Comparison of the unlabeled and labeled pre-mRNA splicing assays in vitro

Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome that catalyzes the removal of non-coding intron sequences to ligate exons into mature mRNA prior to transport and translation. The purpose of our study is to explore whether the in vitro unlabeled pre-mRNA splicing assay could be performed as an alternative method of splicing reaction other than the radiolabeled one. Two different splicing methods in vitro , P labeled and unlabeled pre-mRNA as the substrates in the reaction, were investigated. The radiolabeled products were visualized by autoradiography while the unlabeled products were observed by Ethidium Bromide (EB) staining. As a result, although there are more unspecific bands in the EB staining assay than 32P labeled one, the RNA products of in vitro splicing could be observed clearly. This suggests that the unlabeled pre-mRNA splicing assay can be an optional substitution for the isotope-labeled assay.     

噻托溴铵治疗中重度支气管哮喘30例临床观察

目的:探讨噻托溴铵在中重度支气管哮喘治疗中的意义。方法:将60例中重度支气管哮喘患者随机分为观察组和对照组,两组患者均依据2009 GINA规范治疗3个月,其中观察组每天增加吸入一次噻托溴铵18μg。比较两组患者治疗前后性别、年龄、病程、哮喘控制测试(ACT)评分、血清免疫球蛋白E(IgE)、嗜酸细胞计数(EOS)、吸入支气管扩张剂后一秒钟用力呼气量(FEV1)、用力呼气中期流速(FEF25%~75%)值、用力呼气峰速(PEF)及每周急救药使用次数等指标变化并进行统计学分析。结果:①两组间患者性别、年龄、病程、治疗前各指标差异无统计学意义(P0.05)。②治疗后两组各指标与治疗前比较,差异有统计学意义(P0.01)。③治疗后,观察组的ACT评分、FEV1、PEF、FEF及每周急救药使用喷数与对照组比较,差异有统计学意义(P0.01)。治疗后观察组EOS、IgE与对照组比较,差异无统计学意义(P0.05)。结论:噻托溴铵治疗中重度支气管哮喘有效,且以扩张气道、改善症状和肺功能为主,其是否有非特异性抗炎作用还需进一步研究论证。     

噻托溴铵粉吸入剂治疗148例支气管扩张症的效果评定

目的观察噻托溴铵粉吸入剂应用于临床治疗支气管扩张症的效果。方法选取我院治疗的148例支气管扩张症患者,随机分为观察组和对比组,对比组给予常规的支气管扩张症治疗;观察组在进行常规治疗的同时给予噻托溴铵粉吸入剂进行治疗。结果治疗后观察组和对比组的BODE指数和临床症状均有改善,但观察组优于对比组,P0.05,差异具有统计学意义。结论使用噻托溴铵粉吸入剂治疗支气管扩张症疗效显著。     

苯扎溴铵酊处方改进及苯扎溴铵的含量测定

目的：改进苯扎溴铵酊处方,并建立容量分析法测定苯扎溴铵醇溶液（苯扎溴铵酊去除曙红成分）中苯扎溴铵的含量。方法在建立容量分析法测定苯扎溴铵的含量时,发现曙红在不同程度上均能影响含量测定结果,故将曙红成分从处方中删去。采用四苯硼钠（0.02 mol/L）为滴定液,滴定苯扎溴铵醇溶液中苯扎溴铵的含量。结果苯扎溴铵酊制剂更改为苯扎溴铵醇溶液,在含量测定中,滴定终点变化明显,易于判断。苯扎溴铵在浓度0.4~1.4 mg/mL的范围内与消耗四苯硼钠滴定液体积呈良好线性关系,r=0.9995,回归方程为V=18.3C-0.32。高、中、低3种剂量苯扎溴铵的平均回收率为99.12%,R SD为0.84%。结论本实验方法简便、快捷、准确,可作为苯扎溴铵醇溶液的质量控制标准。     

