卡维地洛对体外柯萨奇病毒B3致心肌细胞凋亡与Bcl-2/Bax蛋白表达的影响

目的探讨卡维地洛对体外柯萨奇病毒B3(CVB3)致心肌细胞凋亡的抑制作用及其机制。方法采用差速贴壁法培养原代Wistar乳鼠心肌细胞。实验分为正常对照组、病毒组、卡维地洛组及美托洛尔组。正常对照组仅加入不含血清的DMEM培养液,后3组于CVB3感染心肌细胞后分别加入不含血清的DMEM培养液、无血清培养基配制的卡维地洛、美托洛尔溶液。72h后在倒置相差显微镜下观察各组心肌细胞的形态、搏动速率;采用黄嘌呤氧化酶法测定其心肌细胞培养液中超氧化物歧化酶(SOD)活性,硫代巴比妥酸法测定其心肌细胞培养液中丙二醛(MDA)水平,流式细胞术检测其心肌细胞凋亡率,免疫细胞化学法检测其心肌细胞内Bcl-2及Bax蛋白表达水平。结果1.与病毒组比较,卡维地洛组心肌细胞搏动速率明显提高(P<0.05)、凋亡率明显降低(P<0.05),心肌细胞培养液中SOD活性显著升高(P<0.05)、MDA水平明显降低(P<0.05);美托洛尔组与病毒组比较,心肌细胞搏动速率、凋亡率、心肌细胞培养液中SOD活性、MDA水平均无统计学差异(Pa>0.05);2.与病毒组及美托洛尔组比较,卡维地洛组心肌细胞内Bcl-2蛋白表达增多,而Bax蛋白表达减...     

钙敏感受体在缺氧/复氧诱导乳鼠心肌细胞凋亡中的作用

目的 探讨钙敏感受体(CaSR)对缺氧/复氧(A/R)所诱导乳鼠心肌细胞凋亡的影响及其机制.方法 原代培养乳鼠心室肌细胞缺氧2 h/复氧24 h,建立A/R心肌细胞损伤模型.心肌细胞随机分为三组:对照组、A/R组和CaSR激动剂氯化钆(GdCl3)组.四氮唑嗅盐法(MTT)检测细胞的存活率,原位缺口末端标记法(TUNEL)分析细胞凋亡率,蛋白印迹法检测CaSR、半胱氨酸天冬酶(caspase)-3、9和细胞色素c(Cytc)蛋白的表达.结果 TUNEL染色显示A/R后心肌细胞凋亡为(17±3)%,与对照相比,心肌细胞凋亡明显增加,CaSR表达也增加.CaSR激动剂GdCl3使心肌细胞凋亡进一步增加至(28±4)%,MTT检测表明心肌细胞存活率仅为(51.2±6.8)%;CaSR、caspase-3、caspase-9和Cytc表达进一步上调,明显高于A/R组.结论 CaSR通过线粒体Cytc-caspase-3途径参与了A/R所诱导的心肌细胞凋亡.     

川芎嗪对柯萨奇B3病毒感染乳鼠心肌细胞的保护作用及信号转导机制研究

目的：临床显示川芎嗪对病毒性心肌炎有治疗作用,但机制不明,该研究拟探讨川芎嗪对病毒感染的心肌细胞的作用及信号转导机制。方法：利用培养的Sprague-Dawley乳鼠心肌细胞,分为空白对照组（心肌细胞正常培养）、阴性对照组（心肌细胞加入川芎嗪至终浓度100 μmol/L后培养）、病毒感染组［心肌细胞感染柯萨奇病毒B3(CVB3)］、川芎嗪干预组(心肌细胞感染病毒后加入终浓度100 μmol/L川芎嗪干预)。观察心肌细胞搏动频率及培养液中乳酸脱氢酶(LDH)活性。采用Western blot测定NF-κB蛋白表达变化,MTT法检测心肌细胞活性。结果：病毒感染组与川芎嗪干预组比较,心肌细胞搏动频率明显降低(32.0±3.6次/min vs 84.3±3.5次/min, P<0.01),细胞病变加重,LDH活性显著升高(P<0.05)。川芎嗪干预组细胞NF-κB蛋白表达明显低于病毒感染组(P<0.01),存活率明显高于病毒感染组［(86.7±2.7）％ vs （35.3±3.4）%, P<0.01］。结论：在病毒性心肌炎时川芎嗪可以抑制NF-κB的表达,起到保护心肌细胞的作用。［中国当代儿科杂志,2009,11（8）：687-690］     

