雷奈酸锶改善成骨不全症模型oim小鼠代谢失衡的双向机制

目的 探讨雷奈酸锶对成骨不全症（OI）模型oim小鼠成骨细胞和破骨细胞的双重作用。方法 取1周龄OI模型纯合子oim/oim小鼠及野生型（wt/wt）小鼠的颅骨,采用Ⅰ型胶原酶通过连续消化法获取成骨细胞;取5～7周龄oim/oim小鼠和wt/wt小鼠的长骨,获取骨髓单核细胞诱导分化成破骨细胞。给予不同浓度（1、10 mmol/L）的雷奈酸锶进行干预,采用实时定量PCR（qRT-PCR）及蛋白质印迹法分别在mRNA及蛋白水平检测成骨细胞分化相关基因Runt相关转录因子2（Runx2）、碱性磷酸酶（ALP）、骨钙蛋白（OCN）,破骨细胞分化相关基因降钙素受体（Calcr）、抗酒石酸磷酸酶（Trap）、组织蛋白酶K（CTSK）,以及破骨分化相关转录因子c-fos、活化T细胞核因子c1（NFATc1）的表达;采用ALP染色及茜素红S染色观察成骨分化及矿化情况;采用Trap染色及骨片陷窝实验评估破骨细胞形成数量及骨吸收活性;采用3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑溴盐（MTT）比色法检测雷奈酸锶对成骨细胞和破骨细胞活性的影响。结果 qRT-PCR及蛋白质印迹法检测结果显示,雷奈酸锶在mRNA及蛋白水平均可促进oim/oim小鼠成骨细胞分化相关基因（Runx2、ALP、OCN）的表达增加,同时抑制破骨细胞分化相关基因及转录因子（Calcr、Trap、CTSK、c-fos、NFATc1）的表达（P均<0.05）,且随浓度增加作用增强。ALP染色及茜素红S染色结果显示,雷奈酸锶可促进oim/oim小鼠成骨细胞分化及矿化（P均<0.05）。Trap染色及骨片陷窝实验结果显示,雷奈酸锶可降低oim/oim小鼠破骨细胞形成数量和破骨细胞骨吸收活性（P均<0.05）。MTT结果显示,1 mmol/L、10 mmol/L雷奈酸锶对oim/oim小鼠成骨细胞和破骨细胞无细胞毒性。结论 雷奈酸锶可有效改善OI模型oim小鼠的骨代谢失衡,其机制可能是促进成骨细胞分化及矿化,同时抑制破骨细胞的形成及骨吸收活性。     

知母皂苷元对成骨细胞活性和破骨细胞分化及功能的影响

目的： 考察知母皂苷元(SAR)对体外培养成骨细胞活性和破骨细胞分化及功能的影响。方法： 培养小鼠胚胎成骨细胞MC3T3-E1,分别采用MTT法、碱性磷酸酶(ALP)试剂盒检测及茜素红染色法观察SAR对MC3T3-E1细胞增殖、ALP活性及矿化结节形成的影响;分别采用皮质骨来源破骨细胞分离及骨髓间充质干细胞诱导破骨细胞形成两种方法培养破骨细胞,利用抗酒石酸酸性磷酸酶(TRAP)阳性细胞计数及骨吸收陷窝计数来观察不同浓度SAR对成熟破骨细胞活性及破骨细胞诱导分化的影响。结果： 与对照组相比,各质量浓度SAR(0.01,0.1,1 μg/mL)对MC3T3-E1细胞均具有明显的促增殖作用(P<0.05,P<0.01);处于快速增殖的MC3T3-E1细胞给药处理后各组间ALP活性未见明显差异,但对于增殖晚期MC3T3-E1细胞,不同浓度SAR均可使ALP活性增高,其中1 μg/mL组效应最强,与对照组相比差异非常显著(P<0.01);连续给药处理细胞15 d,SAR各组矿化结节形成数量与对照组相比均有增多趋势。此外,各浓度SAR对成熟破骨细胞数量及骨吸收能力均无明显影响,但均能抑制骨髓细胞分化形成破骨细胞。结论： SAR能促进体外培养的成骨细胞的增殖与分化成熟;SAR对分化成熟的破骨细胞无明显影响,但可抑制骨髓细胞向破骨细胞的分化,从而减少破骨细胞的产生。     

