animal model. CONCLUSION: Enteroviruses CVA21, CVA21-DAFv, and EV1 are potentially potent oncolytic agents against human prostate cancer.     

培养的人睾丸组织对人绒毛膜促性腺激素的反应

用猝死的正常成年男子睾丸组织，加入含有１０％胎牛血清的ＭＥＭ培养液，培养１４天。观察睾丸组织在培养期间对人绒毛膜促性腺激素（ｈＣＧ）０～５０ＩＵ／ｍｌ的反应，氧耗量测定及形态学观察。结果表明体外培养的人睾丸组织有３天适应期，第４天逐渐恢复分泌功能。在培养前期（０天）、中期（７天）、后期（１４天）对相同剂量ｈＣＧ刺激的睾酮分泌反应及氧耗量无显著差异（Ｐ＞０．０５）。光镜和电镜下可见睾丸组织结构完整。第１４天生精细胞有退行性变，少数赖迪细胞空泡样变     

Patterns of human tumor-infiltrating lymphocytes in 120 human cancers.

Tumor-infiltrating lymphocytes from 120 samples of human cancers, including melanoma, renal cell carcinoma, breast cancer, sarcoma, and colon cancer, were examined. The percentage of lymphocytes recovered from the cancer varied widely; that of renal cell carcinoma was higher than that of breast or colon cancer (65% vs 45%), which was higher than that of melanomas or sarcomas (30% to 35%). The types of lymphocytes before and after interleukin 2 activation showed specific patterns. CD4+ helper T cells predominated in all tumors except melanomas, which had more CD8+ cytotoxic T cells. CD16+ natural killer cells were recovered in renal cell carcinoma and sarcomas. Three different cytotoxic lymphocytes were identified among interleukin 2-activated tumor-infiltrating lymphocytes: (1) CD3+ CD16- cytotoxic T lymphocytes with cytotoxicity restricted to autologous tumor cells in melanomas, (2) CD3-CD16+ natural killer cells with vigorous major histocompatibility complex-nonrestricted cytotoxicity in renal cell carcinoma, and (3) CD3+ CD16- T cells with modest levels of major histocompatibility complex-nonstricted cytotoxicity in all cancers except melanomas. Thus, there was considerable diversity of tumor-infiltrating lymphocytes among these histologically distinct tumors with respect to magnitude of lymphocyte infiltration, phenotypic expression, and functional capacity.     

人前脑啡肽基因修饰人胚胎肾细胞的构建

目的 构建人前脑啡肽基因(hPPE)修饰的人胚胎肾细胞(HEK293细胞).方法 重组质粒pcDNA3.1(+)/hPPE经限制性内切酶HindⅢ和Not Ⅰ进行双酶切获得hPPE基因,运用基因重组技术将hPPE基因与表达载体同源重组,转染293T细胞进行慢病毒包装、扩增、纯化,测定病毒滴度,再将重组的慢病毒载体转染HEK293细胞.用Western blot法检测hPPE基因在HEK293细胞中的表达.结果 重组慢病毒载体阳性克隆测序结果和基因库的hPPE基因序列完全一致.含有hPPE基因的慢病毒载体,滴度为2.07×108TU/ml.转染慢病毒载体后的HEK293细胞,在荧光显微镜下未见到GFP荧光.转染Ubc-GFP-L.V.空病毒载体的HEK293细胞,可见到较强的荧光.Western blot法检测到经慢病毒载体转染后的HEK293细胞中hPPE基因表达呈阳性.结论 成功构建了hPPE基因修饰的HEK293细胞,使hPPE基因可在HEK293细胞中稳定表达.     

Continuous versus intermittent administration of human endostatin in xenografted human neuroblastoma

Purpose

The authors examined whether recombinant human endostatin (rhEndostatin), an antiangiogenic agent, is effective against a human neuroblastoma cell line (designated TNB9) using a human neuroblastoma xenograft model and investigated whether continuous infusion is more effective than intermittent administration.

Methods

In the first experiment, when tumors on the back of nude mice reached a weight of 90 to 95 mg, rhEndostatin, 10 mg/kg/d mouse weight, was administered subcutaneously to the mice (n = 5) every day for 10 consecutive days. In the second experiment, the same daily dose of rhEndostatin was administered continuously to the TNB9-bearing mice (n = 6) via subcutaneous infusion pumps for 3 consecutive days with total dose being 30% of that in the first experiment. Nestin and factor VIII expression levels were studied immunohistochemically to elucidate whether histologic evidence of the effects of rhEndostatin was present on day 4 in the second experiment.

Results

In the first experiment, relative tumor weight in treated mice (n = 5) was significantly less than that in controls (n = 12) on day 2 only after treatment initiation (P < .05). The maximum inhibition rate (MIR) of TNB9 xenograft growth by rhEndostatin was 46.4%, indicating lack of efficacy. In the second experiment, the effects of rhEndostatin were much more marked than those in the first experiment, with an MIR of 60.7%. The mean relative tumor weight in the treated group (n = 6) in the second experiment was significantly less than that in controls (n = 10) on days 2, 4, and 6 (P < .01) as well as on days 8 and 10 (P < .05). Nestin staining in the endothelium of control tumors (n = 2) was marked, whereas it showed a loss of fibrillar structure in rhEndostatin-treated tumors (n = 2). The number of vessels immunostained with antifactor VIII antibody was markedly reduced in tumors (n = 2) from rhEndostatin-treated mice compared with that in tumors from control animals (n = 2).

Conclusions

Continuous administration of rhEndostatin resulted in more significant tumor regression than intermittent administration of the agent in the same model. This indicates that rhEndostatin, if administered in continuous fashion, could become an effective agent for treating patients with neuroblastoma in the future.     

Dissolution of human gallstone

A new water-soluble preparation of terpene derivative, natural limonene with polyoxyethylene hydrogenated castor oil was introduced. This preparation made it possible to dissolve completely 1.412 to 3.250 gm human gallstones, such as pure cholesterol stone, cholesterol pigment-calcium stone in extraordinary short time, about 2 hoursin vitro. And remarkable dissolving effects were shown on 1.925 to 2.801 gm human cholesterol stone placed into the pig gallbladder after only two times of instillation of this preparationin vivo. Histological examination after this instillation was within normal limits on various organ of pig, such as the gallbladder, common bile duct, duodenum, small and large intestines, liver, kidneys, suprarenal glands and sternal-marrow. LD50 value of this new solvent was higher than 5g/kg, therefore it was revealed as non-toxic preparation. These results indicate that this new preparation can be applied clinically with safety especially to cholesterol stone. Moreover dissolving effect was shown even on bile pigment-calcium stone and fatty acid-calcium stone, however the dissolving velocity was not so high as that of cholesterol stone. When this new preparation will be used clinically, the conventional surgical procedures on residual gallstone can be improved.     

pH of human semen

The World Health Organization (WHO) laboratory manual (1992) states the normal values for pH in liquefied semen to be between 7.2 and 8.0. This implies an adjustment compared with the previous version ( WHO, 1987 ) in which the upper limit was 7.8, whereas in the WHO clinical manual (1993 ) the normal range of values is still stated to be in the range of 7.2–7.8. In this study pH was measured in ejaculates from 207 men in couples undergoing infertility examination. The pH was measured within 30 and 60 min after ejaculation with both pH paper and pH meter. The mean pH values were consistently well above 8.0 regardless of analysis method and time after ejaculation. Since semen analysis is part of clinical assessment of male infertility and includes pH measurement, our findings suggest that the range of normal values needs to be revised further.