基因工程抗角蛋白抗体在正常皮肤和几种表皮增生性皮肤病皮损中的反应定位

目的　探索一株基因工程人源性抗角蛋白Fab抗体在正常皮肤及几种表皮增生性皮肤病皮损中的反应定位。方法　利用从噬菌体抗体库中筛选到的特异表达抗角蛋白Fab片段的质粒转化大肠杆菌 ,IPTG诱导表达出Fab抗体 ,纯化鉴定后用此抗体对正常皮肤及银屑病、鳞癌、基底细胞癌和脂溢性角化病皮损进行免疫组化染色。结果　正常皮肤表皮呈阴性染色 ,毛囊呈阳性染色 ,银屑病、鳞癌、基底细胞癌和脂溢性角化病皮损均呈现明显的阳性着色 ,其中银屑病皮损基底细胞层为强阳性。所有细胞染色部位均位于胞质 ,胞核未见着色 ,真皮为阴性。结论　该株人源性抗角蛋白Fab抗体主要与表皮组织结合 ,对银屑病等表皮增生性皮肤病的损害具有较高特异性。     

甲状腺乳头状癌CK19、Mesothelin、CD56表达及临床意义

目的观察甲状腺乳头状癌（PTC）的临床病理特征,检测相关免疫标记并探讨其在PTC中的应用价值。方法收集63例PTC、20例甲状腺良性乳头状增生性病变（BPH）,进行形态学观察,采用免疫组化SABC法检测细胞角蛋白19（CK19）、间皮细胞（Mesothelin）及CD56的表达。结果CK19、Mesothelin、CD56在63例PTC中阳性表达率分别为100．0％、95．2％、3．2％;在20例BPH中三者阳性表达率分别为35．0％、15．0％、90．0％。结论CK19和Mesothelin的阳性表达率在PTC与BPH之间比较差异有统计学意义（P〈0．05）,CD56阴性表达对两种病变的鉴别诊断有重要价值。联合应用三种标记物可以明显提高诊断的准确性和特异性。     

The formation of wrinkles caused by transition of keratin intermediate filaments after repetitive UVB exposure

It has been reported that the formation of wrinkles involves changes in the elastic properties of the dermis due to the denaturation of elastic fibers. Several studies have shown that the hydration condition of the stratum corneum is also important in wrinkle formation. It is, however, still unclear how the stratum corneum contributes to wrinkle formation. Here we investigated the relationship between the formation of wrinkles and changes in the physical properties and condition of the skin after repetitive ultraviolet B (UVB) irradiation of hairless mice (HR/ICR). Repetitive UVB irradiation caused wrinkles on the dorsal skin of the mice. The elasticity (E) of the stratum corneum of UVB-irradiated mice was significantly lower than that of age-matched control (unirradiated) mice. UVB exposure also caused a deterioration of the fibrous ultrastructure of keratin intermediate filaments (KIFs) in the skin. We conclude that the deterioration of KIFs in the stratum corneum caused by repetitive UVB irradiation decreases the elastic properties of the stratum corneum, resulting in the formation of wrinkles.     

PET-CT、血清CEA及CYFRA21-1预测靶向药物对非小细胞肺癌疗效的研究

目的探讨PET-CT的标准摄取值(SUV)、血清癌胚抗原(CEA)、细胞角蛋白19的可溶性片段(CYFRA21-1)对靶向药物治疗非小细胞肺癌疗效的预测作用。方法对39例非小细胞肺癌患者在治疗前,进行EGFR基因突变、PET-CT SUV值、血清CEA、CYFRA21-1检测,治疗2周观察血清CEA、CYFRA21-1下降程度,计算以上各项指标与患者无进展生存期(PFS)及总生存期(OS)的关系。结果 SUV值≤5的患者及治疗2周后CEA水平下降≥50%的患者获得更佳的PFS及OS。但CYFRA21-1水平下降≥50%的患者,并未获得更长的PFS及OS。EGFR突变的患者SUV均值较EGFR野生型患者明显低(P<0.05)。治疗后EGFR基因突变的患者较野生型患者更可能获得CEA下降≥50%(P<0.05)。二元logistic回归分析结果表明,SUV值≤5为EGFR基因突变的独立预测因素(P<0.001)。结论 PET-CT的SUV值、用药前期CEA水平的变化,可以预测靶向药物治疗非小细胞肺癌的疗效。     