尼古丁对血管内皮细胞自噬的影响

目的 初步探讨尼古丁对人脐静脉内皮细胞（human umbilical vein endotheliel cell,HUVEC）自噬的影响。方法 采用蛋白免疫印迹（Western blot）技术分别检测尼古丁不同浓度（0、6×10^-8、6×10^-7、6×10^-6和6×10^-5mol/L）和不同时间（浓度为6×10^-6mol/L时,分别干预0、1.5、3、6和12h）作用后,人脐静脉内皮细胞中自噬标志性蛋白微管相关蛋白轻链3Ⅱ（LC3Ⅱ）蛋白表达水平的变化,以确定尼古丁的最佳刺激浓度和最佳刺激时间。依据尼古丁最佳条件将体外培养的HUVE细胞随机分组：对照组、尼古丁处理组,Western blot法检测LC3Ⅱ、多聚泛素结合蛋白p62/SQSTM1（p62）和溶酶体相关膜蛋白-2（LAMP-2）的蛋白表达,Annexin Ⅴ-FITC/PI双染流式细胞术（flow cytometry,FCM）检测细胞凋亡率。结果 体外实验显示,LC3Ⅱ蛋白表达呈浓度和时间依赖性,且尼古丁浓度为6×10^-6mol/L、时间为6h时,LC3Ⅱ蛋白表达最多（P<0.01）。与对照组相比,尼古丁处理组LC3Ⅱ、p62蛋白表达显著增加（P<0.05）,LAMP2蛋白表达减少（P<0.05）,细胞凋亡率升高（P<0.01）。结论 尼古丁可以引起自噬体与溶酶体融合障碍,使自噬降解异常,导致内皮细胞自噬功能紊乱,促进细胞凋亡。     

迷迭香酸⁃锌配合物的抗氧化和抗菌性研究

目的：研究迷迭香酸（rosmarinic acid,RA）和锌（zinc,Zn）离子络合后的结构、抗氧化性、抗菌性和生物相容性。方法：利用扫描电子显微镜、红外和紫外-可见光谱分析迷迭香酸锌配合物（RA-Zn）的结构。通过细胞增殖实验比较RA和RA-Zn 对成骨细胞增殖活性的影响,通过铁离子还原抗氧化能力实验和2,2-联氮-二（- 3-乙基苯并噻唑-6-磺酸）二铵盐［2,2′-azinobis （3-ethylbenzothiazoline-6-sulfonic acid）ammonium salt,ABTS］自由基清除实验分析两者的抗氧化性,最小抑菌浓度法比较两者对金黄色葡萄球菌的抑制活性。结果：成功合成了RA-Zn。作用第5天,RA-Zn促进成骨细胞增殖的作用优于RA（P < 0.05）, 200 μmol/L RA-Zn的抗氧化能力比RA更强（P < 0.05）。RA浓度≥400 μmol/L、而RA-Zn浓度≥25 μmol/L时能抑制金黄色葡萄球菌活性（P < 0.05）。结论：RA和Zn离子络合后抗氧化性和抗菌性增强,具有促进成骨细胞增殖的作用。     

蛇麻酮通过Fas/FasL途径诱导人肝癌HepG2细胞凋亡机制研究

目的 探讨啤酒花 Humulus lupulus 中成分蛇麻酮 (lupulone, LP) 通过 Fas/FasL 途径诱导 HepG2 凋亡的机制。方法 体外培养人肝癌细胞株 HepG2 4、8、16 μg/mL 剂量的蛇麻酮作用 HepG2 细胞 24 h 后,采用活性检测试剂盒检测 caspase-8 相对活性,采用 Western blotting 技术检测 Fas、FasL、Caspase-3 蛋白表达情况。结果 LP 能显著提高 HepG2 细胞内 caspase-8 活性水平,并且具有剂量依赖性,其中 8、16 μg/mL LP 组与对照组比较有显著性差异 (P＜0.05)。此外,LP 能够上调 HepG2 细胞的 Fas/FasL 及 Caspase-3 蛋白表达水平,并且均具有剂量依赖性,其中 4 μg/mL 组与对照组比较均有显著性差异 (P＜0.05),8、16 μg/mL 组与对照组比较均有非常显著性差异 (P＜0.01)。结论 蛇麻酮可以通过激活 Fas/FasL,启动死亡受体途径诱导 HepG2 细胞凋亡。     

3-乙酰基-11-羰基-β-乙酰乳香酸在Caco-2和MDCK细胞模型中的吸收研究

目的 研究乳香提取物中3-乙酰基-11-羰基-β-乙酰乳香酸（AKBA）在Caco-2、MDCK-MDR1和MDCK-Wild细胞模型中的吸收转运机制。方法 利用Caco-2、MDCK-MDR1和MDCK-Wild细胞模型,研究AKBA由细胞层顶端（AP）→基底端（BL）和BL→AP的双向转运过程;采用LC-MS/MS法测定AKBA的量,计算表观渗透系数（P_app）。结果 在Caco-2细胞模型中,50 μmol/L AKBA 由AP→BL、BL→AP的P_app分别为7.9×10^?7、1.5×10^?7 cm/s,在MDCK-MDR1细胞模型中,50 μmol/L AKBA由AP→BL、BL→AP的P_app分别为2.6×10^?7、0.8×10^?7 cm/s,在MDCK-Wild细胞模型中,50μmol/L AKBA由AP→BL、BL→AP的P_app分别为2.4×10^?7、0.6×10^?7 cm/s,3种细胞模型中外排率均小于2。结论 AKBA在肠道中吸收不良,不是P-糖蛋白的底物,推测其通过摄入型主动转运和被动扩散两种机制透过小肠上皮细胞。     

