Wilson病分子机制研究进展

Wilson病是一种铜代谢缺陷病,其发病与铜转运相关的P型ATP酶(ATP7B蛋白)及X连锁凋亡抑制蛋白(XIAP)功能改变密切相关。ATP7B蛋白及XIAP通过一系列相互独立的分子生物学机制作用于铜转运。ATP7B蛋白的突变和XIAP的异位表达会打乱铜平衡,导致铜过量并沉积于肝、脑和眼角膜,引起相应的器官脏器损伤。本文就ATP7B蛋白和XIAP及其在Wilson病发生、发展中的分子机制予以综述。     

急性冠状动脉综合征患者ST段变化与高敏C反应蛋白的关系

目的观察急性冠状动脉综合征患者ST段变化与高敏C反应蛋白（hs-CRP）的关系。方法急性心肌梗死患者136例,根据入院时的心电图变化分为3个组：ST段上抬组59例,ST段下移组50例,心电图正常组27例。所有患者入院后即检测hs-CRP水平,采用的截点值为3 mg/L。结果ST段下移组hs-CRP水平超过截点值者显著较其它两组多（P=0.009）。ST段下移组hs-CRP水平明显高于ST段抬高组和正常组（P〈0.01）。1年后随访,ST段下移组心血管事件发生率高于ST抬高组和正常组（P〈0.05）。结论ST段下移患者hs-CRP水平增高有较高的发生率,提示炎症诱发临床急性冠状动脉综合征呈明显的不均一性。入院时hs-CRP浓度较高患者预后较差。     

Maternal alcohol ingestion reduces surfactant protein A expression by preterm fetal lung epithelia.

In addition to neurodevelopmental effects, alcohol consumption at high levels during pregnancy is associated with immunomodulation and premature birth. Premature birth, in turn, is associated with increased susceptibility to various infectious agents such as respiratory syncytial virus (RSV). The initial line of pulmonary innate defense includes the mucociliary apparatus, which expels microorganisms trapped within the airway secretions. Surfactant proteins A and D (SP-A and SP-D, respectively) are additional components of pulmonary innate immunity and have an important role in pulmonary defense against inhaled pathogens. The purpose of this study was to determine if chronic alcohol consumption during the third trimester of pregnancy alters the function of the mucociliary apparatus and expression of SP-A and SP-D of fetal lung epithelia. Sixteen, date-mated ewes were assigned to two different groups; an ethanol-exposed group in which ewes received ethanol through surgically implanted intra-abomasal cannula during the third trimester of pregnancy, and a control group in which ewes received the equivalent amount of water instead of ethanol. Within these two groups, ewes were further randomly assigned to a full-term group in which the lambs were naturally delivered, and a preterm group in which the lambs were delivered prematurely via an abdominal incision and uterotomy. Ethanol was administered five times a week as a 40% solution at 1g/kg of body weight. The mean maternal serum alcohol concentration measured 6h postadministration was 16.3+/-4.36 mg/dl. Tracheas from six full-term lambs were collected to assess ciliary beat frequency (CBF). The lung tissue from all (24) lambs was collected for immunohistochemistry analysis of SP-A and SP-D protein production and fluorogenic real-time quantitative polymerase chain reaction analysis of SP-A and SP-D mRNA levels. Exposure to ethanol during pregnancy significantly blocked stimulated increase in CBF through ethanol-mediated desensitization of cAMP-dependent protein kinase. In addition, preterm born/ethanol-exposed lambs showed significantly decreased SP-A mRNA expression when compared with the preterm born/control group (P=.004); no significant changes were seen with SP-D. The full-term/ethanol-exposed lambs had no significant alterations in mRNA levels, but had significantly less detectable SP-A protein when compared with the full-term/control lambs (P=.02). These findings suggest that chronic maternal ethanol consumption during the third trimester of pregnancy alters innate immune gene expression in fetal lung. These alterations may underlie increased susceptibility of preterm infants, exposed to ethanol in utero, to RSV and other microbial agents.     

