Mutation analysis of type Ⅱ hair keratin gene in a pedigree with monilethrix

Objective To investigate the mutation of type Ⅱ human hair basic keratin (hHb) gene in a family with monilethrix.Methods Scanning electron microscopy was used to observe the structure of hair shafts.With informed consent,blood samples were drawn from affected and unaffected membets in this family,as well as from 50 healthy controls.Genomic DNA was isolated from these samples.The exon 1 and exon 7 of hHb1,hHb3 and hHb6 were amplified by polymerase chain reaction (PCR).All the PCR products were sequenced directly using ABI3730 automated sequencer.DNA sequence alignment was carried out with BLAST software.Results A typical beaded appearance was observed in affected hairs by using scanning electron microscopy.There were obvious longitudinal ridges and sulcuses in hair node.and hair cuticles were irregularly shaped.Most cortex and medullary substance were absent in affected hairs of a patient.After sequence alignment,a G1289A point mutation in exon 7 of hHb6 gene,which led to a substitution of arginine for glutamide at codon 430,was detected in affected members of this family,but not in unaffected family members or 50 unrelated human controls.No mutation was observed in exon 1 or exon 7 of hHb1 and hHb3 gene or exon 1 of hHb6 gene.Conclusion The missense mutation of R430Q is a novel mutation.which may be associated with the pathogenesis of monilethrix in this pedigree.     

类胰蛋白酶的检测及临床意义

类胰蛋白酶是主要由肥大细胞分泌的一种炎症介质,具有多重生物活性效应,可启动多种疾病反应的进程,并与其发生和发展有关.在很多肥大细胞相关疾病中,血清类胰蛋白酶都伴随肥大细胞的脱颗粒过程而升高.检测此类疾病时类胰蛋白酶血清水平的变化,有望为疾病的临床诊断和治疗提供重要的依据.     

应用双重免疫组化技术研究肥大细胞亚型与食管癌的相关性

目的：探讨食管癌组织中肥大细胞（mastcell，MC）亚型的变化及其浸润程度与食管癌临床病理特征的关系。方法：采用双重免疫组化染色方法对90例食管癌根治术手术标本中MC进行检测，并探讨MC亚型T型、TC型和C型与食管癌的组织学分级、浸润深度及淋巴结转移之间的关系。结果：食管癌组织内的MC主要分布于癌旁交界区。食管癌中MC、和MCTC两种亚型MC的表达与食管癌浸润深度及淋巴结转移呈显著的负相关性，而MCC型MC的表达与癌组织浸润深度及转移无关。结论：癌间质内MCT和MCTC型MC的浸润可能具有抑制食管癌生长及转移的作用。MC浸润的数量与食管癌的生物学行为有相关性，对食管癌患者预后评估有一定的参考价值。     

革兰阳性菌肽聚糖对人嗜碱性粒细胞中TLR3、TLR9mRNA的上调作用

目的:探讨Toll样受体(TLR)的配体肽聚糖(PGN)、聚肌苷酸_聚胞苷酸(PolyI_C)、脂多糖(LPS)和含有去甲基化的寡核苷酸序列(CpGDNA)对嗜碱性粒细胞中TLR2～4和TLR9mRNA表达的调节作用。方法:用实时定量_逆转录聚合酶链反应检测PGN、PolyI_C、LPS和CpGDNA刺激人嗜碱性粒细胞系(KU812)后,TLR2～4和TLR9mRNA表达的变化。结果:PGN刺激KU812细胞后,TLR3和TLR9mRNA的表达与未刺激组比有统计学意义(P<0.05);而PolyI_C、LPS和CpGDNA刺激KU812细胞后,细胞中TLR2～4和TLR9的mRNA与未刺激组比无统计学意义(P>0.05)。结论:革兰阳性菌的感染可能增加人嗜碱性粒细胞对病毒或其它细菌感染的敏感性。     

肿瘤坏死因子调节的肥大细胞蛋白酶激活受体表达分析

目的 检测肿瘤坏死因子(TNF)引起的肥大细胞蛋白酶激活受体(PAR)-1,2,3,4表达。方法 肥大细胞P815培养后以不同浓度的TNF激发肥大细胞,在不同时间点收集细胞,给予聚乙二醇辛基苯基醚( Triton X-100)非Triton X-100处理后用流式细胞术和激光共聚焦方法检测肥大细胞蛋白酶激活受体的表达。结果 TNF激发肥大细胞2h、6h和16 h后,Triton X-100与非Triton X-100处理后,肥大细胞PAR-1,3表达均无明显变化;但TNF以浓度依赖方式上调P515肥大细胞PAR-2,4表达(P＜0.05);上述各时间点检测PAR-1,2,3表达情况,Triton X-100处理组与非Triton X-100处理组检测结果无明显差异,但PAR-4表达结果显示Triton X-100处理组表达强于非Triton X-100处理组。结论 TNF可以上调P815肥大细胞PAR-2,4表达,但对PAR-1,3表达的调节作用不明显,Triton X-100处理和非Triton X-100处理不影响TNF调节的肥大细胞PARs表达的检测结果。     

抗人蛋白酶激活受体4单克隆抗体的制备与鉴定

目的:制备抗人蛋白酶激活受体4(PAR4)单克隆抗体(mAb).方法:以hPAR4为免疫原免疫BALB/c小鼠,运用杂交瘤技术制备抗PAR4 mAb.采用Ig类与亚类鉴定试剂盒鉴定mAb的Ig类型,采用ELISA、点杂交、免疫组化染色法(人结肠、扁桃体及包皮组织)、流式细胞术、激光共聚焦显微镜(CLSM)技术鉴定mAb的特异性.结果:获得3株可稳定分泌抗PAR4 mAb的杂交瘤细胞,其分泌的mAb均为IgM.免疫组化染色表明,mAb将人结肠组织中的腺上皮细胞、疑似肥大细胞和淋巴细胞,扁桃体组织中的淋巴细胞和包皮组织中的肥大细胞染成褐色.流式细胞术分析显示,3株mAb与A549细胞的PAR4均呈阳性反应.CLSM观察发现,荧光标记的阳性反应物主要位于A549细胞的胞质和胞膜上.结论:成功制备了抗PAR4 mAb,为进一步的研究工作提供了有用的试剂.