二甲双胍与吡格列酮治疗多囊卵巢综合征的疗效和安全性

目的 探讨了二甲双胍或吡格列酮联合炔雌醇环丙孕酮治疗多囊卵巢综合征（PCOS）的临床疗效,为PCOS的联合用药治疗提供更多的循证医学证据。方法 收集笔者医院2014年3月~2016年11月收治的PCOS患者132例,使用数字表法随机分为二甲双胍组和吡格列酮组,每组66例。使用电化学发光法测定卵泡刺激素（FSH）、黄体生成素（LH）、睾酮（T）及雌二醇（E₂）水平。采用比色法测定总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白（LDL-C）、高密度脂蛋白（HDL-C）。稳态模型测定胰岛素分泌指数（HOMA-β）和胰岛素抵抗指数（HOMA-IR）。比较两组临床疗效和不良反应。结果 治疗前两组间FSH、LH、T和E₂激素水平比较,差异无统计学意义（P>0.05）,治疗后两组FSH、LH、T均显著减低和E₂均显著升高（P<0.01）,但治疗后两组FSH、LH、T和E₂激素水平比较,差异无统计学意义（P>0.05）。二甲双胍组有效率为90.9%（60/66）,吡格列酮组有效率为93.9%（62/66）,两组间临床疗效比较,差异无统计学意义（P>0.05）。治疗前两组TC、TG、LDL-C、HDL-C、HOMA-β和HOMA-IR水平比较,差异无统计学意义（P>0.05）,治疗后两组TC、TG、LDL-C、HOMA-β和HOMA-IR均显著减低和HDL-C均显著升高（P<0.05和P<0.01）,但治疗后两组TC、TG、LDL-C、HOMA-β和HOMA-IR水平比较,差异无统计学意义（P>0.05）,治疗后吡格列酮组HDL-C水平显著高于二甲双胍组（P<0.05）。吡格列酮组治疗后BMI为25.64±5.35kg/m²,显著高于二甲双胍组的21.31±4.13kg/m²（P<0.05）,吡格列酮组治疗期间胃肠道不良反应率为13.6%,显著低于二甲双胍组的27.3%（P<0.05）。结论 炔雌醇环丙孕酮联合二甲双胍或吡格列酮治疗PCOS临床疗效显著,但吡格列酮可显著升高HDL-C,具有低的胃肠道不良反应率,而二甲双胍可以减轻患者体重,根据患者的临床特点,可以合理地选择药物联合治疗PCOS。     

十雄角果木树皮的二萜类化学成分研究

目的 研究角果木属植物十雄角果木Ceriops decandra树皮的二萜类化学成分。方法 采用正反相硅胶色谱、高效液相色谱技术进行分离纯化,根据理化性质和波谱数据鉴定化合物的结构。结果 从十雄角果木树皮的95%乙醇提取物中分离得到13个二萜类化合物,分别鉴定为7, 13-松香二烯-3β-醇（1）、18-羟基-8, 11, 13-松香三烯-7-酮（2）、8, 11, 13-松香三烯-3, 7-二酮（3）、3β-羟基-8, 11, 13-松香三烯-7-酮（4）、15, 18-二羟基-8, 11, 13-松香三烯-7-酮（5）、8, 11, 13-松香三烯-7β, 18-二醇（6）、8, 11, 13-松香三烯-3β, 18-二醇（7）、8, 11, 13-松香三烯-7α, 18-二醇（8）、13β-羟基-8(14)-松香烯-3, 7-二酮 （9）、13β, 18-二羟基-8(14)-松香烯-7-酮（10）、ent-8(17), 13E-半日花二烯-15-醇（11）、ent-8(14)-海松烯-15R, 16-二醇（12）、(5S^*, 8S^*, 9S^*, 10R^*, 13S^*)-3-hydroxy-16-nor-2-oxodolar-3-ene-15-oic acid（13）。结论 所有化合物均首次从该植物中分离得到。     

