新生儿缺氧缺血性脑病合并心肌损伤血浆N端脑利钠肽前体的变化及与预后的相关性

目的 探讨N端脑利钠肽前体(NT-proBNP)在缺氧缺血性脑病(HIE)合并心肌损伤患儿中的变化,为临床早期诊断、治疗及预后判断提供依据.方法 选择我院收住的40例HIE患儿为研究对象(心肌损伤16例,非心肌损伤24例),以20例正常新生儿为对照组,应用ELISA方法测定血浆NT-proBNP水平;超声心动图测定患儿的心脏收缩功能.结果 (1)心肌损伤患儿NT-proBNP水平[(350±56)pmol/L]显著高于非心肌损伤组[(135±37)pmol/L]和正常对照组[(117±23)pmol/L],差异有统计学意义(P＜0.05);(2)轻、中、重度HIE组NT-proBNP水平分别为(132±34)pmol/L、(247±43)pmol/L和(343±53)pmol/L,中、重度HIE组显著高于对照组;轻、中、重度HIE组间比较差异有统计学意义(P＜0.05);(3)心肌损伤组急性期血浆NT-proBNP水平[(350±56)pmol/L]明显高于恢复期[(250±78)pmoyl/L],差异有统计学意义(P＜0.05);(4)HIE患儿NT-proBNP水平和肺动脉收缩压、左室收缩功能密切相关.结论 HIE合并心肌损伤急性期血浆NT-proBNP水平升高,有助于早期诊断心肌损伤,判断HIE病情.

Abstract：

Objective To investigate the changes of N-terminal pro-brain natriuretic peptide (NT-proBNP) in neonates with hypoxic-ischemic encephalopathy (HIE) complicated by myocardial ischemic injury. Methods Forty neonates with HIE ( 16 cases with concurrent myocardial injury and 24 cases without) were enrolled. Twenty healthy neonates were used as control. Plasma NT-proBNP levels were measured using enzyme immunoassay. Cardiac function was measured by echocardiography. Results ( 1 ) The mean plasma NT-proBNP levels in patients with myocardial injury[(350 ± 56) pmol/L]were significantly higher than those in patients without myocardial injury[(135 ± 37 ) pmol/L]and in the control group [(117 ±23) pmol/L](P ＜0. 05). (2) The NT-proBNP levels in mild,moderate and severe HIE neonates were ( 132 ±34) pmol/L, (247 ±43) pmol/L and (343 ±53) pmol/L. Compared with the control group,the NT-proBNP levels in the neonates with moderate and severe HIE significantly increased. There were significant differences in the NT-proBNP levels among the mild, moderate and severe HIE neonates ( P ＜ 0. 05 ).(3) In patients with myocardial injury,the NT-proBNP levels significantly decreased in the convalescent phase [(250±78) pmol/L]compared with those in the acute phase[(350±56) pmol/L](P ＜0.05). (4) The NT-proBNP levels were significantly related with left ventricular ejection fraction. Conclusion Plasma NT-proBNP levels increase in neonates with HIE complicated with myocardial ischemic injury in the acute phase.Detection of NT-proBNP levels maybe useful in the diagnosis of myocardial ischemic injury and severe HIE.     

SOX10在先天性巨结肠相关性小肠结肠炎发病中的作用

目的 探讨SOX10、潘氏细胞发育及分泌防御素-5与先天性巨结肠相关性小肠结肠炎的关系.方法 收集50例先天性巨结肠病变肠管,根据术前是否发生小肠结肠炎分为HAFC组(n=14)和HD组(n=36),并以20例正常结肠标本作对照组.采用免疫组织化学方法观察结肠中防御素-5蛋白质表达、潘氏细胞发育情况以及SOX10蛋白质表达情况.采用实时荧光定量PCR技术检测防御素-5 mRNA及Sox 10 mRNA表达情况.结果 防御素-5在正常肠管中不表达,HAEC组和HD组在肠腺隐窝基底处呈不同程度阳性表达.但前者阳性区域平均光密度值明显增高(0.33±0.039比0.10±0.031,P＜0.05),HAEC组防御素-5mRNA亦呈显著增高趋势(2.72±0.80比0.78±0.21,P＜0.05).对结肠组织同层切片进行潘氏细胞特异性产物溶菌酶免疫组化染色发现,对照组肠管中除1例存在弱阳性外其他均无阳性表达.HD组和HAEC组结肠中同样在隐窝基底处存在溶菌酶阳性细胞,可鉴别为化生的潘氏细胞,但HAEC组在发生率(78.6%)和细胞个数(2.97±0.80)明显高于HD组(27.8%,0.43±0.85)(P＜0.05).SOX10免疫产物主要在结肠神经节细胞膜及胞浆中表达,对照组、HAEC组、HD组阳性区域的平均光密度值递减(0.75±0.041,0.61±0.048,0.35±0.025),差异具有统计学意义(P＜0.05),同时RT-PCR检测显示Sox10 mRNA在各组中的表达与蛋白质水平呈平行结果.结论 SOX10可能通过影响潘氏细胞发育及分泌防御素-5在先天性巨结肠相关性小肠结肠炎的发生发展中起一定作用.

