伏立康唑在新西兰白兔眼组织中的浓度测定及眼组织分布研究

目的 建立新西兰白兔眼组织中伏立康唑浓度的测定方法,并应用于玻璃体腔注射伏立康唑的眼组织分布研究。方法 采用高效液相色谱-串联质谱（HPLC-MS/MS）法测定兔眼组织中伏立康唑浓度。以甲苯磺丁脲为内标,色谱柱为Waters X-Bridge BEH C₁₈（50 mm×2.1 mm,2.5 μm）,流动相A：0.2%甲酸-水溶液;流动相B：0.2%甲酸-乙腈溶液;梯度洗脱程序：0～0.30 min,30% B;0.30～1.81 min,30%→90% B;1.81～2.30 min,90% B;2.30～2.31 min,90%→30% B;2.31～2.60 min,30% B;体积流量为0.5 mL·min^-1,柱温30℃,进样量1 μL。使用电喷雾离子源,以多重反应监测方式进行正离子扫描,用于定量分析的离子对分别为m/z 350.1→281.1（伏立康唑）、m/z 271.1→155.0（内标）。新西兰白兔按照体质量、性别随机区间分成5组,每组6只,雌雄各半。无菌条件下,新西兰白兔眼部散瞳,im氯胺酮12 mg·kg^-1、赛拉嗪3 mg·kg^-1麻醉动物,玻璃体腔注入伏立康唑眼用注射剂,分别在行玻璃体腔注射术后的0.5、1.0、4.0、24.0、48.0 h,过量麻醉后放血处死试验兔。冰浴条件下分离兔眼组织（结膜、角膜、房水、虹膜、晶体、玻璃体、视网膜、脉络膜、巩膜）,采用建立的HPLC-MS/MS测定各组织中伏立康唑浓度。结果 伏立康唑质量浓度在0.5～500.0 ng·mL^-1,线性关系良好（R²＞0.98）,定量下限为0.5 ng·mL^-1。批内精密度（RSD）和准确度（RE）均小于15%,伏立康唑各眼组织的提取回收率均大于85%;新西兰白兔单眼单次给予伏立康唑后,其主要在眼后段分布,且视网膜浓度相对玻璃体、脉络膜更高。结论 建立的伏立康唑测定方法分析时间短,能够快速检测眼组织中的药物,准确度和灵敏度高,适用于伏立康唑在眼组织的分布研究。     

神经松动术结合常规康复训练对卒中后不同阶段偏瘫患者手功能恢复的疗效

目的:探讨神经松动术结合常规康复训练对卒中后不同阶段偏瘫患者手功能恢复的疗效。方法:脑卒中偏瘫患者102例,根据卒中后时间分为1月组(46例),2月组(32例),3月组(24例);3组均行神经松动术结合常规康复训练3个月。于治疗前、训练1、3个月分别采用Fugl-Meyer上肢运动量表(FMA-U)、偏瘫上肢功能测试(FTHUE)、改良Barthel指数(m BI)和日常生活活动能力(ADL)评价偏瘫手精细动作、运动协调能力、感觉功能、日常生活能力等。于治疗后1、3月部分患者行静息状态下fMRI扫描检测中央前回第一运动区激活情况。结果:治疗前,3组各量表评分差异无统计学意义(P0.05)。治疗1、3个月后,各组FMA、FTHUE、MBI和ADL评分均较同组治疗前改善(P0.05);其中,1月组各评分统计学差异最显著(P0.01)。治疗1、3个月后,中央前回第一运动区信号强度明显升高,1月组最明显(P0.01)。结论:神经松动术结合常规康复训练能有效的促进脑卒中偏瘫患者手运动功能的恢复,对卒中早期患者疗效更好。     