姜黄素诱导人肝癌细胞系Huh7自噬和凋亡

目的 探讨姜黄素诱导人肝癌细胞Huh7自噬和凋亡的作用,以及抑制自噬对姜黄素诱导凋亡的影响。方法 将姜黄素（5、10、20、40 μmol/L）加入人肝癌细胞系Huh7的培养液中,用CCK-8检测细胞增殖水平变化。Huh7细胞用5~40 μmol/L姜黄素培养48 h,通过蛋白质印迹法检测自噬微管相关蛋白1轻链3（LC3）-Ⅱ/LC3-Ⅰ的比值,免疫荧光法检测自噬小体的形成,评估Huh7细胞自噬水平的变化。通过流式细胞术检测Huh7细胞凋亡情况。加入5 mmol/L自噬抑制剂3-甲基嘌呤（3-MA）和20 μmol/L姜黄素培养Huh7细胞,检测Huh7细胞凋亡水平和自噬水平的变化。结果 CCK-8检测显示姜黄素能抑制Huh7细胞的增殖（P<0.05,P<0.01）,且细胞凋亡率随着姜黄素浓度的升高而上升;蛋白质印迹检测显示加入姜黄素后LC3-Ⅱ/LC3-Ⅰ的比值上升（P<0.05,P<0.01）,免疫荧光检测显示加入20 μmol/L姜黄素后自噬小体增多。与单独使用20 μmol/L姜黄素培养的Huh7细胞相比,姜黄素联用3-MA培养的Huh7细胞LC3-Ⅱ/LC3-Ⅰ的比值下降（P<0.01）,自噬小体减少。流式细胞术检测发现5~40 μmol/L姜黄素促进了Huh7细胞凋亡（P<0.05,P<0.01）,而联用3-MA后与单独使用20 μmol/L姜黄素培养相比引起的Huh7细胞凋亡减少（P<0.05）。结论 姜黄素能诱导Huh7细胞凋亡、抑制细胞增殖并促进细胞自噬,抑制自噬能减弱姜黄素对Huh7细胞的诱导凋亡效应。     

131I和甲巯咪唑对Graves病患者外周血Treg细胞及IL-10水平的影响

目的 观察¹³¹I和甲巯咪唑治疗Graves病（GD）前后调节性T细胞（Treg）和白细胞介素-10（IL-10）的变化,并探讨其相关的临床意义。方法 选取2013年4月~2015年11月笔者医院收治的GD患者88例,随机分为¹³¹I治疗组和甲巯咪唑治疗组,每组44例。选择同期笔者医院健康体检者40例作为正常对照。血清IL-10的检测使用ELISA法,流式细胞仪检测Treg细胞。比较两组治疗前后FT₃、FT₄和TSH。结果 GD患者Treg细胞比例为3.25%±0.84%,显著低于正常对照组的7.14%±2.13%（P<0.01）,GD患者IL-10因子表达为8.22±1.73μmol/L,也显著低于正常对照组的15.64±3.17μmol/L（P<0.01）。¹³¹I和甲巯咪唑组治疗前GD患者Treg细胞比例和IL-10因子表达差异无统计学意义（P>0.05）,治疗后¹³¹I组Treg细胞比例和IL-10因子表达出现显著升高（P<0.05）,但甲巯咪唑组上述指标治疗前后无显著性差异（P>0.05）。¹³¹I和甲巯咪唑组治疗前GD患者FT₃、FT₄和TSH差异无统计学意义（P>0.05）,治疗后两组FT₃和FT₄均显著减低（P<0.01）,TSH均显著升高（P<0.05）。但¹³¹I组对FT₃和FT₄的减低作用以及对TSH的升高作用显著优于甲巯咪唑组（P<0.05）。结论 ¹³¹I可提高Graves病患者Treg细胞比例和IL-10因子表达,临床疗效显著。     

