Research Advances in the Role of Keratins in Gastrointestinal Cancer

The incidence and mortality rates of gastrointestinal(GI)cancer remain high.Despite constant improvements in diagnostic and therapeutic techniques,the early diagnosis,mid-and late-stage treatment,drug tolerance,and cancer recurrence and metastasis in GI cancer remain challenging.In this review article we summarize the recent research advance in the roles of keratins in GI cancer,with the hope that they will become efficient biomarkers for the prediction,diagnosis,or treatment of these malignancies.

Keratin17联合HPV E6/E7 mRNA对宫颈低度鳞状上皮内病变转归的预测价值

目的研究Keratin17(Krt17)联合人乳头瘤病毒(human papillomavirus,HPV)E6/E7 mRNA对宫颈低度鳞状上皮内病变(low-grade squamous intraepithelial lesions,LSIL)转归的预测价值。方法选择2020年1月~2021年9月期间诊断为LSIL并完成24个月随访的患者,根据第24个月复诊时病变组织学检查结果进行分组,LSIL消退或维持的患者为病变未进展组,LSIL进展至更高级别的患者为病变进展组。比较2组患者首次确诊时临床资料、Krt17 mRNA表达水平、E6/E7拷贝数的差异,采用多因素logistic逐步回归分析LSIL病变进展的危险因素,采用ROC曲线分析Krt17联合E6/E7对LSIL病变进展的预测价值。结果98例LSIL患者中病变进展者19例、占19.39%,病变未进展者79例、占80.61%;病变进展组的LSIL患者年龄、吸烟比例、绝经比例、Krt17 mRNA表达水平、E6/E7拷贝数高于病变未进展组(P<0.05);多因素logistic逐步回归分析显示,年龄≥45岁、吸烟、Krt17 mRNA表达水平增加、E6/E7拷贝数增加是LSIL病变进展的危险因素;ROC曲线分析显示Krt17和E6/E7对LSIL病变进展具有预测价值,两指标联合预测的灵敏度和特异度分别为81.01%和94.74%。结论Krt17联合HPV E6/E7对LSIL病变进展具有一定预测价值。

Artificial nerve graft constructed by coculture of activated Schwann cells and human hair keratin for repair of peripheral nerve defects

Studies have shown that human hair keratin(HHK) has no antigenicity and excellent mechanical properties. Schwann cells, as unique glial cells in the peripheral nervous system, can be induced by interleukin-1β to secrete nerve growth factor, which promotes neural regeneration. Therefore, HHK with Schwann cells may be a more effective approach to repair nerve defects than HHK without Schwann cells. In this study, we established an artificial nerve graft by loading an HHK skeleton with activated Schwann cells. We found that the longitudinal HHK microfilament structure provided adhesion medium, space and direction for Schwann cells, and promoted Schwann cell growth and nerve fiber regeneration. In addition, interleukin-1β not only activates Schwann cells, but also strengthens their activity and increases the expression of nerve growth factors. Activated Schwann cells activate macrophages, and activated macrophages secrete interleukin-1β, which maintains the activity of Schwann cells. Thus, a beneficial cycle forms and promotes nerve repair. Furthermore, our studies have found that the newly constructed artificial nerve graft promotes the improvements in nerve conduction function and motor function in rats with sciatic nerve injury, and increases the expression of nerve injury repair factors fibroblast growth factor 2 and human transforming growth factor B receptor 2. These findings suggest that this artificial nerve graft effectively repairs peripheral nerve injury.

Close-loop recyclable and flexible halide perovskite@wool keratin sensor with piezoelectric property

