泪腺上皮细胞凋亡与丙酸睾酮的抑制效应

目的:分析丙酸睾酮抑制泪腺上皮细胞凋亡的机制,为干眼症的性激素防治提供科学的理论依据。方法:实验于2003-09/2004-01在重庆医科大学附属第一医院中心实验室完成。①实验材料:二三月龄新西兰健康白色家兔30只,无眼疾,雌性,体质量1.5～2.5kg。丙酸睾酮(产品批号:020902,上海通用药业股份有限公司产品),用DMSO配成储存液,-70℃保存。分析纯过氧化氢购自北方同正生物技术发展有限公司。②实验方法:经耳缘静脉注射空气处死家兔,按常规无菌操作行泪腺摘除术,酶消化法分离兔泪腺上皮细胞。取二三代培养的泪腺上皮细胞,按约1×108L-1密度接种于预置小玻片的24孔板内,培养4d后,加入浓度为1×10-8,1×10-7,1×10-6,1×10-5mol/L丙酸睾酮,继续培养24h,另以未加丙酸睾酮的培养液培养的同期传代的泪腺上皮细胞作空白对照。培养24h后,再分别加入终浓度为100μmol/LH2O2于不同浓度丙酸睾酮组继续培养60min,另以未加丙酸睾酮的DMEM/F12培养液培养的同期传代的泪腺上皮细胞作对照。③实验评估:应用TUNEL法检测泪腺上皮细胞凋亡,采用免疫细胞化学法检测泪腺上皮细胞Bax和Bcl-2表达。结果:①丙酸睾酮对过氧化氢诱导的泪腺上皮细胞凋亡的影响:与单纯加入100μmol/LH2O2组比较,加入不同浓度丙酸睾酮组泪腺上皮细胞凋亡率均明显下降且丙酸睾酮的抗凋亡作用呈现一定范围内的剂量依赖性[(15.15±1.05)%,(13.94±0.73)%,(12.51±0.91)%,(10.63±0.78)%,(8.19±0.85)%,P<0.01]。②免疫细胞化学法检测Bcl-2和Bax蛋白表达:与单纯加100μmol/LH2O2组相比较,同时加入1×10-8,1×10-7,1×10-6,1×10-5mol/L丙酸睾酮各组均可使泪腺上皮细胞Bcl-2的表达明显增强和Bax的表达明显减弱,且与丙酸睾酮的剂量呈正相关(Bcl-2:0.92±0.10,1.01±0.08,1.12±0.06,1.24±0.12,1.37±0.11;Bax:2.28±0.09,2.17±0.11,2.06±0.07,1.91±0.13,1.77±0.10,P<0.01)。结论:丙酸睾酮抑制泪腺上皮细胞凋亡的机制可能与凋亡调节相关基因Bcl-2表达上调和Bax表达下调有关。     

大鼠眼外肌成肌细胞的增殖及分化特征

目的:完善眼外肌成肌细胞体外培养、鉴定的方法及观察其生物学特性。方法:实验于2005-02/08在青岛大学医学院附院中心实验室(省级实验室)完成。取出生后3～7d的大鼠,通过大鼠眼外肌细胞的取材、分离、消化、培养等技术,观察细胞的形态、生长曲线、细胞融合率,观察大鼠眼外肌卫星细胞的增殖与分化能力,利用成肌细胞标记物α-横纹肌肌动蛋白、中间丝结蛋白免疫细胞化学染色对所获得的细胞进行鉴定。结果:①成肌细胞的生长情况:在生长培养基作用下,细胞增殖旺盛;在分化培养基条件下,细胞分化良好,可融合成肌管。②成肌细胞融合率:在24h和48h融合率提高明显,72h达高峰,之后不再变化。③细胞免疫化学检测结果:α-横纹肌肌动蛋白和中间丝结蛋白免疫细胞化学染色阳性。结论:体外培养的大鼠眼外肌卫星细胞具有良好的增殖与分化能力,其生物学特征同骨骼肌卫星细胞。     

