成纤维细胞生长因子受体3与人乳头瘤病毒2型E2对角质形成细胞分化的初步研究

目的研究成纤维细胞生长因子受体3(FGFR3)与人乳头瘤病毒2型(HPV2)E2对人角质形成细胞系HaCaT和正常人表皮角质形成细胞系NHEK分化的调控效应。方法在HaCaT和NHEK细胞中, 采用慢病毒转染法构建HPV2 E2稳转细胞系(HPV2 E2转染组), 采用质粒转染法构建过表达FGFR3或FGFR3突变体K650E的细胞, 即FGFR3-WT转染组、FGFR3-K650E转染组, 均以转染空载体的细胞为空载体对照组。采用实时荧光定量PCR检测HPV2 E2 mRNA表达, Western印迹法检测HPV2 E2、FGFR3及角质形成细胞分化标志性分子兜甲蛋白、聚丝蛋白、内披蛋白的表达。激光共聚焦显微镜观察HPV2 E2与FGFR3的细胞空间定位。两组数据比较采用独立样本t检验, 多组间比较采用单因素方差分析, 组间两两比较采用Dunnett-t检验。结果成功构建HPV2 E2稳转细胞系, HaCaT和NHEK细胞中HPV2 E2转染组HPV2 E2 FLAG蛋白表达量均显著高于空载体对照组(t值分别为13.71和25.91, 均P < 0.001);在HaCaT和NH...     

CD147在角质形成细胞分化过程中的表达和作用

目的 明确CD147在正常角质形成细胞和鳞状细胞癌细胞分化过程中的表达和作用。方法 运用免疫组化法检测不同分化水平的寻常疣以及良性、癌前和恶性表皮肿瘤中CD147的表达。运用免疫印迹法观察CD147在培养角质形成细胞(HaCaT)和鳞状细胞癌细胞(HSC-5)钙诱导分化过程中的表达变化。并观察高钙培养和CD147抗体对HaCaT和HSC-5细胞分化相关形态学的影响。结果 寻常疣及脂溢性角化病的CD147表达方式与正常表皮相同。光线性角化病及Bowen病中部分病例阳性。鳞状细胞癌中CD147的表达随分化降低而显著升高。HaCaT及HSC-5细胞中CD147的表达均随钙诱导的分化过程而降低。CD147抗体可与高钙培养一样诱导HaCaT及HSC-5细胞的分化。结论 CD147分子是一种新的低分化角质形成细胞标志蛋白,并可能抑制正常角质形成细胞和鳞状细胞癌细胞的分化。     

基因工程人抗角蛋白抗体在角质形成细胞内的反应定位

目的：探索一株基因工程人源性抗角蛋白Fab抗体在培养的角质形成细胞内的反应定位方法：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，纯化鉴定后将该抗体作用于培养的人角质形成细胞，在不同浓度和不同的作用时间应用免疫荧光细胞染色法及共聚焦显微镜观察陔抗体在角质形成细胞内的反应定位，同时以基因工程抗HBsAg人Fab和1％BSA为对照：结果：基因工程人源性抗角蛋白Fab抗体作用于角质形成细胞的胞浆，呈明显的绿色荧光，着色均匀，细胞核未见着色，两种对照均未见着色结论：基因工程人源性抗角蛋白Fab抗体，可与培养状态下的角质形成细胞胞浆中其特异性的抗原相结合，这为其临床应用奠定了基础。     

马尔尼菲青霉基因功能研究工具载体pSilent-GFP的构建

目的 建立一种基于载体pSilent-1介导的对马尔尼菲青霉基因进行功能研究的工具。方法 将绿色荧光蛋白（GFP）基因整合于丝状真菌表达质粒pSilent-1中,构建pSilent-GFP载体,将构建成功的pSilent-GFP载体通过电转化方法转化马尔尼菲青霉酵母细胞,经潮霉素阳性筛选及PCR方法鉴定阳性克隆子,并在荧光显微镜下观察GFP在马尔尼菲青霉中的表达。结果 成功构建了pSilent-GFP表达载体,该载体能转化马尔尼菲青霉酵母细胞并在菌体内稳定表达GFP。结论 pSilent-GFP载体在马尔尼菲青霉中稳定表达GFP,pSilent-1载体可应用于马尔尼菲青霉基因的功能研究。     