常染色体显性多囊肾病中泛素连接酶β-TrCP的表达及其作用和调控机制

目的观察常染色体显性多囊肾病(ADPKD)患者及动物模型中泛素连接酶β-TrCP的表达,初步探讨人囊肿衬里上皮OX161细胞中β-TrCP的作用和调控机制。方法在OX161细胞内转染β-TrCP的siRNA,采用蛋白免疫印迹法检测增殖细胞核抗原(PCNA)的表达,MTT比色法检测吸光度;在OX161细胞中抑制JAK2/STAT3通路或转染STAT3质粒,采用蛋白免疫印迹法检测JAK2、p-JAK2、STAT3、p-STAT3和β-TrCP的表达。收集ADPKD行肾脏切除术患者的肾脏组织和肾旁距癌组织5 cm以上的正常肾脏组织(正常对照组)、雄性Han:SPRD(cy/+)大鼠(ADPKD模型)和野生型Han:SPRD(+/+)大鼠肾组织(对照),均采用蛋白免疫印迹法检测β-TrCP的表达。收集Pkdlflox/flox;tamoxifen-Cre+小鼠(ADPKD模型)和Pkdlflox/flox;tamoxifen-Cre-小鼠(对照)肾组织,采用免疫组织化学法检测β-TrCP的表达和亚定位。结果与UCL93细胞相比,OX161细胞中JAK2/STAT3通路明显活化,β-TrCP表达量增加;β-TrCP敲减后细胞增殖减慢(P<0.05);WP1006抑制JAK2/STAT 3通路后,β-TrCP表达量减低,转染STAT 3质粒后,β-TrCP的表达量增加。与正常对照组、野生型Han:SPRD(+/+)大鼠及Pkdlflox/flox;tamoxifen-Cre-小鼠比较,ADPKD患者肾组织、Han:SPRD(Cy/+)大鼠肾组织及Pkdlflox/flox;tamoxifen-Cre+小鼠肾组织中β-TrCP表达量明显增加。结论在人囊肿衬里上皮OX161细胞株中β-TrCP表达量增多,且受JAK2/STAT3通路调控;在ADPKD患者及其模型鼠肾脏组织中β-TrCP表达量也增加。β-TrCP可能参与促进囊肿的发生和发展。     

罗格列酮对多囊肾囊肿衬里上皮细胞p38促分裂原活化蛋白激酶信号通路的影响

Objective To investigate the effect of rosiglitazone on p38 mitogen-activated protein kinase (p38MAPK) pathway in polycystic kidney cyst-lining epithelial cells. Methods The cyst-lining epithelial cells (PKD cells) from human polycystic kidney were treated with rosiglitazone (10 μmol/L), peroxisome proliferator-activated receptor-γ (PPARγ) inhibitor GW9662 (10 μmol/L), rosiglitazone (10 μmol/L) +GW9662 (10 μmol/L), p38MAPK specific inhibitor SB203580 (10 μmol/L), SB203580 (10 μmol/L)+ rosiglitazone(10 μmol/L) for 2 hours followed by epidermal growth factor (EGF) stimulation. Protein expressions of p38, phuspho-p38 (p-p38) and proliferating cell nuclear antigen (PCNA) were detected by Western blot. p38 mRNA was examined by RT-PCR. Expression of c-fos and c-jun was observed by immunocytochemistry. Results (1) EGF markedly up-regulated the expressions of p38, p-p38, PCNA, c-fos anti c-jun compared with control group (P<0.01). (2) Compared with EGF treated group, rosiglitazone significantly reduced p38 activation and mRNA expression (P<0.01, respectively). Rosiglitazone, rosiglitazone+SB203580 could significantly down-regulated p-p38, PCNA, c-fos and c-jun expression (P<0.01, respectively) with no significant difference between these two groups. (3) GW9662 partially reversed the reduction effect of rosiglitazone. Conclusions Rosiglitazone can inhibit proliferation of autosomal dominant polycystic kidney disease cyst-lining epithelial cells partially through down-regulating p38 activation and reducing c-fos, c-jun and PCNA expression. The above effect of rosiglitazone is in part PPARγ-independcnt.     