仙茅酚苷类成分促进成骨细胞骨形成和抑制破骨细胞骨吸收

目的 以新生大鼠颅盖骨成骨细胞和由骨髓单核细胞诱导的破骨细胞为模型,观察仙茅代表性酚苷类成分仙茅苷、仙茅素A、苔黑酚葡萄糖苷和苔黑酚龙胆二糖苷的抗骨质疏松作用.方法 MTT法测定成骨细胞的增殖;磷酸苯二钠法测定成骨细胞碱性磷酸酶(alkaline phosphatase,ALP)和破骨细胞抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)的活性;茜素红染色观察成骨细胞骨矿化结节的形成;TRAP染色测定破骨细胞的数目;罗丹明-鬼笔环肽染色和激光共聚焦显微镜观察成骨细胞细胞骨架和破骨细胞肌动蛋白环的结构和形态;将破骨细胞与骨片共同培养,计算机图像处理测定破骨细胞在骨片上形成的骨吸收陷窝的面积.结果 仙茅苷在10-9和10-8 mol/L的浓度下可促进成骨细胞的增殖(P＜0.05),10-7～10-5 mol/L浓度时抑制破骨细胞TRAP的活性(P＜0.05).仙茅苷、苔黑酚葡萄糖苷和苔黑酚龙胆二糖苷可减少破骨细胞的数目,抑制破骨细胞的形成(P＜0.05).仙茅苷在10-10mol/L,仙茅素A、苔黑酚葡萄糖苷和苔黑酚龙胆二糖苷在10-9 mol/L浓度时均可增加成骨细胞ALP的活性和骨矿化结节的形成(P＜0.01),在一定程度上使1,25-二羟维生素D3损伤的成骨细胞细胞骨架的结构得以恢复;减少破骨细胞在骨片上形成的骨吸收陷窝面积,破坏破骨细胞伪足和F-actin的结构.结论 仙茅酚苷类成分仙茅苷、仙茅素A、苔黑酚葡萄糖苷和苔黑酚龙胆二糖苷均可促进成骨细胞的骨形成,抑制破骨细胞的骨吸收,具有显著的抗骨质疏松作用.     

呼出气一氧化氮检测在哮喘-慢阻肺重叠综合征治疗中的应用价值

目的 探讨呼出气一氧化氮(FeNO)测定在哮喘-慢阻肺重叠综合征(ACOS)治疗中的应用价值.方法 收集2015年5月至2015年10月在苏州九龙医院确诊但未规范使用药物治疗的ACOS患者28例,给予为期12周的吸入性糖皮质激素/长效β受体激动剂(ICS/LABA)吸入治疗,检测治疗前后患者的FeNO值、FEV1％pred、诱导痰嗜酸粒细胞(EOS)、血清总IgE、超敏C反应蛋白(hs-CRP)的水平;Pearson相关系数法分析FeNO值与其他指标的相关性.将患者按不同年龄和吸烟状况进行分组,比较各组患者在治疗前后各项指标的变化.同时选取28例健康受试者作为对照,检测其FeNO水平.结果 治疗前、后ACOS患者的FeNO水平[(32.04±8.34)×10-9 mol/L vs (25.56±4.13)×10-9 mol/L,P＜0.05]、诱导痰EOS[(18.51±5.36)％ vs (13.18±1.56)％,P＜0.05]、血清总IgE[(251.91±42.24) ng/mL vs (204.65±28.52) ng/mL,P＜0.05]差异有统计学意义;而FEV1％pred[(52.03±7.03)％ vs (55.16±8.20)％,P=0.391]、hs-CRP水平[(10.86±4.92) mg/L vs (9.16±1.82) mg/L,P=0.077]差异无统计学意义.治疗前、后ACOS组患者的FeNO水平均高于健康对照组[(32.04±8.34)×10-9 mol/L、(25.56±4.13)×10-9 mol/L vs (17.04±0.97)×10-9 mol/L,P＜0.05].不同年龄和吸烟状况的患者在ICS/LABA治疗前、后的FeNO值、诱导痰EOS、血清总IgE差异也均有统计学意义.ICS/LABA吸入治疗前、后ACOS组患者的FeNO水平与诱导痰EOS、血清总kE均呈正相关(治疗前:r=0.924,P＜0.01;r=0.945,P＜0.01.治疗后:r=0.247,P＜0.01;r=0.443,P＜0.01),而与血清hs-CRP、FEV1％ pred则无相关性.结论 ACOS患者气道存在EOS性炎症,可使用ICS/LABA吸入治疗,其疗效不受年龄及吸烟状况的影响.FeNO检测可作为检测和评估ACOS使用ICS/LABA治疗疗效的有效手段,且其与诱导痰EOS、血清总IgE相关.     

Effect of propyl gallate on activity of cyclooxygenase 1 and 2 in mice??s peritoneal macrophages

Objective: To investigate the effect of Red Peony 801 (propyl gallate,PrG) on cyclooxygenase (COX) activity in murine peritoneal macrophages.Methods: A screening model for COX inhibitors in vitro based on murine peritoneal macrophages was used. COX-1 activity was reflected by the level of 6-ketoprostaglandin F_1α(6-keto-PGF_1α) in supernatants of cultured macrophages which were stimulated with calcium ionophore A23187 for a short-term, while COX-2 activity was reflected by the level of prostaglandin E₂ (PGE₂) in supernatants of cultured macrophages which were stimulated with lipopolysaccharide (LPS) for a long-term.Results: PrG did not affect A23187-induced, COX-1-derived 6-keto-PGF_1α synthesis at the concentrations of 1 × 10^-6, 5 × 10^-6 mol/L (P>0.05), but enhanced 6-keto-PGF_1α synthesis at the concentrations of 1 × 10^-6, 5 × 10^-7, 1 × 10^-7 mol/L (P<0.01) in vitro, and showed a good dose-dependent manner. It inhibited LPS-induced, COX-2-derived PGE₂ synthesis at the concentrations of 1 × 10^-6, 1 × 10^-6 mol/L (P< 0.05).Conclusion: Within the range of 1 × 10^-5 to 1 × 10^-7 mol/L, PrG activated COX-1 at lower concentrations and inhibited COX-2 at higher concentrations in murine peritoneal macrophages. This work is supported by the National Natural Science Foundation (item number: 90209054)     