慢病毒介导shRNA沉默Nicastrin基因的人永生化角质形成细胞模型构建

目的 构建Nicastrin(NCT)基因的RNA干扰慢病毒表达载体,并在人永生化角质形成细胞(HaCaT细胞)上鉴定其沉默效率,构建NCT基因稳定下调的人永生化角质形成细胞模型,为后续研究NCT基因下调对角质形成细胞的生物学行为影响奠定实验基础.方法 设计3个靶向NCT基因特异性短发卡RNA(shRNA)表达序列并连接到慢病毒表达质粒pGLV3/H 1/GFP+ Puro中,成功构建慢病毒重组表达质粒,并通过测序鉴定.将构建的重组表达质粒与包装质粒共转染293T细胞,产生慢病毒颗粒,并测其滴度.将HaCaT细胞分为空白组(未感染病毒)、阴性对照组(NC组,感染空载病毒LV3-shNC)和干扰组(感染NCT-shRNA1、2、3慢病毒).流式细胞仪检测慢病毒转染效率,实时荧光定量PCR(qRT-PCR)、Western印迹检测靶基因的沉默效率,筛选出沉默效果最佳的干扰序列.结果 测序表明,NCT-shRNA慢病毒重组表达质粒构建成功.重组表达质粒与包装质粒共转染293T细胞后产生的慢病毒颗粒滴度为109 TU/ml.慢病毒感染HaCaT细胞后,流式细胞仪检测,慢病毒转染效率大于95％.qRT-PCR结果,与NC组相比,干扰组NCT mRNA表达量明显下降,其中NCT-shRNA1组NCT基因沉默效果最佳,干扰效率达到75％.Western印迹结果,与NC组相比,shRNA1组NCT蛋白表达抑制率为71.7％.结论 成功筛选出高效NCT-shRNA干扰序列,构建NCT-shRNA慢病毒重组表达载体,并构建NCT基因稳定下调的人永生化角质形成细胞模型.     

角质形成细胞中的自噬现象

自噬是细胞内溶酶体降解胞内大分子或细胞器的过程。最近研究提示,角质形成细胞中存在自噬现象。自噬参与角质形成细胞的分化过程,也是老化的角质形成细胞的死亡方式。部分上皮源性肿瘤中自噬被诱导,可能与其侵袭性、转移有关。自噬减轻角质形成细胞中的炎症。某些药物如卡泊三醇、氨基酮戊酸、5-氟尿嘧啶可诱导自噬而发挥相关作用,自噬还可能参与角质形成细胞、黑素细胞中巨大黑素颗粒或巨大黑素小体的形成。概述自噬在角质形成细胞中的作用。     

蛋白激酶C抑制剂GF109203X诱导角质形成细胞去分化形成表皮干细胞的初步研究

目的探讨蛋白激酶C(PKC)抑制剂GF109203X诱导角质形成细胞去分化形成表皮干细胞的可能性。方法采用酶消化法结合Ⅳ型胶原快速贴壁法获得人原代表皮干细胞及角质形成细胞,倒置相差显微镜下观察细胞生长状况,免疫细胞化学染色法检测GF109203X诱导培养后角质形成细胞的表型及功能改变。同期分离培养的人在体表皮干细胞作为本次实验的阳性对照,原代角质形成细胞培养2 d后加等体积二甲基亚砜为阴性对照。结果表皮干细胞快速黏附,培养4 d后细胞呈圆形,形态规则,折光性强,明显克隆,β1整合素、CK19及CK14呈阳性表达,CK10阴性表达;已分化角质形成细胞不能快速黏附,培养4 d细胞呈类圆形,大小不一,折光性较差,无明显克隆,β1整合素、CK19及CK14呈阴性表达,CK10呈阳性表达。角质形成细胞经GF109203X诱导培养2 d后,实验组与阳性对照组细胞群β1整合素、CK19、CK14均呈阳性表达,但实验组较阳性对照组中细胞β1整合素、CK19、CK14阳性细胞数少,CK10均呈阴性表达;阴性对照组中细胞群β1整合素、CK19及CKl4呈阴性表达,CKl0呈阳性表达。结论 GF109203X能够诱导角质形成细胞去分化形成表皮干细胞。     