Halide perovskites with excellent piezoelectric properties,but their poor stability hinders their largescale application.Herein,a sandwich-structured halide perovskite flexible sensor with good stability was developed according to a three-step procedure as follows:(ⅰ) in-situ growth of wool keratinCsPbBr_(3)(WK-CsPbBr_(3)) using wool keratin in interfacial passivation and coating,(ⅱ) electrospinning of a wool keratin-CsPbBr_(3)/polyacrylonitrile(WCP) nanofiber film,and(ⅲ) coating of the WCP nanofiber with polydimethylsiloxane(PDMS) to obtain a sensor(WCPP).The sensor could generate a piezoelectric voltage of 7.8 V at a pressure of 6 kPa in the stages of pressing and releasing,and the output characteristics did not decline even after 10,000 cycles.Compared to the 4-month stability of the perovskite sensor,WCPP sensor exhibited the output performance even after 16 months,which indicated that wool keratin as a multidentate improved the stability of the halide perovskite.Additionally,the sensor displayed a self-cleaning property and could also light up 14 commercial LEDs.The close-loop recycling of the lead halide perovskite was achieved by dissolving the WCP nanofiber film in DMF and then reelectrospinning.Therefore,the method proposed is a step forward for achieving the commercialization of WK-CsPbBr_(3) and providing new avenues for further utilization of wool waste.

慢性异体肾移植病人肾纤维化组织中MMP-2、TIMP-1、Vimentin、Keratin免疫组化定量分析

目的:探讨慢性异体肾移植病人肾纤维化组织中基质金属蛋白酶-2(MMP-2)、金属蛋白酶抑制物(TIMP-1)、波形蛋白(Vimentin)和角蛋白(Keratin)的表达与肾纤维化间的关系。方法:收集我院18例肾移植后不同程度肾纤维化病人及10例正常肾组织,进行MMP-2、TIMP-1、Vimentin及Keratin的免疫组化定量检测分析。结果:肾纤维化早期,与正常肾组织对比,抗MMP-2和Keratin呈显较强免疫阳性反应,且量增加;随肾纤维化程度增加,抗MMP-2和Keratin免疫染色阳性反应减弱,量减少,甚至出现阴性;抗TIMP-1和Vimentin免疫染色阳性增强,并随着纤维化进展而量增加。结论:由肾纤维化组织局部Vimentin阳性与相关细胞产生并分泌的细胞因子以诱导MMP-2、TIMP-1的活性改变在慢性肾移植肾病患者的肾纤维化过程中具有关键性作用。

人细胞角蛋白Keratin 14的生物信息学分析

人细胞角蛋白Keratin 14是I型中间丝蛋白家族的亚群,参与形成上皮细胞的细胞骨架。利用生物信息学分析Keratin 14基因的启动子区、CpG岛及其所编码的蛋白质的理化性质、结构特点和功能特征。结果表明,Keratin 14基因存在两个启动子区,启动子区不存在CpG岛;Keratin 14全长472个氨基酸,其等电点为5.09,是一个无跨膜结构的亲水性蛋白质;角蛋白Keratin 14二级结构具7个α螺旋和5个β折叠,预测三级结构的拉曼图分析显示其结构稳定;与Keratin 14相互作用的蛋白质主要是角蛋白,而且Keratin 14还参与细胞的生长和分裂,有促进细胞分化的作用;此外,Keratin 14的氨基酸序列在哺乳动物间的同源性较高。上述对角蛋白Keratin 14的表达、结构和功能的预测可以为研究其在生命过程中的作用提供重要的信息。

Isolation,Screening and Primary Identification of a Keratin-degrading Fungus

[Objective] The paper was to provide new germplasm sources for efficient and economical degradation and utilization of animal keratin.[Method] The keratin-degrading fungus was isolated,screened and primarily identified by using the combination method of traditional isolation and screening,solid culture-medium degradation and animal test.[Result] A strain of non-pathogenic filamentous fungi with high degradation efficiency was obtained,which was preliminarily identified to be a species in Mucoraceae.[Conclusion] The discovery of the strain enriched the family members of keratin-degrading fungus,and provided new germplasm resources for degradation and utilization of animal keratin.

角蛋白5基因在内蒙古绒山羊皮肤毛囊发育周期中的表达模式分析

为揭示角蛋白5(Keratin 5)基因与内蒙古绒山羊皮肤毛囊发育周期的关联作用,实验采用RT-q PCR和West-blotting方法,从m RNA水平和蛋白质水平探索周岁母羊体侧部皮肤组织一年12个月毛囊发育周期中Keratin 5表达量的变化。结果表明:Keratin 5在皮肤毛囊发育周期中的表达量在m RNA水平和蛋白质水平上均有明显差异,在10月皮肤毛囊发育兴盛期表达量显著增高(P<0.05),在3月毛囊发育休止期表达量显著降低(P<0.05)。结果显示,Keratin 5与绒山羊皮肤毛囊发育周期有关联作用。