鸡胚胎素对小鼠红细胞数量及免疫器官质量的影响

目的:建立血虚和免疫抑制动物模型,观察鸡胚胎低温提取物对其红细胞造血以及免疫器官质量的影响。方法:实验于2001-04/2002-09在新乡医学院药物研究室完成。①实验材料:健康昆明种小鼠50只,雌雄各半。鸡胚胎素[中国发明专利公开(公告)号:CN1748713],符合研究者申报专利时提出的质量检验标准。②鸡胚胎素对血虚模型小鼠红细胞数值及血红蛋白含量的影响:取20只小鼠,随机排列表法分为鸡胚胎素组、模型对照组,10只/组,建立失血性血虚动物模型。失血后24h当红细胞数<3.2×1012L-1、血红蛋白含量<84g/L,且小鼠外观出现皮色苍白、食欲不振等现象时,代表造模成功。次日,鸡胚胎素组给予鸡胚胎素5g/(kg·d)灌胃,模型对照组给予生理盐水20mL/(kg·d)灌胃,连续14d。分别于失血前、失血后24h、末次给鸡胚胎素后2h尾部采血测定两组红细胞数量及血红蛋白含量的变化。③鸡胚胎素对免疫抑制模型小鼠免疫器官质量的影响:取30只小鼠,随机排列表法分为鸡胚胎素组、模型对照组、正常对照组,10只/组。鸡胚胎素组给予鸡胚胎素5g/(kg·d)灌胃,模型对照组和正常对照组均灌服等量生理盐水,连续14d。在第11天上午鸡胚胎素灌胃2h后,鸡胚胎素组、模型对照组腹腔注射环磷酰胺60mg/(kg·d),连续4d,复制免疫抑制动物模型。末次给予环磷酰胺2h后颈椎脱位法处死小鼠,计算胸腺、脾脏质量指数。结果:50只小鼠均进入结果分析。①失血前及失血后24h鸡胚胎素组与模型对照组的红细胞数值、血红蛋白含量基本相似(P>0.05)。末次给鸡胚胎素后2h与模型对照组比较,鸡胚胎素组红细胞数值、血红蛋白含量均明显升高(t=3.39,P<0.01;t=2.52,P<0.05)。②末次给环磷酰胺2h后与模型对照组比较,鸡胚胎素组、正常对照组的胸腺质量指数和脾脏质量指数均明显升高(t=6.62,P<0.01;t=2.47,P<0.05)。结论:鸡胚胎素灌胃对血虚小鼠具有较好的促红细胞造血功能,同时对环磷酰胺造成的免疫器官质量下降具有明显的增重作用。     

High expression levels of IKK alpha and IKK beta are necessary for the malignant properties of liver cancer

IKK-NF-kappa B signaling is regarded as an important factor in hepatocarcinogenesis and a potential target for liver cancer therapy.Therefore,in this study,we analyzed the expression of mRNAs encoding components and targets of NF-kappa B signaling including IKK alpha,IKK beta,RANK,RANKL,OPG,CyclinD3,mammary serine protease inhibitor(Maspin),CyclinD1,c-FLIP,Bcl-x1,Stat3,Cip1 and Cip2 by real-time PCR in 40 patients with liver cancer.After statistical analysis,7 indices including IKK alpha,IKK beta,RANK,Maspi...     