syndecan-4对碱性成纤维细胞生长因子诱导人肾小球系膜细胞增殖及细胞外基质分泌的影响

目的 研究syndecan-4对碱性成纤维细胞生长因子（bFGF）诱导人肾小球系膜细胞(HMC)增殖及细胞外基质（ECM）分泌的影响,并探讨syndecan-4-蛋白激酶Cα（PKCα）途径在其中的作用。 方法 免疫荧光方法观察syndecan-4在HMC上的表达。筛选有效的syndecan-4-siRNA转染HMC,噻唑蓝（MTT）比色法检测HMC在bFGF不同作用时间下的增殖差异;ELISA法检测细胞上清液中的Ⅰ、Ⅳ型胶原和纤连蛋白（FN）含量;荧光定量PCR观察syndecan-4和PKCα含量的变化。 结果　syndecan-4蛋白在HMC有表达。bFGF可促进HMC的增殖及FN、Ⅳ型胶原的分泌,使得每百万看家基因中PKCα拷贝数明显增加。转染syndecan-4 siRNA后,显著降低了HMC的增殖速度（48~60 h时点,P < 0.01）、ECM分泌（FN：24 h时点,P < 0.01,48~96 h,P < 0.05;Ⅳ型胶原：72~96 h时点,P < 0.05）及PKCα含量 （0 h时点,P < 0.05,12 ~48 h时点,P < 0.01）。 结论 syndecan-4参与调控bFGF诱导HMC的增殖及ECM分泌过程。syndecan-4-PKCα途径可能在其中扮演重要作用。     

罗格列酮对多囊肾囊肿衬里上皮细胞p38促分裂原活化蛋白激酶信号通路的影响

Objective To investigate the effect of rosiglitazone on p38 mitogen-activated protein kinase (p38MAPK) pathway in polycystic kidney cyst-lining epithelial cells. Methods The cyst-lining epithelial cells (PKD cells) from human polycystic kidney were treated with rosiglitazone (10 μmol/L), peroxisome proliferator-activated receptor-γ (PPARγ) inhibitor GW9662 (10 μmol/L), rosiglitazone (10 μmol/L) +GW9662 (10 μmol/L), p38MAPK specific inhibitor SB203580 (10 μmol/L), SB203580 (10 μmol/L)+ rosiglitazone(10 μmol/L) for 2 hours followed by epidermal growth factor (EGF) stimulation. Protein expressions of p38, phuspho-p38 (p-p38) and proliferating cell nuclear antigen (PCNA) were detected by Western blot. p38 mRNA was examined by RT-PCR. Expression of c-fos and c-jun was observed by immunocytochemistry. Results (1) EGF markedly up-regulated the expressions of p38, p-p38, PCNA, c-fos anti c-jun compared with control group (P<0.01). (2) Compared with EGF treated group, rosiglitazone significantly reduced p38 activation and mRNA expression (P<0.01, respectively). Rosiglitazone, rosiglitazone+SB203580 could significantly down-regulated p-p38, PCNA, c-fos and c-jun expression (P<0.01, respectively) with no significant difference between these two groups. (3) GW9662 partially reversed the reduction effect of rosiglitazone. Conclusions Rosiglitazone can inhibit proliferation of autosomal dominant polycystic kidney disease cyst-lining epithelial cells partially through down-regulating p38 activation and reducing c-fos, c-jun and PCNA expression. The above effect of rosiglitazone is in part PPARγ-independcnt.     

罗格列酮对多囊肾囊肿衬里上皮细胞p38促分裂原活化蛋白激酶信号通路的影响

Objective To investigate the effect of rosiglitazone on p38 mitogen-activated protein kinase (p38MAPK) pathway in polycystic kidney cyst-lining epithelial cells. Methods The cyst-lining epithelial cells (PKD cells) from human polycystic kidney were treated with rosiglitazone (10 μmol/L), peroxisome proliferator-activated receptor-γ (PPARγ) inhibitor GW9662 (10 μmol/L), rosiglitazone (10 μmol/L) +GW9662 (10 μmol/L), p38MAPK specific inhibitor SB203580 (10 μmol/L), SB203580 (10 μmol/L)+ rosiglitazone(10 μmol/L) for 2 hours followed by epidermal growth factor (EGF) stimulation. Protein expressions of p38, phuspho-p38 (p-p38) and proliferating cell nuclear antigen (PCNA) were detected by Western blot. p38 mRNA was examined by RT-PCR. Expression of c-fos and c-jun was observed by immunocytochemistry. Results (1) EGF markedly up-regulated the expressions of p38, p-p38, PCNA, c-fos anti c-jun compared with control group (P<0.01). (2) Compared with EGF treated group, rosiglitazone significantly reduced p38 activation and mRNA expression (P<0.01, respectively). Rosiglitazone, rosiglitazone+SB203580 could significantly down-regulated p-p38, PCNA, c-fos and c-jun expression (P<0.01, respectively) with no significant difference between these two groups. (3) GW9662 partially reversed the reduction effect of rosiglitazone. Conclusions Rosiglitazone can inhibit proliferation of autosomal dominant polycystic kidney disease cyst-lining epithelial cells partially through down-regulating p38 activation and reducing c-fos, c-jun and PCNA expression. The above effect of rosiglitazone is in part PPARγ-independcnt.     