Localization of keratins 13 and 14 in the lingual mucosa of rats during the morphogenesis of circumvallate papillae

We used fluorescence immunohistochemistry, analysis of differential interference contrast (DIC) images and confocal laser-scanning microscopy in the transmission mode, after staining specimens with toluidine blue, to examine the localization of keratin 13 (K13) and keratin 14 (K14) in the lingual epithelium of fetal and juvenile Sprague-Dawley rats during the prenatal and postnatal morphogenesis of circumvallate papillae. No immunoreactivity specific for K13 and K14 was detected in the lingual epithelium of fetuses on day 15 after conception (E15), at which time the primitive rudiment of the circumvallate papillae was detectable by the thickening of several layers of cuboidal epithelial cells. On E17 and E19, the developing circumvallate papillae were clearly recognizable, consisting of a central papilla and the surrounding sulcus. No immunoreactivity specific for K13 and K14 was evident in the lingual epithelium around these structures at this time. K14-specific immunoreactivity was first detected in the basal layer of the epithelium of the circumvallate papillae on postnatal day 0 (P0) and K13-specific immunoreactivity was detected on P7. Morphogenesis of the circumvallate papillae progressed significantly from P0 to P14, and immunoreactivity specific for K13 and K14 was clearly recognizable after P7. The respective patterns of K13-specific and K14-specific immunoreactivity differed during the development of the circumvallate papillae: K13-specific immunoreactivity was generally evident in cells of the intermediate layer of the epithelium, while K14-specific immunoreactivity was detected in cells of the basal and suprabasal layers. The present results are discussed in the context of the previously determined localization of K13 and K14 in the dorsal epithelium of the anterior part of the rat tongue during its morphogenesis.     

深Ⅱ度烧伤创面磨痂治疗后残留皮肤组织中表皮干细胞标记物

背景：大面积烧伤往往需要削痂清除痂皮,但削痂手术往往会导致人为因素除去过多的残留再生皮肤组织。 目的：观察磨痂治疗深Ⅱ度烧伤创面对残留皮肤组织中表皮干细胞标记物角化蛋白19表达的影响。 设计、时间及地点：随机分组对比观察,于2002-10/2004-01在广西医科大学完成。 对象：烧伤整形外热烧伤科患者40例,年龄18~37岁,平均烧伤面积为15%~45%,深Ⅱ度烧伤面积为15%~30%。随机分组方法分为磨痂组(n=20)、削痂组(n=20)。 方法：磨痂组采用电动磨痂仪对创面进行磨痂,由浅入深磨去坏死组织,至创面基底呈现红色充血,有珠状的小出血点为止。削痂组采用滚轴削痂刀对创面进行削痂,削至创面基底呈瓷白色、组织致密,湿润面有光泽,无网状血管栓塞和呈灰暗棕色的无光泽组织,放松止血带后可见密集点状出血较均匀,但有时因操作原因削痂过深,基底露出脂肪组织。两组创面术后用辐照猪皮覆盖。 主要观察指标：取术前、术后小块创面组织以免疫组织化学SP法检测标皮肤再生组织中标记物角化蛋白19的表达,在100倍光学显微镜下,任意选取5个视野计数细胞团的表达数;观察两组创面愈合时间,记录超过4周不愈合的肉芽创面,需要再次手术植皮修复创面。 结果：磨痂保留了较多的真皮组织,毛囊,汗腺,皮脂腺等皮肤附件。削痂后创面基底组织有薄层网状组织残留及部分毛囊及汗腺,有些标本视野中可见无真皮组织,为脂肪组织。细胞团的表达数比较结果显示,两组前及术后创面均有创面残留皮肤组织中标记物角化蛋白19表达,磨痂组术前、术后数量无明显变化(P > 0.05),削痂组术后较术前数量减少(P < 0.05);磨痂组创面较削痂组提前愈合(P < 0.05)。需要手术植皮创面磨痂组2处,削痂组8处(P < 0.05)。 结论：应用磨痂术治疗烧伤深Ⅱ度创面能有效掌握磨痂深度,对组织损伤小,与削痂比较保留更多的再生皮肤组织,通过表皮干细胞的再生,利于创面再上皮修复,缩短创面愈合时间。     