Abstract：

Objective To study the expression of SOX10 and Human α-defensins-5(HD-5 )in Hirschsprung's disease associated enterocolitis(HAEC) and expore the possible relationship between SOX10 and HAEC.Methods Fifty pathological colons of Hirschsprung's disease (HD) were divided into HAEC group (n = 14) and HD group(n = 36) according to the presence of preoperative enterocolitis,Twenty normal colons as control group.The protein and mRNA expression of HD-5 and SOX 10 were measured by immunohistochemical staining and real-time quantitative PCR Results Normal colons did not express HD-5 but positive expression of HD-5 was detected at the base of the crypts of Lieberkuhn in HAEC and HD groups in different degree.The mean optical density of HD-5 immunohistochemical staining (0.33 ± 0.039 vs 0.10 ± 0.031 )and HD-5 mRNA expression (2.72 ± 0.80 vs 0.78 ± 0.21 ) in HAEC group was apparently higher than those in HD group(P＜0.05).The expression of lysozyme,a specific product by Paneth Cell,on sequential sections was negative in control group except one sample.In HAEC group and HD group,positive expression of lysozyme can be seen in the crypts of Lieberkuhn,where the cells can be identified as Metaplastic Paneth cell,but the incidence (78.6% vs 27.8%) and the number of cells 2.97 ± 0.80 vs 0.43 ± 0.85) in HAEC group were obviously higher than those in HD group(2.97 ± 0.80 vs 0.43 ± 0.85) (P＜0.05).SOX10 was mainly located in the plasmalemma and cytoplasm of ganglion cell and its mean optical density in control group (0.75 ±0.041 ),HAEC group (0.61 ± 0.048)and HD group(0.35 ± 0.025) were decrement,the difference between three groups were statistically significant (P＜0.05).Meanwhile,Sox10mRNA detected by real-time quantitative PCR indicated the parallel result.Conclusions SOX10 may be an important factor in the pathogenesis and development of HAEC.     

新生儿经皮小时胆红素百分位曲线预测新生儿高胆红素血症价值的系统评价

目的 对不同国家新生儿经皮小时胆红素百分位曲线预测新生儿高胆红素血症价值的文献进行系统评价,为新生儿高胆红素血症的诊治提供依据。方法检索Cochrane 图书馆、PubMed、OVID、Springer数据库、中国期刊全文数据库、维普数据库、万方数据库和中国生物医学文献数据库,检索时间均从1990年1月至2011年3月。由2名系统评价员进行资料提取和质量评价。结果 10项研究进入分析,偏倚评价为B级6项、C级4项。不同国家制备的新生儿小时胆红素百分位曲线值有一定的差异,印度的小时胆红素百分位曲线值最高,希腊、以色列、中国次之,美国最低。在高度危险区（P95）,胆红素上升速度在生后24 h内最快;中度（P75）和低度（P40或P50）危险区,生后36 h内胆红素上升速度最快,其中印度上升速度最快最高,中国、美国、以色列次之,希腊上升速度最低。新生儿经皮小时胆红素百分位曲线预测高胆红素血症,高度危险区的敏感度为26.9%～75.0%,特异度为89.4%～97.3%;中度危险区的敏感度为78.7%～100.0%,特异度为48.3%～82.3%;低度危险区的敏感度为98.7%～100.0%,特异度为24.4%～56.1%;曲线下面积为0.766～0.990,其中泰国预测的准确性最高,印度、中国次之,美国最低。结论 经皮小时胆红素百分位曲线图预测新生儿高胆红素血症有较高的准确性,具有临床推广应用价值。不同国家经皮小时胆红素百分位曲线有一定的差异,各国应根据本国新生儿小时总胆红素的变化趋势制备各自相对应的曲线图作为新生儿高胆红素血症的干预参考标准。     