Mutation-specific effects of lidocaine in Brugada syndrome

Brugada syndrome (BrS) is a hereditary cardiac disease characterized by right bundle-branch block, an elevation of the ST-segment in leads V1 through V3 on the electrocardiogram, and ventricular fibrillation that can lead to sudden cardiac death. Mutations in the cardiac sodium channel gene SCN5A, which encodes the alpha-subunit of the human cardiac voltage-dependent Na+ channel (Na(v)1.5), are identified in 15-30% of patients with BrS. Most SCN5A mutations lead to a 'loss-of-function' phenotype, reducing the Na+ current during the early phases of the action potential. Anti-arrhythmic drugs that affect Na+ channels typically block these Na+ channels, thereby exaggerating the ECG abnormalities and arrhythmogenicity in the BrS. However, the N406S mutation in SCN5A causes distinct gating defects and enhanced intermediate inactivation of Na+ channels, which led to unexpected pharmacological effects of lidocaine in a patient carrying this mutation. In the presence of the N406S mutation, use-dependent block by lidocaine is reduced and recovery from intermediate inactivation is hastened by lidocaine. These findings suggest that lidocaine may improve the Brugada phenotype in patients with N406S by increasing the availability of Na+ channels.     

Ryanodine receptors as pharmacological targets for heart disease

Calcium release from intracellular stores plays an important role in the regulation of muscle contraction and electrical signals that determine the heart rhythm. The ryanodine receptor （RyR） is the major calcium （Ca^2＋） release channel required for excitation-contraction coupling in the heart. Recent studies have demonstrated that RyR are macromolecular complexes comprising of 4 pore-forming channel subunits, each of which is associated with regulatory subunits. Clinical and experimental studies over the past 5 years have provided compelling evidence that intracellular Ca^2＋ release channels play a pivotal role in the development of cardiac arrhythmias and heart failure. Changes in the channel regulation and subunit composition are believed to cause diastolic calcium leakage from the sarcoplasmic reticulum, which could trigger arrhythmias and weaken cardiac contractility. Therefore, cardiac RyR have emerged as potential therapeutic targets for the treatment of heart disease. Consequently, there is a strong desire to identify and/or develop novel pharmacological agents that may target these Ca^2＋ signaling pathways. Pharmacological agents known to modulate RyR in the heart, and their potential application towards the treatment of heart disease are discussed in this review.     

9-(2-膦酰甲氧乙基)腺嘌呤及其位置异构体3-(2-膦酰甲氧乙基)腺嘌呤的合成和抗病毒活性研究

Phosphonylmethoxyethyl)adenine (1),PMEA,an acyclic nucleotide withbroad-spectrum antiviral activity was synthesized with some modifications of Holy's procedure.Simutaneously,an N-3 regioisomer(2)of PMEA and a by-preduct, formaldehyde di-[2-(9-adenyl)ethyl] acetal(7)were seperated by silica gel chromatography in the ratio of 50:10:1.Compound(2)and(7) are new compounds that we have not yet found in literatures. The structure of them weredetermined with ¹HNMR,2DNMR, MS and Spot test.Antiviral test showed that N-3 isomer(2)completely lost activity against both HIV-1 and HSV-1 in vitro. It seems that regiospecificity of theacyclic nucleotide structure is important for antiviral activity.     

5种注射剂的细菌内毒素检查法初步考察

目的：考察５种注射剂盐酸林可霉素注射液，盐酸精氨酸注射液，注射用玻璃酸酶，肝素钠注射液和顺铂注射液的细菌内毒素检查法可行性。方法：中国药典细菌内毒素检查法。结果；除ｄ外，其余原液及稀释液均于有干扰作用。结论：ｄ的原液及ａ的２０倍以上稀释液可用灵敏度为０．５ＥＵ／ｍｌ的鲎试剂作细菌内毒素检查，而ｂ，ｃ和ｅ尚需进一步探讨。     

d-生物素的立体专一性全合成研究

对confaione的d-生物素(1)全合成进行了改进,从半胱氨酸计算,总收率3.7%。5与4-溴代三苯膦丁酸甲酯进行Wittig-Schlosser缩合反应可立体专一性地转化成反式-烯(6)。以叠氮三甲基硅烷代替叠氮化锂亲核进攻9分子中C3-溴原子,可显著地提高顺式-叠氮内酰胺10的收率。