抗角蛋白自身抗体对培养角朊细胞产生白细胞介素8的影响

为探讨抗角蛋白自身抗体（AKantoAb）对角朊细胞免疫功能的影响，实验以IL-1β刺激无血清培养角朊细胞及AKautoAb作用后的角朊细胞，用夹心ELISA法检测培养上清中IL－8的产量。结果表明IL-1β可刺激角朊细胞产生IL－8，并对IL－1β有浓度依赖性，AKautoAb对角朊细胞产生IL－8有明显抑制作用。提示AKautoAb在炎性皮损角朊细胞中的沉着，可能存在有益的调节作用。     

PCR-DNA直接测序检测1例单纯型大疱性表皮松解症Weber-Cockayne亚型(WC-EBS)患者角蛋白K5基因点突变

单纯型大疱性表皮松解症（ＥＢＳ）是一组常染色体显性的遗传性疾病，研究表明本病存在角蛋白Ｋ５／Ｋ１４基因点突变。ＥＢＳ的各个亚型突变发生部位有一定差异，其中Ｗｅｂｅｒ－Ｃｏｃｋａｙｎｅ亚型（ＷＣ－ＥＢＳ）突变多位于Ｋ５／Ｋ１４的连接区Ｌ１－２。本研究设计了扩增Ｋ５基因Ｌ１－２区ＤＮＡ片段的引物，应用ＰＣＲ对－ＷＣ－ＥＢＳ家系的患者及未发病成员进行扩增。ＰＣＲ产物测序发现患者Ｋ５第３４６密码子发生了Ａ→Ｃ的碱基替换，导致色氨酸（ＴＡＴ）变成丝氨酸（ＴＣＴ），而未发病成员则未见有碱基突变。结果表明，通过ＰＣＲ结合ＤＮＡ直接测序不失为快速、准确检测基因突变的方法。此外，连接区在角蛋白结构中不如螺旋区重要，因而此区基因突变对角蛋白二聚体形成的影响不大，这与临床上ＷＣ－ＥＢＳ病情相对较轻是一致的。     

Cloning and characterization of antikeratin human antibodies using a semisynthetic phage antibody library

Antikeratin autoantibodies (AK auto Abs) are very important elements of the human immune system. To improve the outcome of studies on AK auto Abs, it is necessary to generate antikeratin human monoclonal antibodies. The purpose of present study was to isolate antikeratin human monoclonal antibodies by panning a phage antibody library. A semisynthetic phage antibody library with capacity of 4.0×10⁸ members was previously constructed. Panning of the library was performed against human epidermal keratin extracted from psoriatic scales. At the last round of the panning, individual colonies were grown in culture for expression of phage antibodies. Their binding activities and specificities to keratin were determined by ELISA, and positive clones were analyzed by DNA fingerprinting. The selected clones were induced with IPTG to express soluble Fab fragments, which were further characterized by ELISA, immunohistochemistry and Western blotting. Finally, DNA sequencing of the variable regions was performed. A human antibody clone which was able to express soluble Fab fragments and recognize Mr 46,000 keratin (K17) was isolated. DNA sequencing demonstrated that the VH and VL of the antibody came from the human VH1 and V2 families, respectively. We conclude that phage antibody library technology is a powerful way to generate human monoclonal antibodies. The antikeratin antibody we isolated in the present study would be useful in the research on AK auto Abs.