黏多糖贮积症Ⅰ型二例基因突变检测及产前诊断

目的 对黏多糖贮积症Ⅰ型病例进行基因检测,对再孕母亲进行产前诊断.方法 用酶学检测方法 确诊2例黏多糖贮积症Ⅰ型先证者;采用PCR扩增技术结合序列分析的方法,检测2例先证者血液及其再孕母亲羊水细胞中IDUA基因外显子及其两侧内含子.结果 在2例经酶学检测确诊的先证者中共检测出4种基因突变:p.L238R、c.883InsC、c.531InsT、p.L346R,2种为插入突变,2种为错义突变.在先证者1母亲羊水细胞IDUA基因中未发现致病基因突变,羊水细胞中IDUA酶活性为9.0 nmol/(h·mg蛋白);在先证者2母亲羊水细胞IDUA基因中检测到与先证者相同的两个致病突变,羊水细胞中IDUA酶活性为0.5 mol/(h·mg蛋白).结论 从黏多糖贮积症Ⅰ型先证者发现的4种突变中,p.L346R为已知突变,p.L238R,c.883InsC,c.531InsT为首次发现的新突变.胎儿1未获得与先证者相同致病基因,为正常胎儿.胎儿2获得与先证者相同致病基因,为受累胎儿.产前基因诊断结果 与酶学产前诊断结果 相符合.

Abstract：

Objectiye Mucopolysaccharidosis type Ⅰ(MPS Ⅰ; MIM# 252800)is an autosomal recessive disease that results from the deficiency in the lysosomal enzyme α-L-iduronidase(IDUA).IDUA is one of the enzymes involved in degradation of glycosaminoglycans heparan sulphate and dermatan sulphate.The deficiency of IDUA leads to widespread accumulation of partially degraded mucopolysaccharides inside lysosomes,resulting in progressive cellular and multiorgan dysfunction. Up to now there is no definitely effective treatment for this disorder,therefore it is important to provide an accurate genetic diagnosis and prenatal diagnosis for the MPS Ⅰ families.This study was conducted to detect IDUA gene mutation in patients with MPS Ⅰ and make a definite diagnosis of homozygote or heterozygote and make first trimester prenatal diagnosis.Method The 2 male probands included in this study were diagnosed as MPS Ⅰ patients in Peking Union Medical College Hospital,case 1 was 2 years old and case 2 was 5 years old.Genomic DNA was extracted from leucocytes in the 2 patients and 2 mothers' cultured amniocytes.IDUA gene DNA sequence was amplified by polymerase chain reaction(PCR)and the PCR products were sequenced directly.Novel mutations were analyzed in 100 normal chromosomes.Result The genotype of case 1 was p.L238R/c.883InsC,while of case 2 was c.531InsT/p.L346R.The fetal case 1 did not inherit the same pathogenic inherited the same pathogenic mutations with the proband,the genotype of fetal 2 was c.531InsT/p.L346R,in 2 MPS Ⅰ patients,p.L238R,c.883InsC,c.531InsT were novel.The fetal case 1 was diagnosed as normal fetus while the fetus 2 was diagnosed as affected.The results of the two kinds of prenatal diagnostic methods were correspondent with each other.     

肿瘤坏死因子-α对人脂肪细胞STEAP4蛋白膜转位的影响及其机制

目的 探讨肿瘤坏死因子-α (TNF-α)对人脂肪细胞STEAP4蛋白膜转位的影响,并分析其可能的机制.方法 应用1-甲基3-异丁基黄嘌呤(MIX)、地塞米松、胰岛素、罗格列酮方案诱导人前体脂肪细胞分化,以人重组TNF-α或加用细胞外信号调节蛋白激酶( ERK)抑制剂PD-98059干预人成熟脂肪细胞24 h,采用Western blot技术检测细胞膜蛋白与总蛋白中STEAP4蛋白的表达.结果TNF-α干预能显著促进人成熟脂肪细胞中STEAP4蛋白向细胞膜转位;ERK抑制剂PD-98059联合TNF-α与单独应用TNF-α诱导人成熟脂肪细胞的细胞膜蛋白和总蛋白中STEAP4蛋白的表达水平比较均无显著差异.结论 TNF-α干预显著促进人成熟脂肪细胞中STEAP4蛋白的膜转位,初步排除丝裂原活化蛋白激酶信号途径参与这一调控机制.     

广州地区小儿乙肝无症状携带者及其父母的HBV DNA基因型研究

目的扩增HBV DNA S基因,建立 HBV DNA的分型方法,研究家庭中小儿乙肝无症状携带者与其父母的基因型.方法利用PCR-RFLP基因分型方法,扩增HBV DNA S基因,以限制性内切酶AvaⅡ,MboⅠ酶切分型;结果 12个家庭共27例乙肝无症状携带者血清,其中两个家庭儿童与父母的基因型相同,均为B型;9个家庭儿童与母亲的基因型相同(7个B型,2个C型),父亲HBV DNA阴性;1个家庭儿童与母亲为B型,父亲为C型,结论本文利用的分型方法经济实用,分型率高,广州地区的基因型以B型,HBV DNA的传播具有家庭聚集性特点及母婴传播的特点.     