FTY720对柯萨奇B3病毒感染大鼠心肌细胞的保护作用及信号转导机制研究

目的　观察新型免疫抑制剂FTY72 0对病毒性心肌炎 (VM)的保护作用 ,探讨其治疗VM的分子机制。方法　建立实验性VM模型。采用Westernblot法分别测定ERK1 / 2蛋白 ,磷酸化ERK1 / 2 (P ERK1 / 2 )的表达变化。结果　FTY72 0能明显减少柯萨奇B3病毒 (CVB3)感染后心肌酶释放 ,心肌细胞搏动较病毒感染组维持时间长 ,心肌细胞病变发展亦较缓慢 ,病毒滴度显著低于病毒感染组。而且感染CVB3后FTY 72 0治疗组的心肌细胞内P ERK1 / 2活性持续降低。结论　FTY72 0对CVB3病毒感染的乳鼠心肌细胞有明显的保护作用 ,它可以通过抑制ERK1 / 2信号通路使病毒复制能力降低 ,从而抑制CVB3基因的表达和复制。     

骨膜蛋白在实验性病毒性心肌炎小鼠的表达及意义

目的 探讨骨膜蛋白(periostin)在病毒性心肌炎(VMC)中的表达及可能作用.方法 应用柯萨奇病毒B3(CVB3)感染4周龄Balb/c小鼠,建立VMC小鼠模型.100只小鼠分为正常对照组(n ＝ 40)和病毒组(n ＝ 60).在CVB3感染后第0、7、14、28、56天留取标本,测定各实验组不同时间点心肌胶原容积分数(CVF)值(Masson染色)、血清血管紧张素(Ang)Ⅱ水平(ELISA法)、心肌组织中骨膜蛋白和转化生长因子(TGF)β1 mRNA相对表达量(RT-PCR).将骨膜蛋白 mRNA相对表达量与其他指标进行直线相关性分析.结果 VMC组小鼠骨膜蛋白mRNA表达量从病毒感染后第7天开始增加,第28天持续升高,第56天时表达量最高(P ＜ 0.01).骨膜蛋白mRNA表达量与CVF、血清AngⅡ、TGFβ1 mRNA相对表达量均呈直线正相关(r ＝ 0.870 ～ 0.943,P均＜ 0.01).结论 骨膜蛋白的异常表达在VMC的发生、发展中起重要作用,可能参与了VMC心肌纤维化的发生过程.同时骨膜蛋白与AngⅡ及TGF-β1表达相关,可能成为抗VMC心肌纤维化的新靶点.     

结缔组织生长因子在病毒性心肌炎心肌组织中的变化

目的 观察结缔组织生长因子(connective tissue growth factor,CTGF)在病毒性心肌炎( viral myocarditis,VMC)小鼠心肌组织中的表达情况.方法 实验选取BALB/C小鼠100只,分为对照组、VMC组各50只.应用柯萨奇B3病毒(coxsackie virus B3,CVB3)感染小鼠建立VMC模型.在CVB3感染后第4、7、14、21天各随机抽取8只采集心脏标本,行HE染色测定心肌病理积分,应用Masson染色法观察小鼠心肌胶原纤维分布,测量胶原容积分数(collagen volume fraction,CVF),检测血清CK-MB水平,应用免疫组织化学方法检测转化因子β1(transforming growth factor-β1,TGF-β1)、CTGF蛋白在小鼠心肌中的分布;应用RT-PCR方法检测小鼠心肌组织TGF-β1、CTGF mRNA的相对表达量;并作相关性分析.结果 (1)VMC组小鼠CK-MB水平于第7天达高峰,后逐渐下降(各时间点分别为455.45±37.95,606.95±35.64,573.62±42.90,308.60±20.49),其中第4、7、14天时高于对照组,差异有统计学意义(t值分别为6.144,12.558,11.182,P均＜0.01);(2)VMC组小鼠第14、21天时CVF明显高于对照组(CVF值分别为8.22±1.95,9.46±1.87,t值分别为4.486,5.552,P均＜0.01);(3)免疫组织化学法检测VMC组小鼠各时间点心肌CTGF蛋白(各时间点分别为171.50±10.25,141.70±10.863,110.35±11.051,81.05±10.190)及TGF-β1蛋白(184.90±11.480,150.25±9.915,103.50±10.455,84.15 ±9.848)阳性表达均较对照组高(P均＜0.01);(4)VMC小鼠心肌组织中CTGF mRNA(0.4728±0.0328,0.5750±0.0439,0.6228±0.0458,0.7265±0.0469)及TGF-β1mRNA(0.5757 ±0.0426,0.6922 ±0.0408,0.7447±0.0510,0.8513±0.0505)表达随病毒感染时间延长而增强(P均＜0.01);(5)VMC小鼠心肌组织中CTGF的表达与TGF-β1的表达呈显著正相关(r=0.987,P＜0.01);VMC小鼠心肌组织中CTGF的表达与CVF密切相关(r=-0.901,P＜0.01),但CTGF的表达早于心肌纤维化的出现.结论 CTGF的表达伴随VMC的心肌纤维化程度而增加,提示CTGF的异常表达可能参与了VMC中心肌纤维化的发展过程.     