Flow cytometry evaluation of in vitro cellular necrosis and apoptosis induced by silver nanoparticles

Particles possess unique properties in the nanoscale, e.g., enhanced catalytic activity, high surface area, and light emission/absorption properties, that might result in interference with colorimetric in vitro cytotoxicity assays such as MTT, XTT or MTS. Alternatively, assays that do not use spectrophotometric detection, such as trypan blue exclusion or flow cytometry (FC) based assays, are less likely to be influenced by nanoparticle interference. The aim of this study was to evaluate FC assays to assess the cytotoxicity of three different sizes (10, 100, or 200 nm) of silver nanoparticles (AgNPs) at different mass concentrations (1, 25, or 50 ug/ml) in L-929 fibroblast cells. After 4 h and 24 h exposure, cell necrosis and apoptosis were assessed using 7-AAD and Annexin V dyes, respectively, with FC. The data indicate that cell necrosis and apoptosis in AgNP-exposed fibroblasts depends on dose, exposure time, and AgNP size. The data indicate that AgNPs produced a dose- and time-dependent decrease in cell viability; however, 10 nm AgNPs were significantly more toxic than larger-sized particles. Thus, standard FC assays can be utilized to assess apoptosis and necrosis in response to nanomaterial exposure.     

联合应用支气管扩张剂治疗慢性阻塞性肺疾病合并呼吸衰竭的疗效观察

目的：探讨联合应用支气管扩张剂治疗慢性阻塞性肺疾病（ chronic obstructive pulmonary disease,COPD）合并呼吸衰竭的临床效果。方法：将126例COPD合并呼吸衰竭患者以数字随机表法分为2组各63例,对照组患者给予噻托溴铵与盐酸氨溴索吸入治疗,观察组患者在对照组的基础上辅以沙丁胺醇吸入治疗,对比观察2组患者的临床疗效。结果：观察组患者的有效率为92．06％（58／63）,明显高于对照组的79．37％（50／63）,差异有统计学意义（P＜0．05）;治疗后,观察组患者的氧分压、一秒用力呼气容积及其占预计值百分比均明显高于对照组,二氧化碳分压明显低于对照组,差异均有统计学意义（ P＜0．05）。结论：联合应用支气管扩张剂治疗COPD合并呼吸衰竭,能纠正呼吸衰竭,改善肺功能。     

米氮平联合奥替溴铵及复合乳酸菌胶囊治疗难治性腹泻型肠易激综合征的疗效观察

目的：观察米氮平联合奥替溴铵及复合乳酸菌胶囊治疗难治性腹泻型肠易激综合征（RIBS-D）的临床疗效。方法：82例RIBS-D患者随机分为试验组（43例）与对照组（39例）,两组均给奥替溴铵及复合乳酸菌胶囊,试验组加服米氮平,对照组加服安慰剂,疗程4周。结果：试验组与对照组的总有效率分别为95.35％、12.82％（P＜0.01）;试验组焦虑及抑郁评分较对照组显著降低,生活质量评分较对照组显著升高,差异均有统计学意义（P＜0.01）。结论：米氮平联合奥替溴铵及复合乳酸菌胶囊治疗RIBS-D患者,能明显改善症状、焦虑抑郁状态及生活质量,十分安全。     

噻托溴铵治疗慢性阻塞性肺疾病作用机制研究进展

噻托溴铵是一种新型长效抗胆碱类药物,是目前第一个能每日用药1次的吸人性药物,能高度选择性作用于胆碱M1和M3受体,表现出强大的支气管扩张作用,吸入一次疗效持续24 h以上.临床实验结果表明该药对中、重度慢性阻塞性肺疾病具有良好疗效,与其他常用抗胆碱能药物和β2受体激动剂相比较,噻托溴铵可显著改善肺功能、减少急性发作和改善生命质量,耐受性和安全性较好,主要不良反应为口干.     