sprouty4蛋白对角质形成细胞增殖及分化的影响

 目的:探证sprouty4(SPRY4)蛋白对角质形成细胞增殖及分化的影响。方法：以人永生化表皮细胞株HaCaT细胞作为实验对象,实验组HaCaT细胞采用基因敲降技术进行SPRY4蛋白抑制物的转染,对照组HaCaT细胞不作任何处理。运用实时荧光定量PCR技术（RT-qPCR）检测实验组与对照组中sprouty4蛋白对HaCaT细胞分化指标Involucrin、CK1与CK10的影响,运用CCK-8实验检测HaCaT细胞的增殖功能。结果：RT-qPCR结果显示,实验组HaCaT细胞中SPRY4基因敲降成功,敲降率约为94.75%。与对照组相比,实验组HaCaT细胞中分化指标Involucrin、CK1与CK10表达水平降低;CCK-8实验结果显示,与对照组相比,实验组HaCaT细胞增殖能力增强。结论：SPRY4蛋白表达下降对细胞增殖起到促进作用,对细胞分化起到抑制作用。     

亚砷酸对角质形成细胞增殖及凋亡的影响

目的：研究亚砷酸对不同角质形成细胞增殖及凋亡的影响。方法：以体外培养的角质形成细胞株为对象，不同浓度的亚砷酸处理细胞后，用MTT比色分析法反映细胞增殖变化，用光镜和电镜观察细胞形态，流式细胞仪测定细胞周期分布及凋亡峰的出现，Annexin-V染色荧光照相证实凋亡的发生。结果：0.5-10μmol/L亚砷酸对良性角质形成细胞株HaCaT细胞有明显抑制作用，并呈时间和剂量依赖关系，而该浓度砷剂对A431细胞无明显影响。光镜及HE染色后观察均显示随浓度升高、时间延长，凋亡细胞增多。高于上述范围HaCaT细胞变性死亡增多，流式细胞仪检测细胞周期分布变化，G2M期细胞比例及亚G1期凋亡明显升高，Annexin-V法显示有绿色荧光的阳性细胞。而A431细胞无明显形态和细胞周期分布变化。结论：一定浓度的亚砷酸可抑制HaCaT细胞增殖，并具有诱导凋亡作用，对皮肤鳞状细胞癌A431细胞无明显影响，提示良性表皮角质形成细胞株HaCaT对亚砷酸的处理比鳞状细胞癌A431细胞株更敏感。     

靶向角蛋白17基因的小干涉RNA对角质形成细胞增生与凋亡的影响

目的利用RNA干涉技术诱导角蛋白17(K17)基因沉默,观察其对角质形成细胞(KC)增生和凋亡等生物学活性的影响。方法合成两条含有针对人K17mRNA序列的正义和反义寡核苷酸,退火后与表达载体psilencer3.1-H1neo相连接,经鉴定后转染人角质形成细胞系HaCaT,分别以逆转录聚合酶链反应(RT-PCR)和免疫印迹法(W est-ern b lot)检测转染细胞K17 mRNA与蛋白水平的改变,用流式细胞仪检测转染细胞的细胞周期及凋亡情况,并通过透射电镜观察细胞的凋亡。结果成功构建了靶向人K17基因的siRNA表达载体psilencer3.1/K17,检测到瞬时转染的HaCaT细胞中K17的蛋白水平及mRNA水平均明显下降。流式细胞仪检测表明转染细胞的细胞周期发生了明显的G1期阻滞并证实凋亡的存在,电镜下观察到凋亡小体。结论对于增生活跃的角质形成细胞,K17的表达对其增生、分化和凋亡等生物学活性具有重要影响。靶向K17的siRNA能够抑制角质形成细胞增生,诱导其凋亡。     