Characterization of CD133^+ parenchymal cells in the liver:Histology and culture

AIM： To reveal the characteristics of CD133^＋ cells in the liver, METHODS： This study examined the histological characteristics of CD133^＋ cells in non-neoplastic and neoplastic liver tissues by immunostaining, and also analyzed the biological characteristics of CD133^＋ cells derived from human hepatocellular carcinoma （HCC） or cholangiocarcinoma cell lines. RESULTS： Immunostaining reveated constant expression of CD133 in non-neoplastic and neoplastic biliary epithelium, and these cells had the immunophenotype CD133^＋/CK19^＋/HepPar-1. A small number of CD133^＋/CK19/HepPar-1^＋ cells were also identified in HCC and combined hepatocellular and cholangiocarcinoma. In addition, small ductal structures, resembling the canal of Hering, partly surrounded by hepatocytes were positive for CD133. CD133 expression was observed in three HCC （HUH7, PLC5 and HepG2） and two cholangiocarcinoma cell lines （HuCCT1 and CCKS1）. Fluorescence-activated cell sorting （FACS） revealed that CD133^＋ and CD133 cells derived from HuH7 and HuCCT1 cells similarly produced CD133^＋ and CD133 cells during subculture. To examine the relationship between CD133^＋ cells and the side population （SP） phenotype, FACS was performed using Hoechst 33342 and a monoclonal antibody against CD133. The ratios of CD133^＋/CD133 cells were almost identical in the SP and non-SP in HUH7. In addition, four different cellular populations （SP/CD133^＋, SP/CD133, nonP/CD133^＋, and non- SP/CD133） could similarly produce CD133^＋ and CD133- cells during subculture. CONCLUSION： This study revealed that CD133 could be a biliary and progenitor cell marker in vivo. However, CD133 alone is not sufficient to detect tumor-initiating cells in cell lines.

Rational design of water-soluble,homotypic keratins self-assembly with enhanced bioactivities

Recombinant keratins possess strong hemostatic and wound healing properties but suffer from poor water solubility that restricts their bioactivities in biomedical applications.Herein,we report the rational design and synthesis of water-soluble keratins using a simple methodology named the QTY code.In vitro biophysical analyses and molecular dynamic simulation demonstrated a 200-fold increase in the water solubility of QTY variant keratins without apparent structural changes compared to native proteins.Homotypic self-assembly was observed for the first time in recombinant keratins in an aqueous environment,without urea and after QTY modification.Cell and animal experiments showed the in situ gel-forming capability of QTY variant keratins with superior hemostatic and wound healing activities at the wound sites compared to native recombinant keratins.Our work not only presented a simple and feasible pathway to produce large amounts of water-soluble keratins using QTY modification but also validated the enhanced self-assembly,hemostasis,and wound healing properties of these novel keratin species that may open up new venues for biomedical applications.

北京大学第一医院杨勇、林志淼课题组发现皮肤角蛋白代谢新基因

2016年10月Nature Genetics在线发表了北京大学第一医院皮肤性病科杨勇、林志淼课题组与清华大学谭旭课题组合作的研究成果,题为＂Stabilizing mutations of KLHL24 ubiquitin ligase cause loss of keratin 14 and human skin fragility＂。该研究在国际上确定了遗传性大疱性表皮松解症（epidermolysis bullosa,EB）的一种新致病基因KLHL24及其全新发病机制，同时，研究还发现，KLHL24是皮肤结构分化形成及蛋白代谢稳态的重要调节基因，揭示了皮肤角蛋白泛素化的信号传导通路，为角蛋白异常性疾病治疗提供了新途径。

Keratin和CEA在前列腺病变组织的分布

本文用CEA和Keratin单克隆抗体,对前列腺正常组织、结节性增生和癌各15例进行标记,其中15例前列腺癌随访2年以上,标记方法为BA法。结果,CEA在正常前列腺组织、结节性增生和癌中含量低,阳性率低,提示CEA非识别前列腺癌的理想标记物。Keratin在正常前列腺组织、结节性增生和癌的所有病例中均为阳性。三者在染色强度上无显著差异。不同分化程度的癌在染色强度上也无显著差异。Keratin广泛分布于前列腺癌的上皮细胞内,间质标记均为阴性,对鉴别前列腺上皮性和非上皮性肿瘤是有意义的。