The functional impact of adding salmeterol and tiotropium in patients with stable COPD

The aim of this double-blind, double-dummy, crossover, randomised, pilot study was to explore the acute effects of adding salmeterol and tiotropium in patients with stable COPD. A total of 20 outpatients with stable COPD were enrolled. Single doses of 18-microg tiotropium, 50-microg salmeterol, and 18-microg tiotropium+ 50-microg salmeterol were given. Serial measurements of forced expiratory volume in 1 s (FEV1) were performed over 24h. The mean maximum increases in FEV1 from pre-dosing value on each of the dosing days were 0.165l (95% CI: 0.098-0.232) for tiotropium, 0.241 l (95% CI: 0.151-0.332) for salmeterol, and 0.290 l (95% CI: 0.228-0.353) for the combination and occurred 4 h after inhalation of tiotropium or salmeterol and 3 h after the combination. At 12h, the mean increases in FEV1 from pre-dosing value were 0.071 l (95% CI: 0.001-0.141; P = 0.047) for tiotropium, 0.069 l (95% CI: 0.018-0.120; P = 0.010) for salmeterol, and 0.108 l (95% CI: 0.047-0.170; P = 0.001) for the tiotropium + salmeterol combination. Only the difference between salmeterol and tiotropium + salmeterol was statistically significant (P = 0.009). At 24h, the mean FEV1 value was still higher than the mean pre-dosing value for tiotropium (0.042 l; 95% CI: -0.012-0.097; P=0.119) and the tiotropium+salmeterol combination (0.051 l; 95% CI: 0.01 5-0.087; P = 0.007), but not for salmeterol alone (-0.013 l; 95% CI: -0.041-0.014; P = 0.324). The FEV1 area under the curve (AUCs0-12h) were 1.657 l (95% CI: 1.152-2.162) for tiotropium, 2.068 (95l CI: 1.385-2.752) for salmeterol, and 2.541 l (95% CI: 1.954-3.129) for tiotropium + salmeterol. Only the difference between tiotropium and the tiotropium +salmeterol combination was statistically significant (P = 0.01). The FEV1 AUCs0-24h were 2.854 l (95% CI: 1.928-3.780) for tiotropium, 2.786 l (95% CI: 1.913-3.660) for salmeterol, and 3.640 l (95% CI: 2.674-4.605) for tiotropium + salmeterol. ALL differences between treatments were not statistically significant (P> 0.05). These results seem to indicate that the use of the tiotropium + salmeterol combination is more efficacious than the single agents alone, but the once-daily administration of the two drugs is inadvisable due to the broncholytic profile of salmeterol.     

Tiotropium bromide inhibits relapsing allergic asthma in BALB/c mice

Recurrent relapses of allergic lung inflammation in asthmatics may lead to airway remodeling and lung damage. We tested the efficacy of tiotropium bromide, a selective long-acting, muscarinic receptor antagonist as an adjunct therapy in relapses of allergic asthma in mice. We compared the effectiveness of local intranasal administration of tiotropium and dexamethasone in acute and relapsing allergic asthma in BALB/c mice. Although tiotropium at low doses is a potent bronchodilator, we tested higher doses to determine effectiveness on inflammation and mucus hypersecretion. A 5-day course of twice daily intranasal tiotropium or dexamethasone (1 mg/kg (b.w.)) suppressed airway eosinophils by over 87% during disease initiation and 88% at relapse compared to vehicle alone. Both drugs were comparable in their capacity to suppress airway and parenchymal inflammation and mucus hypersecretion, though tiotropium was better than dexamethasone at reducing mucus secretion during disease relapse. Despite treatment with either drug, serum antigen-specific IgE or IgG1 antibody titres remained unchanged. Our study indicates that tiotropium at higher doses than required for bronchodilation, effectively suppresses inflammation and mucus hypersecretion in the lungs and airways of mice during the initiation and relapse of asthma. Tiotropium is currently not approved for use in asthma. Clinical studies have to demonstrate the efficacy of tiotropium in this respiratory disease.