rhLEDGF P52基因原核表达载体的构建、诱导表达及蛋白纯化

目的构建rhLEDGFp52基因原核表达载体,获得rhLEDGFp52蛋白。方法利用基因重组技术将rhLEDGFp52基因构建于原核表达载体pET30a(+)中,经酶切、测序鉴定证实构建完全正确后,通过IPTG诱导在大肠杆菌BL21(DE3)中获得表达,经免疫蛋白印迹试验对rhLEDGFp52进行鉴定并用Ni-NTAHis.Bind.Resin方法进行纯化。结果成功构建rhLEDGFp52基因原核表达载体,在大肠杆菌BL21(DE3)中获得可溶性形式表达,rhLEDGFp52蛋白表达量占菌体蛋白总量的34.63%.WesternBlot结果显示rhLEDGF2蛋白能够特异性与LEDGF-ab结合。Ni-NTAHis.Bind.Resin方法进行纯化后的rhLEDGFp52,最终质量浓度达520mg·L-1,分析其纯度达87.93%.结论成功获得rhLEDGFp52蛋白,这为深入研究其生物学功能奠定了基础。     

PRAK不同突变体的构建、表达及细胞内定位的研究

目的:构建p38调节/激活蛋白激酶(PRAK)不同突变体的绿色荧光蛋白融合表达载体,并在真核细胞中表达后,观察这些突变体在细胞内的定位情况.方法:将克隆在绿色荧光蛋白载体pEGFP-C2上的野生型PRAK通过定点突变技术构建其无活性突变体PRAK(T182A)、活性突变体PRAK(T182D)、无激酶活性突变体PRAK(KM)、核定位信号缺失突变体PRAK(NLSm)及核输出信号缺失突变体PRAK(NESm),随后转染NIH3T3细胞,并在荧光显微镜下观察其表达和细胞内定位情况.结果:经测序鉴定各种PRAK不同突变体正确无误后,在NIH3T3细胞中得到高量表达;融合蛋白发出的绿色荧光表明,在静息状态下,野生型PRAK(WT)、PRAK(182A)、PRAK(182D)、PRAK(KM)和PRAK(NESm)主要分布于细胞核内,而PRAK(NLSm)在细胞中均匀分布;在受到NaAsO2刺激后,PRAK(WT)和PRAK(KM)移位出核,而PRAK(182A)、PRAK(182D)和PRAK(NESm)失去了移位出核的能力,PRAK(NLSm)则与刺激前无明显差异.结论:成功构建了PRAK不同突变体的绿色荧光蛋白融合表达载体并在真核细胞中进行了表达,且不同的突变体能够影响PRAK的细胞内定位.     

早期丰富环境干预对未成熟大鼠脑室周围白质软化脑功能的影响

目的探讨早期丰富环境干预对未成熟大鼠脑室周围白质软化(PVL)损伤后神经行为及生长相关蛋白(GAP-43)表达的影响。方法选择2日龄SD大鼠制作PVL模型,随机分为干预组、非干预组、假手术组、对照组,各40只。假手术组仅分离左侧颈总动脉,不予结扎和缺氧,非干预组和假手术组均饲养于标准环境中,不进行丰富环境干预。干预组于术后第4天行触摸和丰富环境干预,总干预时间为28d。干预结束后行神经行为学检测,同时分别于术后第4、11、18、25、32天取各组大鼠海马脑组织,采用RT-PCR技术检测GAP-43 mRNA表达。结果干预组大鼠于生后5周龄感觉运动功能及学习记忆能力较非干预组明显改善[(悬吊试验:(4.5±1.5)minvs(3.1±0.9)min;斜坡试验:(2.3±0.7)svs(3.9±1.1)s;逃避潜伏期:(4.86±2.94)svs(15.0±11.23)s;空间探索能力:(62.89±8.69)%vs(43.99±8.29)%Pa<0.05]。干预组海马GAP-43 mRNA表达于术后第11、18、25、32天的光密度比值较非干预组明显增高[(3.21±0.45)vs(2.63±0.32);(2.74±0.34)vs(2.47±0.25);(2.14±0.29)vs(1.75±0.19);(1.96±0.25)vs(1.63±0.15)Pa<0.01]。结论早期适度丰富环境干预可增强未成熟大鼠PVL脑功能的恢复,GAP-43增多可能是其脑功能恢复机制之一。     