钙敏感受体对乳鼠心肌细胞凋亡的诱导作用

探讨在缺氧/复氧所诱导乳鼠心肌细胞凋亡中,钙敏感受体（calcium - sensing receptor,CaSR）对Fas/Fas配体（Fas L）途径的影响。方法对8只2日龄Wistar大鼠心肌细胞进行原代培养,后随机分为三组：(1)对照组：细胞不经缺氧/复氧处理,在其他组复氧开始时培养基内加入与用药组等体积的生理盐水;(2)缺氧/复氧组：心室肌细胞缺氧2h/复氧24h,细胞培养基内于复氧开始时加入与用药组等体积的生理盐水;(3) CaSR激动剂氯化钆(GdCl3)组：细胞培养基内于复氧开始时加入300μmol/L CaSR激动剂GdCl3。流式细胞仪分析细胞凋亡率,透射电镜观察细胞超微结构,蛋白印迹法检测CaSR、Fas和FasL蛋白的表达。结果流式细胞仪检测显示,缺氧/复氧组心肌细胞凋亡为12.18％±1.54％,与对照组相比,心肌细胞凋亡明显增加(P＜0.01)。CaSR激动剂GdCl3使心肌细胞凋亡进一步增加至20.25％±2.87％ (P＜0.01)。透射电镜下可见线粒体絮状变、线粒体嵴溶解、空泡样变;CaSR激动剂使CaSR、Fas和FasL表达进一步上调,明显高于缺氧/复氧组(P＜0.05)。结论CaSR激活参与了缺氧/复氧所诱导的乳鼠心肌细胞凋亡,CaSR可通过激活Fas/FasL死亡受体途径导致乳鼠心肌细胞凋亡。     

缺氧缺血性脑损伤新生大鼠心肌细胞凋亡及凋亡蛋白Bax、Bcl-2表达的变化

目的 了解缺氧缺血性脑损伤新生大鼠心肌细胞凋亡及Bax、Bcl-2蛋白表达的变化.方法 通过结扎7 日龄新生大鼠一侧颈总动脉,吸入低浓度氧复制缺氧缺血性脑损伤(HIBD)的动物模型,并没假手术对照(NC)组,两组分别于HIBD后1 h、4 h、12 h、24 h、48 h、72 h、7 d取心脏组织,应用TUNEL法检测心肌细胞凋亡,SP法检测心肌细胞Bax、Bcl-2蛋白的表达.结果 NC组偶见心肌阳性凋亡细胞1～4个/mm~2,各时间点阳性细胞数差异无统计学意义(F=0.5,P>0.05).HIBD组12 h心肌阳性凋亡细胞开始增多,为(14.40±3.21)个/mm~2;72h达高峰,为(26.80 ±2.28)个/mm~2,12～72 h各时间点阳性凋亡细胞明显高于NC组(P<0.05);7 d阳性细胞数减少,但仍高于NC组.NC组大鼠心肌组织Bax呈阳性表达,Bcl-2呈弱阳性表达.HIBD组Bax于24 h开始增高.72 h表达最明显,7 d时表达有所降低,24 h～7 d表达高于NC组(P<0.05);Bel-2于24 h表达开始增高,以后缓慢增高,72 h达高峰,24 h～7 d较NC组表达增高,差异有统计学意义(P<0.05).HIBD组Bcl-2/Bax比值逐渐降低.Bax、Bcl-2的表达与凋亡细胞数之间均星直线正相关(r=0.921、0.980,P均<0.01).Bcl-2/Bax的比值与凋亡细胞数无相关性(r=0.702,P>0.05).结论 HIBD新生大鼠心肌细胞存在凋亡,心肌细胞Bax、Bcl-2蛋白表达增加,且与心肌细胞凋亡有关.     