Keratin expression in basal cell carcinomas

The keratin phenotype of 15 cases of basal cell carcinoma was assayed immunohistochemically using a panel of monospecific antibodies to single keratin polypeptides. Whilst tumour tissue strongly expressed primary keratins 5 and 14 (normally synthesized in basal keratinocytes) no expression of secondary keratins 1 and 10 (characteristic of skin-type differentiation) was detected. Keratin 17, characteristic of the outer hair root sheath, was strongly expressed in all tumours. Keratin 19 was also normally expressed in parts of the hair follicle and was detected in four cases. The 'high cell turnover' keratin 16 was frequently induced in the overlying epidermis, but was rare within tumour tissue. No expression of simple epithelial keratins 8 and 18 was detected. Whilst the keratin phenotype of tumour cells is similar to that of basal cells within part of the hair root sheath, in keeping with suggestions of a follicular origin for basal cell carcinomas, the findings are also compatible with an origin from interfollicular pluripotent stem cells differentiating towards follicular structures.     

Keratin 16 expression in epidermal melanocytes of normal human skin

Although the prevailing dogma states that keratin filaments are the hallmark of keratinocytes and other epithelial cells, recent publications suggest that they may be expressed by a variety of normal and malignant cells of different embryonic origin. Keratin expression has been reported in fibroblasts and endothelial cells as well as in various sarcomas. Also, some human melanomas express keratins in addition to the traditional diagnostic markers of differentiation, such as S-100 and melanocyte-specific antigens. Many studies have shown that cultured cells obtained from various melanomas express keratin. Most recently, keratin expression has also been shown in cultured melanocytes of normal skin. We now report that normal human melanocytes in vivo express keratin 16 (K16) but not keratins 1, 5, 8, 10, 14, or even keratin 6, the type II partner that is normally expressed with K16 in keratinocytes. Similarly, melanocytes in vitro express K16 but not K6. Keratin 16 expression in vivo was present in basal melanocytes in specimens derived from donors (0-77 years) and from different anatomic locations, suggesting that keratin 16 is constitutively expressed by all melanocytes. It appears that keratin expression may be more prevalent than previously assumed, and that these cytoskeletal filaments may play important roles in tissues and cells other than epithelia.     

Study of normal and pathological keratinization using anti-keratin polypeptide sera (author's transl)

Anti-keratin polypeptide sera were obtained against the different bands of polyacrylamide gel electrophoresis of fibrous proteins of stratum corneum derived from normal human epidermis. The sera were tested by indirect immunofluorescence of immunoperoxidase techniques. It was demonstrated that antibodies against P1 and P2 polypeptides, of MW 67,000 and 62,000 daltons respectively, were directed towards cytoplasmic antigens of keratinocytes of the upper Malpighian layers, while no labelling could be detected in the basal cell layer. Anti-P3 polypeptide sera and the anti-whole keratin serum labelled the whole epidermis, including the basal cell layer. Ultrastructural immuno-labelling performed on free epidermal cells obtained after trypsinization demonstrated that receptors for anti-P1 polypeptide sera were tonofilaments. These results showed that some keratin components (P1 and P2 polypeptides) might be absent in basal cell tonofilaments. This in in favour of various differentiation stages of the keratinizing cells. Keratin polypeptide 1 could be a useful marker of keratinization. According to preliminary studies, the expression of this keratin antigens was markedly disturbed in tumors such as basal and squamous cell carcinoma, in warts and in ichthyosis.     