Involvement of M3 cholinergic receptor signal transduction pathway in regulation of the expression of chemokine MOB-1, MCP-1 genes in pancreatic acinar cells

Summary Whether M3 cholinergic receptor signal transduction pathway is involved in regulation of the activation of NF-κB and the expression of chemokine MOB-1, MCP-1 genes in pancreatic acinar cells was investigated. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, atropine and PDTCin vitro. The MOB-1 and MCP-1 mRNA expression was detected by using RT-PCR. The activation of NF-κB was monitored by using electrophoretic mobility shift assay. The results showed that as compared with control group, M3 cholinergic receptor agonist (10⁻³ mol/L, 10⁻⁴ mol/L carbachol) could induced a concentration-dependent and time-dependent increase in the expression of MOB-1, MCP-1 mRNA in pancreatic acinar cells. After treatment with 10⁻³ mol/L carbachol for 2 h, the expression of MOB-1, MCP-1 mRNA was strongest. The activity of NF-κB in pancreatic acinar cells was significantly increased (P<0.01) after treated with M3 cholinergic receptor agonist (10⁻³ mol/L carbachol)in vitro for 30 min. Either M3 cholinergic receptor antagonist (10⁻⁵) mol/L atropine) or NF-κB inhibitor 10⁻² mol/L PDTC) could obviously inhibit the activation of NF-κB and the chemokine MOB-1, MCP-1 mRNA expression induced by carbachol (P<0.05). This inhibitory effect was significantly increased by atropine plus PDTC (P<0.01). The results of these studies indicated that M3 cholinergic receptor signal transduction pathway was likely involved in regulation of the expression of chemokine MOB-1 and MCP-1 genes in pancreatic acinar cellsin vitro through the activation of NF-κB. ZHENG Hai, male, born in 1972, M. D., Ph. D.     

P16INK4A联合HPV L1壳蛋白检测在宫颈鳞状上皮内病变诊断中的应用

目的 初步探讨P16^INK4A联合HPV L1壳蛋白检测在宫颈鳞状上皮内病变诊断中的应用价值。方法 选择在温州市人民医院就诊患者,收集69例宫颈鳞状上皮内病变标本,行P16^INK4A、HPV L1壳蛋白检测,分析P16^INK4A、HPV L1壳蛋白及P16^INK4A联合HPV L1壳蛋白在各级宫颈病变中的表达差异。结果 P16^INK4A在LSIL组、HSIL组、SCC组中的阳性率分别为45.5%、85.2%、100.0%。同LSIL组比较,P16^INK4A在HSIL组及SCC组阳性率明显升高（P<0.01）。HPV L1壳蛋白在LSIL组、HSIL组、SCC组中的阳性率分别为63.6%、18.5%、0%,HPV L1壳蛋白在LSIL组中阳性率明显低于HSIL组及SCC组（P<0.01）。随着宫颈病变加重,P16+/L1-阳性率有升高趋势（P<0.01）,P16-/L1+阳性率有下降趋势（P<0.01）。LSIL组中P16+/L1-阳性率明显低于HSIL组和SCC组（P<0.01）,SCC组P16+/L1-阳性率较HSIL组有明显提高（P<0.05）。LSIL组中P16-/L1+阳性率明显低于HSIL组和SCC组（P<0.01）。P16+/L1-较P16^INK4、HPV L1壳蛋白单一检测筛查HSIL+特异性明显升高（P<0.01）。结论 P16^INK4A联合HPV L1壳蛋白有望成为诊断宫颈鳞状上皮内病变的有效指标。     

Effect of Morphine and Naloxone on Release of the Excitatory Amino Acids of Spinal Astrocytes Induced by TNF-α