病毒性心肌炎心肌组织中骨桥蛋白表达的研究

病毒性心肌炎(viral myocarditis,VMC)是常见的心血管疾病之一,近年来发病呈上升趋势.研究表明大量的单个核细胞(T、NK、MΦ等)在局部浸润及其造成的后续免疫损伤在VMC的发病中占有重要的地位[1].骨桥蛋白(OPN)是一种基质功能性非胶原蛋白.已证实OPN在炎症浸润、细胞免疫、组织修复、血管重塑、细胞凋亡等方面扮演着重要角色[2-4].为此,我们以柯萨奇病毒B3(CVB,)感染BALB/c小鼠制作VMC模型,通过实时定量逆转录聚合酶链反应(RT-PCR)分析CVB3感染后0、4、7、9、14和21 d OPN的表达水平,分析其表达与炎症细胞浸润的关系,为VMC发病机制的研究提供依据.     

TNF配体相关分子1A在病毒性心肌炎小鼠心肌中的表达及意义

目的 探讨TNF配体相关分子1A(TLlA)在实验性小鼠病毒性心肌炎(VM)发病中的作用.方法 80只小鼠随机分为实验组和对照组,实验组小鼠腹腔注射CVB3,建立病毒性心肌炎实验模型,对照组注射生理盐水.分别于实验第3、7、15、30天随机选取存活小鼠10只处死,对照组小鼠在接种后30 d处死作为总体对照,取其心肌,采用免疫组织化学技术及RT-PCR技术检测心肌细胞TLlA表达水平,检测心肌病理变化,并对11L1A水平与心肌病变程度行相关性分析.结果 VMC小鼠在接种病毒后3 d心肌TL1A mRNA及蛋白表达水平升高,7 d表达水平最高,此后逐渐下降,但30 d仍维持在一个较高水平,各时点TLlA mRNA及蛋白表达水平与对照组相比,差异均有统计学意义(P＜0.05或0.01).结论 CVB3病毒感染可引起TLlA表达上调,TLlA表达增加可能在CVB3病毒性心肌炎发病机制中起重要作用.     

Allergic factors associated with the development of asthma and the influence of cetirizine in a double-blind, randomised, placebo-controlled trial: First results of ETA®

There is a common progression known as the allergic march from atopic dermatitis to allergic asthma. Cetirizine has several antiallergic properties that suggest a potential effect on the development of airway inflammation and asthma in infants with atopic dermatitis. Methods. Over a two year period, 817 infants aged one to two years who suffered from atopic dermatitis and with a history of atopic disease in a parent or sibling were included in the ETAC^® (Early Treatment of the Atopic Child) trial, a multi-country, double-blind, randomised, placebo-controlled trial. The infants were treated for 18 months with either cetirizine (0.25mg/ kg b.i.d.) or placebo. The number of infants who developed asthma was compared between the two groups. Clinical and biological assessments including analysis of total and specific IgE antibodies were performed. Results. In the placebo group, the relative risk (RR) for developing asthma was elevated in patients with a raised level of total IgE (≥ 30 kU/I) or specific IgE (≥ 0.35 kUA/I) for grass pollen, house dust mite or cat dander (RR between 1.4 and 1.7). Compared to placebo, cetirizine significantly reduced the incidence of asthma for patients sensitised to grass pollen (RR = 0.5) or to house dust mite (RR = 0.6). However, in the population that included all infants with normal and elevated total or specific IgE (intention-to-treat - ITT), there was no difference between the numbers of infants developing asthma while receiving cetirizine or placebo. The adverse events profile was similar in the two treatment groups. Discussion. Raised total IgE level and raised specific IgE levels to grass pollen, house dust mite or cat dander were predictive of subsequent asthma. Cetirizine halved the number of patients developing asthma in the subgroups sensitised to grass pollen or house dust mite (i.e. 20% of the study population). In view of the proven safety of the drug, we propose this treatment as a primary pharmacological intervention strategy to prevent the development of asthma in specifically sensitised infants with atopic dermatitis.     