用mRNA RT-PCR差异显示技术克隆拟除虫菊酯抗性家蝇细胞色素P450基因片段

细胞色素P450单加氧酶系是一类广泛分布在生物有机体中的重要酶系,具有多样的功能。细胞色素P450介导的杀虫剂代谢解毒作用的增强是昆虫对杀虫剂产生抗性的普遍机制。为揭示家蝇对拟除虫菊酯类杀虫剂的分子基础,我们利用mRNA差异显示技术(DDRTPCR)从溴氰菊酯抗性品系家蝇Muscadomestica中分离出2个新的基因片段(M1,M2)。序列比对(BlastP)结果表明,M1与黑腹果蝇Drosophilamelanogaster中的Cyp6A9在氨基酸水平上具有54%的相似性,M2分别与果蝇Drosophilasimulans中的Cyp6g1有65%的相似性。M1和M2包含P450的特征序列,并与细胞色素P450具有最高程度的序列相似性,是为细胞色素P450基因片段。     

家蝇抗药性的分子遗传机制

综述了近年来家蝇抗药性分子机制的研究进展。脂族酯酶基因(MdaE7)在位置137(Gly)和251(Trp)的氨基酸突变可能与家蝇对有机磷抗性有关;谷胱苷肽S转移酶(GSTs)超量表达在家蝇对有机磷农药的抗性中起重要作用;有机磷抗性家蝇的AChE基因普遍存在Gly262Ala和Phe327Tyr突变。击倒抗性(kdr)和P450介导的代谢抗性是家蝇对DDT和拟除虫菊酯类杀虫剂抗性的重要机制,电压门控钠离子通道的L1041F突变产生Kdr,而M918T和L1041F双突变导致超击倒抗性(superkdr);P450介导的代谢抗性表现出进化可塑性的特点。有关家蝇抗性机制的阐明,可为抗性的检测和监测提供准确和方便的手段。     

骨髓基质干细胞移植选择下调大鼠心肌梗死细胞外基质基因表达

目的心肌纤维化是心肌梗死后左室重构的主要组成部分，本实验主要观察骨髓基质干细胞移植对缺血性心肌左室重构细胞外基质的基因表达以及对心脏左室功能的影响。方法将骨髓基质干细胞经体外培养、5-氮杂胞苷（5-azacytidine）诱导、扩增及标记后，植入急性心肌梗死大鼠动物模型心肌中，于28d测定大鼠心脏功能后取出心脏并称量，对缺血性心肌的纤维化程度及注入的骨髓基质干细胞进行观察，应用逆转录聚合酶链反应（RT-PCR）及原位杂交技术，检测Ⅰ型胶原、Ⅲ型胶原、基质金属蛋白酶1（MMP-1）和基质金属蛋白酶抑制剂1（TIMP-1）等细胞外基质基因表达情况，并观察了转化生长因子（TGF）-β1基因表达情况。结果在心肌中骨髓基质干细胞存活；梗死大鼠心脏功能明显改善；心肌梗死面积明显减小（10％）；Ⅰ胶原、Ⅲ型胶原、TIM-1以及TGF-β1基因表达水平与未注入骨髓基质干细胞对照组相比显著降低，且心肌细胞纤维化被有效抑制。结论骨髓基质干细胞移植可以抑制心肌纤维化，降低细胞外基质基因表达，改善心脏功能，有望成为心肌梗死治疗的一条新途径。     

检测膀胱肿瘤Survivin蛋白及其mRNA表达的临床意义

目的为明确Survivin作为膀胱肿瘤患者的肿瘤诊断和监测标志物的可能性,我们检测了Survivin在膀胱肿瘤和非肿瘤患者病变组织的Survivin蛋白及其mRNA表达情况。方法从我科住院病人和膀胱镜检患者中获取31例膀胱癌患者(实验组)和24例非肿瘤患者(对照组)的组织标本,用免疫组织化学法(IHC)和RT-PCR检测Survivin蛋白和基因在肿瘤组织和非肿瘤组织中的表达情况。并对结果行相应的统计学分析。结果IHC检测实验组和对照组Survivin蛋白的阳性率分别为74%(23/31)和4%(1/24),χ2检验P<0.001,IHC的阳性率与肿瘤的分级分期有一定的关系;实验组和对照组RT-PCR检测Survivin mRNA的表达阳性率分别为90%(30/31)和4%(1/24),χ2检验P<0.001。结论膀胱肿瘤组织Survivin蛋白和基因的表达量高于对照组,因此,Survivin有望成为膀胱瘤高危人群筛选,肿瘤诊断和监测的工具之一。