干细胞因子受体基因在再生障碍性贫血患儿中的表达

目的探讨再生障碍性贫血（再障）患儿造血干细胞因子受体（C-KIT）基因Exon9、11、13的表达和序列构象及与发病机制的关系。方法应用逆转录-聚合酶链反应（RT-PCR）和聚合酶链反应单链构象多态分析（PCR-SSCP）和序列测定技术,测定再障与正常对照儿童各15例C-KIT基因Exon9、11、13的表达水平和结构是否存在变异。结果1.再障组C-KIT基因Exon9、11、13阳性表达率（40.0%）与正常对照组（46.67%）比较无显著性差异（P〉0.05）;2.经PCR-SSCP分析和基因序列测定C-KIT基因Exon9、11、13均未见碱基序列改变或碱基数目增多、缺失。结论C-KIT基因Exon9、11、13表达与儿童再障发病关系不大,提示儿童再障的发病可能不是由于Exon9、11、13突变引起。     

前降钙素和C反应蛋白对儿科细菌感染的诊断价值

目的 评价前降钙素（PCT）、C反应蛋白（CRP）对儿童细菌感染的诊断价值。方法 检测51例患儿（21例细菌感染、17例病毒感染、13例非感染）入院时的血清PCT和外周血CRP水平，并比较他们对细菌感染的敏感度和特异性。结果 细菌感染患儿血清PCT阳性率高于病毒感染和非感染患儿。血清PCT对细菌感染的特异性优于外周血CRP，但敏感性低于CRP。结论 联合应用血清PCT和外周血CRP可提高诊断细菌感染的敏感性和特异性，对临床诊治细菌感染有一定的指导作用。     

慢性鼻-鼻窦炎中鼻甲黏膜环氧合酶2与上游丝裂原激活蛋白激酶及核因子κB信号通路相关性探讨

目的探讨慢性鼻-鼻窦炎（chronic rhinosinusitis，CRS）患者中鼻甲黏膜环氧合酶2（cyclooxygenase 2，COX-2）与丝裂原激活蛋白激酶（mitogen-activated protein kinase，MAPK）及核因子KB（nuclear factor kappaB，NF-KB）通路关键酶的表达及其相关性，明确其在黏膜炎性反应机制中的作用。方法将24例CRS患者中鼻甲外侧黏膜按海口分型分期标准分为3组（组1为Ⅰ型2期，组2为Ⅱ型2期，组3为Ⅲ型，每组8例），8例正常鼻黏膜作为对照。应用免疫组化和荧光实时定量多聚酶链反应检测COX-2、p38丝裂原激活蛋白激酶（p38MAPK）、细胞外信号调节蛋白激酶（ERK）、c-Jun氨基端激酶（JNK）、NF-KBp65和p50的表达，并分析其相关性。结果COX-2、p38MAPK、ERK和NF-KB在3组CRS中均有表达，强度高于对照组，差异有统计学意义（P值均〈0．05）；3组CRS之间表达差异无统计学意义（P值均〉0．05）；JNK在CRS各组和对照组中均无阳性表达。相关性分析表明：CRS各组中COX-2、p38MAPK、ERK、NF-κB p65及050的蛋白质表达量同mRNA表达水平呈直线正相关（P值均〈O．05）；在同一组CRS中COX-2与p38MAPK、ERK的蛋白质和mRNA表达呈正相关（P值均〈0．05）；CRS各组中COX-2表达与NF-κB p65、p50的核内表达正相关（P〈0．05）。结论CRS中COX-2表达上调，与上游p38MAPK、ERK、NF-κB等信号转导通路具有相关性，提示COX-2在CRS鼻黏膜炎性反应中可能受后者调节。