罗格列酮对多囊肾囊肿衬里上皮细胞p38促分裂原活化蛋白激酶信号通路的影响

Objective To investigate the effect of rosiglitazone on p38 mitogen-activated protein kinase (p38MAPK) pathway in polycystic kidney cyst-lining epithelial cells. Methods The cyst-lining epithelial cells (PKD cells) from human polycystic kidney were treated with rosiglitazone (10 μmol/L), peroxisome proliferator-activated receptor-γ (PPARγ) inhibitor GW9662 (10 μmol/L), rosiglitazone (10 μmol/L) +GW9662 (10 μmol/L), p38MAPK specific inhibitor SB203580 (10 μmol/L), SB203580 (10 μmol/L)+ rosiglitazone(10 μmol/L) for 2 hours followed by epidermal growth factor (EGF) stimulation. Protein expressions of p38, phuspho-p38 (p-p38) and proliferating cell nuclear antigen (PCNA) were detected by Western blot. p38 mRNA was examined by RT-PCR. Expression of c-fos and c-jun was observed by immunocytochemistry. Results (1) EGF markedly up-regulated the expressions of p38, p-p38, PCNA, c-fos anti c-jun compared with control group (P<0.01). (2) Compared with EGF treated group, rosiglitazone significantly reduced p38 activation and mRNA expression (P<0.01, respectively). Rosiglitazone, rosiglitazone+SB203580 could significantly down-regulated p-p38, PCNA, c-fos and c-jun expression (P<0.01, respectively) with no significant difference between these two groups. (3) GW9662 partially reversed the reduction effect of rosiglitazone. Conclusions Rosiglitazone can inhibit proliferation of autosomal dominant polycystic kidney disease cyst-lining epithelial cells partially through down-regulating p38 activation and reducing c-fos, c-jun and PCNA expression. The above effect of rosiglitazone is in part PPARγ-independcnt.     

中心夜间透析和传统透析患者循环成纤维细胞生长因子23水平差异及影响因素

目的 比较中心夜间血液透析(INHD)和传统血液透析(CHD)患者循环成纤维细胞生长因子23(FGF23)水平差异并探讨影响患者FGF23水平的因素.方法 收集第二军医大学长征医院44例INHD及64例CHD患者空腹静脉血,测定患者钙、磷、全段甲状旁腺素(iPTH)、尿素清除指数(Kt/V)、25-羟维生素D、铁蛋白、血红蛋白、血脂、FGF23等指标并比较两组各指标的差异,采用多重线性回归分析探究FGF23的影响因素,Pearson相关性分析探究FGF23与血钙、血磷、钙磷乘积、iPTH、25-羟维生素D、Kt/V的相关性.结果 INHD患者血磷较CHD患者低(P＜0.05),INHD透析充分性明显高于CHD,INHD患者循环FGF23水平低于CHD患者(P＜0.05).FGF23与钙、磷、钙磷乘积相关(P＜0.01),与iPTH及Kt/V不相关.结论 INHD比CHD更能有效改善慢性肾脏病-矿物质和骨异常(CKD-MBD)参数.钙磷乘积是血液透析患者FGF23水平的独立影响因素,INHD通过减少钙磷乘积降低血清FGF23水平可能是其改善患者临床预后的机制之一.     

“三点”注射神经阻滞麻醉法在下肢手术中的应用和体会

<正> 三点神经阻滞麻醉包括:股神经、股外侧皮神经、坐骨神经,本组于一九八三年中应用此麻醉方法对下肢十八例患者施行了手术,现介绍如下: 一、解剖复习:1、股神经:第二三四脊神经向下在第四腰椎横突处组成腰丛,股神经是腰丛中最大的分支,位于腰大肌及髂