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孤独症谱系障碍(autistic-spectrum disorders,ASDs)近年来患病率逐年攀升至1%左右,其症状往往伴随终生,成为严重威胁儿童健康和发展的神经发育性疾患;注意缺陷多动障碍(attention deficit hyperactivity disorder,ADHD)是儿童期最常见的精神障碍,国内报道患病率为4.13%～5.83%,其症状可延续至青少年期,甚至到成年期[1]。这两类精神障碍在成年期的临床表现、共患病、治疗策略和预后与儿童期有哪些不同呢?本文通过回顾相     

EXCITING NEW TREATMENT APPROACHES FOR PATHYPHYSIOLOGIC MECHANISMS OF SICKLE CELL DISEASE

During the past several decades, our understanding of the complex pathophysiology of vasoocclusion associated with sickle cell disease has improved greatly. Interaction of genes, hemoglobin molecules, red cell membrane and metabolic changes, cell-cell interactions and cell-plasma interactions, red cell adhesion to vascular endothelium, activation of coagulation, and vascular reactivity play a role in vaso occlusion. Penicillin prophylaxis of pneumococcal infections and appropriate use of blood transfusions and other supportive measures improved survival of sickle cell patients. Hydroxyurea made a major impact on sickle cell therapy when it was shown to decrease acute painful episodes, acute chest syndrome, and the need for blood transfusion in adults. Significant experience in the use of hydroxyurea has been accumulated in older children. The benefits and risks of hydroxyurea for younger children and long-term risks in all patients will be evaluated in future investigations. Other promising therapies include butyrate compounds, clotrimazole, magnesium supplementation, poloxamer 188, antiadhesion agents, anticoagulant approaches, and nitric oxide. Hemopoietic transplantation remains the only curative therapy. However, several transgenic mouse models are available for studies of gene therapy or other treatment approaches on biochemical, cellular, and pathologic effects of mutant genes.     

Infiltrations of Epstein-Barr virus-carrying cells in granular lymphocyte-proliferative disorders corresponding to chronic active Epstein-Barr virus infection

A 21-year-old man with granular lymphocyte-proliferative disorders (GLPD) associated with chronic active Epstein-Barr virus (EBV) infection is described. Chromosomal analyses revealed several clonal abnormalities and two of them were mainly repetitious. High copy numbers of monoclonal EBV genome were also detected in the proliferative large granular lymphocytes (LGLs), indicating the monoclonal expansion of EBV-infected LGLs. The patient had an indolent course for several years, and there was no evidence of infiltrations of his bone marrow until the end stage. At autopsy, microscopic studies revealed marked infiltrations of LGL in the liver and spleen, and the infiltrating cells were NK-cell immunophenotype. The infiltrated LGLs showed latency I.     

LUTEINIZING HORMONE RECEPTOR MUTATIONS IN DISORDERS OF SEXUAL DEVELOPMENT AND CANCER

Human male sexual development is regulated by chorionic gonadotropin (CG) and luteinizing hormone (LH). Aberrant sexual development caused by both activating and inactivating mutations of the human luteinizing hormone receptor (LHR) have been described. All known activating mutations of the LHR are missense mutations caused by single base substitution. The most common activating mutation is the replacement of Asp-578 by Gly due to the substitution of A by G at nucleotide position 1733. All activating mutations are present in exon 11 which encodes the transmembrane domain of the receptor. Constitutive activity of the LHR causes LH releasing hormone-independent precocious puberty in boys and the autosomal dominant disorder familial male-limited precocious puberty (FMPP). Both germline and somatic activating mutations of the LHR have been found in patients with testicular tumors. Activating mutations have no effect on females. The molecular genetics of the inactivating mutations of the LHR are more variable and include single base substitution, partial gene deletion, and insertion. These mutations are not localized and are present in both the extracellular and transmembrane domain of the receptor. Inactivation of the LHR gives rise to the autosomal recessive disorder Leydig cell hypoplasia (LCH) and male hypogonadism or male pseudohermaphroditism. Severity of the clinical phenotype in LCH patients correlates with the amount of residual activity of the mutated receptor. Females are less affected by inactivating mutation of the LHR. Symptoms caused by homozygous inactivating mutation of the LHR include polycystic ovaries and primary amenorrhea.     