血清CYFRA21-1、CEA、TSGF联合检测对恶性肿瘤的诊断价值

目的 联合检测细胞角蛋白19片段(CYFRA21-1)、癌胚抗原(CEA)、恶性肿瘤特异性生长因子(TSGF)在恶性肿瘤诊断中的价值。方法 采用放免、化学发光及生化方法检测198例恶性肿瘤病人血清CYFRA21-1、CEA、TSGF的含量,并与对照组进行比较、分析。结果 恶性肿瘤组的血清CYFRA21-1、CEA、TSGF含量明显高于正常对照组,差异有显著性(P<0.05)。CYFRA21-1、CEA、TSGF测定的阳性率分别为29.7％、8.0％、53.6％,各组间差异有高度显著性(P<0.001),联检阳性率明显提高。结论 联合检测血清CYFRA21-1、CEA、TSGF能明显提高恶性肿瘤的诊断阳性率,同时,CYFRA21-1、TSGF对肿瘤普查具有一定的价值。

C-type lectins and human epithelial membrane protein1:Are they new proteins in keratin disorders?

Here we report a family with a clinical spectrum of Pachyonychia Congenita Tarda (PCT) encompassing two generations via a balanced chromosomal translocation between 4q26 and 12p12.3. We discuss the effects of chromosomal translocations on gene expression through involved breakpoints and structural gene abnormalities detected by array CGH. We believe that the family we present gives further insight to the better understanding of molecular and structural basis of keratin disorders, and to the late onset and genetic basis of PCT through the possible role of C-type lectins and human epithelial membrane protein1 (EMP1). Better understanding of the molecular basis of keratin disorders is the foundation for improved diagnosis, genetic counseling and novel therapeutic approaches to overcome the current treatment limitations related to this disease.

结直肠癌中Keratin 17的表达及其临床预后价值

目的分析角蛋白17(Keratin 17,KRT17)在结直肠癌(colorectal cancer,CRC)中的表达及其临床预后价值。方法采用生物信息学方法分析KRT17在CRC数据库中的表达及预后价值;进一步,采用免疫组织化学技术检测KRT17蛋白在95对CRC临床样本组织中的表达水平,并分析其表达与患者临床病理特征及预后的相关性。结果生物信息分析发现,KRT17在CRC组织中的表达水平显著高于正常组织,其表达与肿瘤病理类型、淋巴结转移及TNM分期相关,并预示较差的总生存期(P均<0.05)。免疫组化验证显示,与癌旁组织相比,蛋白在CRC组织中的表达显著上调(t=45.704,P<0.001),与生物信息学分析结果保持一致;KRT17高表达与肿瘤分化程度、浸润深度、淋巴结转移、TNM分期、淋巴脉管浸润及神经侵犯显著相关(P_(均 )0.05);生存分析显示,KRT17高表达组CRC患者的总生存期显著低于低表达组(χ^(2)=8.510,P=0.004);进一步的单因素和多因素分析结果表明,KRT17高表达是影响CRC患者总生存期的一个独立危险因素(HR=2.015,95%CI:1.219~4.457,P=0.018)。结论KRT17在CRC组织中表达显著上调,且其高表达预示着肿瘤进展及不良预后,有望成为CRC患者预后评估的生物标志物。

增生性筋膜炎异常表达角蛋白的临床病理及免疫组织化学分析并文献复习

增生性筋膜炎是发生于皮下的一种形成肿物的增生性病变,除了有类似于结节性筋膜炎的肥胖的纤维母/肌纤维母细胞增生之外,可见到数量不等的神经节样大细胞。本文报道发生于臀部皮下的增生性筋膜炎1例,鉴于本例患者有直肠癌手术病史,首先需排除转移癌可能性,有关纤维母细胞/肌纤维母细胞异常表达角蛋白查阅复习相关文献。

Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis

A new feather-degrading bacterium was isolated from a local feather waste site and identified as Bacillus subtilis based on morphological, physiochemical, and phylogenetic characteristics. Screening for mutants with elevated keratinolytic activity using N-methyl-N＇-nitro-N-nitrosoguanidine mutagenesis resulted in a mutant strain KD-N2 producing keratinolytic activity about 2.5 times that of the wild-type strain. The mutant strain produced inducible keratinase in different substrates of feathers, hair, wool and silk under submerged cultivation. Scanning electron microscopy studies showed the degradation of feathers, hair and silk by the keratinase. The optimal conditions for keratinase production include initial pH of 7.5, inoculum size of 2% （v/v）, age of inoculum of 16 h, and cultivation at 23 ℃. The maximum keratinolytic activity of KD-N2 was achieved after 30 h. Essential amino acids like threonine, valine, methionine as well as ammonia were produced when feathers were used as substrates. Strain KD-N2, therefore, shows great promise of finding potential applications in keratin hydrolysis and keratinase production.

Keratin13基因在喉癌发生中的作用

目的 探讨keratin 13基因在喉癌发生中的作用。方法 在keratin 13基因内部及附近选择 5个微卫星引物进行LOH分析 ,于DNA水平间接检测 10 0例喉癌患者中该基因的缺失情况。结果 5个STR位点均存在LOH ,其中D17S196 4E、D17S2 0 92、D17S791、D17S16 6 5及D17S80 8位点的LOH频率分别为 30 4 8%、2 6 0 2 %、2 1 6 2 %、37 6 6 %和 2 1 5 1%,以D17S16 6 5位点的LOH频率最高 ,杂合性丢失与临床分期无显著相关。结论 Keratin 13基因在喉癌的发生中具有重要作用 ,具体机制有待进一步研究。

The Sandfish's Skin:Morphology,Chemistry and Reconstruction

The sandfish is a lizard having the remarkable ability to move in desert sand in a swimming-like fashion. The most outstanding adaptations to this mode of life are the low friction behaviour and the extensive abrasion resistance of the sandfish skin against sand, outperforming even steel. We investigated the topography, the composition and the mechanical properties of sandfish scales. These consist of glycosylated keratins with high amount of sulphur but no hard inorganic material, such as silicates or lime. Remarkably, atomic force microscopy shows an almost complete absence of attractive forces between the scale surface and a silicon tip, suggesting that this is responsible for the unusual tribological properties. The unusual glycosylation of the keratins was found to be absolutely necessary for the described phenomenon. The scales were dissolved and reconstituted on a polymer surface resulting in properties similar to the original scale. Thus, we provide a pathway towards exploitation of the reconstituted scale material for future engineering applications.

In vitro tissue engineering of lamellar cornea using human amniotic epithelial cells and rabbit cornea stroma

AIM:To reconstruct the lamellar cornea using human amniotic epithelial(HAE) cells and rabbit cornea stroma in vitro using tissue engineering technology.·METHODS:Human amnia taken from uncomplicated caesarean sections were digested by collagenase to obtain HAE cells,and the cells were cultured to proliferate.Rabbit corneal epithelial cells were removed by n-heptanol to make lamellar matrix sheets.The second passage of HAE cells were cultured on the corneal stroma sheets for 1 or 2 days,then transferred to an air-liquid interface environment to culture for 2weeks.Tissue engineered lamellar cornea(TELC)morphology was observed by Hematoxylin-eosin(HE)staining;its ultrastructure was observed by transmission electron microscopy(TEM) and scanning electron microscopy(SEM);corneal epithelial cell-specific keratin3 and keratin 12 were detected with immunofluorescence microscopy.·RESULTS:HAE cells grew on the rabbit corneal stroma,forming a monolayer after 1-2 days.About 4-5 layers of epithelial cells developed after 2 weeks of air-liquid interface cultivation,a result similar to normal corneal epithelium.Rabbit corneal stromal cells were significantly reduced after one week,then almost completely disappeared after 2 weeks.TEM showed desmosomes between the epithelial cells;hemidesmosomes formed between the epithelial cells and the basement membrane.SEM revealed that the HAE cells which grew on the lamellar cornea had abundant microvilli.The tissue-engineered cornea expressed keratin 3 and keratin 12,as detected by immunofluorescence assay.·CONCLUSION:Functional tissue-engineered lamellar corneal grafts can be constructed in vitro using HAE cells and rabbit corneal stroma.