The effect of morphine and naloxone on release of the excitatory amino acids (EAAs) of spinal astrocytes induced by TNF-α was studied. Astrocytes were purified from 2- to 3-day old SD rats and divided into 8 groups: group 1 (without any stimulatants); group 2 (10 ng/ml TNF-α); group3 (10 ng/ml TNF-α+0.5 μmol/L morphine); group 4 (10 ng/ml TNF-α+1.0 μmol/L morphine); group 5 (10 ng/ml TNF-α+2.0 μmol/L morphine); group 6 (10 ng/ml TNF-α+0.5 μmol/L naloxone); group 7 (10 ng/ml TNF-α+1.0 μmol/L naloxone); group 8 (10 ng/ml TNF-α +2.0 μmol/L naloxone). In group 2, 3, 4 and 5, 0, 0.5, 1.0 or 2.0 μmol/L morphine was added to the cells cultured with serum-free Neurobasal/B27 medium containing 10 ng/ml TNF-α respectively, while in group 6, 7 and 8, 0.5, 1.0 or 2.0 μmol/L naloxone was added respectively to the cells cultured with serum-free Neurobasal/B27 medium containing 10 ng/ml TNF-α. After 30 min incubation, high-pressure liquid chromatography (HPLC) was used to measure the levels of EAAs in all cultured cells. The results showed the level of EAAs in group 2 was significant higher than in group 1 (P<0.01). As compared with group 2, the levels of EAAs in group 3, 4 and 5 were decreased with the difference being significant between group 5 and group 2 (P<0.01) or between group 4 and group 2 (P<0.05). The levels of EAAs in group 6, 7 and group 8 was significantly lower than in group 2 (P<0.05 orP<0.01). It was concluded that TNF-α could promote the release of glutamate and aspartate from astrocytes, and morphine and naloxone might reduce the release of EAAs in cultured spinal astrocytes induced by TNF-α.     

Effects of lptakalim on intracellular free calcium concentration of cultured rabbit pulmonary arterial smooth muscle cells

Objective：To explore the effects of Iptakalim on intracellular free calcium concentration and on the proliferation of cultured rabbit pulmonary arterial smooth muscle cells induced by endothelin-1 （ET-1） in vitro. Methods：A cell culture model, [^3H]-thymidine（[^3H]-TdR） incorporation test and confocal microscope were used to observe proliferation and intracellular free calcium concentration（[Ca^2±]） of rabbit PASMC induced by ET-1 in vitro. Results：The value of [^3H]-TdR incorporation in ET-1 group was increased 1.468 times higher than that in control group. Iptakalim at the concentration of 10^-7mol/L, 10^-6mol/L ,10^-5 mol/L lowered [^3H]-TdR incorporation by （19.8 ± 4.6）%, （41.2 ± 9.5）%, （54.7 ± 10.1）%, respectively, compared with the value of the cells treated with ET-1（P〈 0.01）; The intracellular fluorescence intensity of PASMC in ET-1 group was increased from 73.70 ± 10.12 to 143.84 ± 28.23, significantly higher than that in control group（P 〈 0.01）; whereas with Iptakalim,the fluorescence intensity（FI） was only increased from 74.30 ± 10.20 to 86.03 ± 9.82, significantly lower than that in ET-1 group（P 〈 0.01）. Conclusion：Iptakalim inhibited proliferation of PASMC and decreased intracellular free calcium concentration of cultured rabbit PASMC induced by ET-1.     

Effects of Total Flavonoids ofHippophae Rhamnoides L. on intracellular free calcium in cultured vascular smooth muscle cells of spontaneously hypertensive rats and Wistar-Kyoto rats

Objective: To explore the effects of total flavonoids ofHippophae rhamnoides L. (TFH), quercetin (Que) and isorhamnetin (Isor) on the intracellular free calcium ([Ca²⁺ ]_i) in vascular smooth muscle cells (VSMC) of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY).Methods: Fluo 3-acetoxymethylester(Fluo-3/AM) was used to observe the effects of TFH (100mg/L) and its essential monomers, namely Que (l0^-4 mol/L) and Isor (l0^-4 mol/L) on changes of [Ca²⁺]_i in cultured SHR and WKY VSMC (abbr. to Ca-SHR & Ca-WKY) following exposure to high K⁺, norepinephrine (NE) and angiotensin II (Ang II), and to compare with the effects of verapamil (Ver).Results: (1) TFH, Que and Isor had inhibitory effects on resting Ca-SHR (P<0.05), but had no significant effects on Ca-WKY (P>0.05). (2) High K⁺ could increase Ca-SHR more significantly than Ca-WKY (P<0.05); TFH, Que and Isor could inhibit the elevation of [Ca²⁺]_i induced by high K⁺-depolarization, with the effects similar to that of Ver, and the effect on Ca-SHR was more significant than that on Ca-WKY (P<0.05). (3) NE and Ang II could increase Ca-SHR more significantly than Ca-WKY (P<0.05), TFH, Que and Isor had remarkably inhibitory effect on the elevation of Ca-SHR and Ca-WKY induced by NE or Ang II. (4) In the absence of extracellular Ca²⁺, TFH, Que and Isor also had certain inhibitory effect on Ca-SHR and Ca-WKY induced by NE, and the effect on the former was more significant than that on the latter(P<0. 05).Conclusion: TFH, Que and lsor might decrease the levels of [Ca²⁺]_i in VSMCs by blocking both voltage-dependent calcium channels (VDC) and receptor-operated calcium channels (ROC) in physiological or pathological state, which may be one of the important mechanisms of their hypotensive and protective effects on target organs in patients with hypertension. Supported by One-hundred-people Plan of Hygiene System in Shanghai (No. 990122)     