Resurgence of measles in Singapore: profile of hospital cases

OBJECTIVE: To ascertain the profile of cases of measles seen at a general hospital during a recent outbreak that occurred despite a measles vaccination program. METHODOLOGY: A retrospective study from January 1991 to March 1998. All patients with measles (ICD code 055. 9) seen at the emergency unit or as inpatients were included. RESULTS: There were 87 cases identified. The diagnosis was clinical in all and proven serologically in 71%. Eighty-five per cent of the cases occurred between January 1997 and March 1998. There was a bi-modal age distribution with peaks in the very young (相似文献   

Personality profiles and heart rate variability (vagal tone) in children with recurrent abdominal pain

The aim of the study was to explore psychological factors and autonomic activity in children with recurrent abdominal pain and to compare them with those in a control group of healthy children. The Personality Inventory for Children was used for assessment of developmental, emotional and psychosocial factors in 25 children with recurrent abdominal pain (age, 7-15 y). Parasympathetic and sympathetic functions in these children and in 23 healthy control subjects (age, 7-13 y) were also investigated, non-invasively using a computerized polygraph. Vagal tone (parasympathetic function) was indexed by calculation of respiratory sinus arrhythmia in beats/min. Skin conductance (sympathetic function) was recorded by the constant current method. On the Personality Inventory for Children, 16 patients had high scores on somatic concern. Several patients had scores in the clinical range for depression, withdrawal and anxiety, but the mean scores for these personality profile scales were well within the normal range of healthy children. Interestingly, there was a spike on the L (Lie)-scale for most of the patients and 15 patients had scores above or close to the clinical cut-off value. As compared with the scores in healthy children, vagal tone and sympathetic tone were normal. Conclusion: Many children with recurrent abdominal pain have scores in the clinical range for depression, withdrawal, anxiety and L-scale indicating coping problems, denial and a trend towards somatic concern that may contribute to the evolution of abdominal pain. Autonomic nerve activity was not disturbed in these children.     

Overcoming surfactant inhibition with polymers

Inhibition of the function of pulmonary surfactant in the alveolar space is an important element of the pathophysiology of many lung diseases, including meconium aspiration syndrome, pneumonia and acute respiratory distress syndrome. The known mechanisms by which surfactant dysfunction occurs are (a) competitive inhibition of phospholipid entry into the surface monolayer (e.g. by plasma proteins), and (b) infiltration and destabilization of the surface film by extraneous lipids (e.g. meconium-derived free fatty acids). Recent data suggest that addition of non-ionic polymers such as dextran and polyethylene glycol to surfactant mixtures may significantly improve resistance to inhibition. Polymers have been found to neutralize the effects of several different inhibitors, and can produce near-complete restoration of surfactant function. The anti-inhibitory properties of polymers, and their possible role as an adjunct to surfactant therapy, deserve further exploration.     

Understanding infant formula

The World Health organisation recommends breast feeding infants for the first six months of life. When this breast feeding does not occur either through parental choice or medical need, infant formulas will be required. There is a bewildering array of formulas on the UK market for many different requirements. When faced with an unsettled infant many parents (and healthcare professionals) will experiment with the infant formula available and then attend the paediatric clinic looking for help and advice. It is therefore essential that paediatricians understand what milks are available and what the key differences between different products are. This review attempts to provide a simple guide through many of the formulations currently available in the UK; and offers advice for the dietary management of the child with extra calorie requirements, infants with cow's milk protein allergy, gastro oesophageal reflux disease, apparent unresolved hunger and infantile colic. Whatever the underlying condition, there is likely to be an infant formula that is suitable in this generation of ever expanding formulations.