Relationship between carbachol hyperstimulation-induced pancreatic acinar cellular injury and trypsinogen or NF-κB activation in ratsin vitro

Summary The relationship between M3 cholinergic receptor agonist (carbachol) hyperstimulation-induced pancreatic acinar cellular injury and trypsinogen activation or NF-κB activation in rats was studiedin vitro. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor (pefabloc), and NF-κB inhibitor (PDTC)in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The cellular injury was evaluated by measuring the leakage of LDH from pancreatic acinar cells. The results showed that as compared with control group, 10⁻³ mol/L carbachol induced a significant increase of the intracellular trypsin activity and the leakage of LDH from pancreatic acinar cells. Pretreatment with 2 mmol/L pefabloc could significantly decrease the activity of trypsin and the leakage of LDH from pancreatic acinar cells (P<0.01) following the treatment with a high concentration of carbachol (10⁻³ mol/L)in vitro. The addition of 10⁻² mol/L PDTC didn't result in a significant decrease in the activity of trypsin and the leakage of LDH from pancreatic acinar cells treated with a high concentration of carbachol (10⁻³ mol/L)in vitro (P>0.05). It was concluded that intracellular trypsinogen activation is likely involved in pancreatic acinar cellular injury induced by carbachol hyperstimulationin vitro. NF-κB activation may not be involved in pancreatic acinar cellular injury induced by carbachol hyperstimulationin vitro. Zheng Hai, male, born in 1972, Doctor in Charge, M. D., Ph.D.     

不同钙、镁比例饮用水对相对高草酸尿症大鼠肾结石形成及代谢的影响

[摘要] 目的 探讨低于硬水标准的不同钙、镁比例饮用水对相对高草酸尿症大鼠肾结石形成及代谢的影响。 方法 将42只SPF级雄性Sprague-Dawley大鼠随机分成7组（n=6）：空白组、模型组、高钙低镁组、中钙低镁组、低钙低镁组、低钙中镁组和低钙高镁组,空白组饮用纯净水,模型组饮用0.1%乙二醇（EG）配制水,高钙低镁组、中钙低镁组、低钙低镁组、低钙中镁组、低钙高镁组各干预组在模型组基础上给予不同钙、镁浓度比例的饮用水,浓度比例分别为360/10、120/10、10/10、10/40、10/80（mg/L）。各组大鼠在相同环境下饲养8周后,收尿液、血液及双肾标本,左肾做H&E染色石蜡切片,光学显微镜观察草酸钙结晶情况;分别测定大鼠24h尿量、尿钙、尿镁、尿草酸、尿枸橼酸排泄量及血钙、血镁、血肌酐以及血尿素氮浓度。 结果 大鼠肾重量低钙低镁组、低钙中镁组均较空白组比较显著增加（P<0.05）。低钙中镁组血尿素氮浓度较空白组、模型组比较均显著升高（P<0.05）。各组大鼠体重变化、摄水量、24h尿量、血钙、血镁和血肌酐等的比较无统计学差异（P>0.05）。24h尿镁排泄量低钙中镁组较空白组显著增加（P<0.05）。24h尿草酸排泄量模型组、低钙低镁组、低钙中镁组均显著高于空白组（P<0.01）,中钙低镁组也较空白组显著升高（P<0.05）;除低钙低镁组外,其余干预组24h尿草酸排泄量均显著低于模型组（P<0.01）。尿钙、24h尿枸橼酸排泄各组均无显著性差异。血钙、血镁、血肌酐各组间均无显著性差异。各组尿结晶均为阴性。各组肾组织病理学检查各组均未见结晶形成,肾小球、小管细胞大小正常,排列整齐、规则,肾小管管腔无扩张,管腔内无坏死脱落样物质。结论 相对高草酸尿症造模对大鼠体内钙和镁的代谢、尿结晶及成石没有影响;在饮用水硬度低于硬水标准下,随钙镁总量（硬度）升高24h尿草酸排泄量减低;单纯高草酸尿症成石草酸浓度或量需要达到一定水平才能显示成石作用;而尿枸橼酸作为结石的保护性因素,它是相对独立的,不受造模及不同钙、镁比例饮用水影响。