Keratin expression in normal skin and epidermal neoplasms demonstrated by a panel of monoclonal antibodies

The tissue labelling of a panel of monoclonal antikeratin antibodies (LL001, LL002, LL003, LP2K, BA17, LP34, CAM5.2, and LH1) recognising keratins 1, 5, 8, 10, 14, 18, and 19 were investigated in frozen and formalin-fixed normal skin. Antibodies LL001, LL003, BA17, LP34, CAM5.2, and LH1 were found to be reactive in formalin-fixed material and were used to study 23 basal cell carcinomas, 8 squamous cell carcinomas, 5 keratoacanthomas, 5 Bowen's disease, and 6 clear cell acanthomas. All these tumours demonstrated a loss of keratin 10 expression as demonstrated by loss of labelling with LH1. Keratin 14 expression, as demonstrated by LL001, was reduced but present in all the tumours except squamous cell carcinomas and keratoacanthomas where increased labelling was observed in the more differentiated areas of these tumours. Simple epithelial keratin expression was demonstrated by positive labelling with CAM5.2 and keratin 19 by BA17 in a third of basal cell carcinomas and squamous cell carcinomas. Three of the five keratoacanthomas labelled with BA17, indicating the presence of keratin 19 in these lesions. These results support the concept that keratin expression is a phenotypic marker of the state of differentiation or malignant transformation and that patterns of keratin expression are not specific to any particular premalignant or malignant disorder.     

Transduction of the E6 and E7 genes of epidermodysplasia-verruciformis-associated human papillomaviruses alters human keratinocyte growth and differentiation in organotypic cultures

Epidermodysplasia-verruciformis-associated human papilloma virus DNA has been detected in skin cancers, in premalignant and benign skin lesions, and in plucked hairs from immunocompetent and immunosuppressed patients. The role of epidermodysplasia-verruciformis-associated human papilloma virus in the pathogenesis of nonmelanoma skin cancer is still enigmatic. In organotypic cultures we investigated the effects of retroviral transduction of the E6 and E7 genes of epidermodysplasia-verruciformis-associated human papilloma virus types 5, 12, 15, 17, 20, and 38 on the growth and differentiation of human keratinocytes. Differentiation was disturbed to different degrees as revealed by histology and by the expression patterns of differentiation markers keratin 10 and small proline rich protein 2. Conversely, proliferating cell nuclear antigen was induced in some of the suprabasal, differentiated cells to varying extent. No unscheduled DNA synthesis was detected in these cells, however, as probed by 5'-bromo-2'-deoxyuridine incorporation. Most intriguingly, when the E6 and E7 genes of epidermodysplasia-verruciformis-associated human papilloma virus types 15 and 17 were transduced, a broadening layer of basal cells and an accelerated differentiation were observed. In addition, "papilla-like structures" comprising basal-like keratinocytes arose from the basal layer into the differentiated layers. These cells did not express the differentiation markers keratin 10 and small proline rich protein 2, but did actively replicate DNA. These observations warrant further research by using this system to elucidate the replication strategy of epidermodysplasia-verruciformis-associated human papilloma virus types in keratinocytes and to shed light on the role of these human papilloma virus types in the pathogenesis of skin cancer.     

原发性皮肤淀粉样变淀粉样蛋白组织来源的研究

对30份原发性皮肤淀粉样变标本石腊切片进行抗角蛋白抗体免疫组化及复染荧光刚果红的对比观察,发现淀粉样蛋白的沉积非常广泛,而角蛋白的沉积仅发生在病变中期及后期少数标本的个别乳头内。在大多数有淀粉样蛋白沉积处并不存在角蛋白成分,电镜下也未见表皮角朊细胞破坏变性,而真皮乳头内有角蛋白沉积的部位均有淀粉样蛋白的沉积及基底细胞破坏。说明乳头内角蛋白的沉积是在淀粉样蛋白沉积之后发生的,是后果而不是原因。     

Identification of a de novo keratin 1 mutation in epidermolytic hyperkeratosis with palmoplantar involvement

Epidermolytic hyperkeratosis (EHK) (OMIM 113800) is a generalized skin disease with mostly autosomal dominant inheritance, caused by mutations in keratin 1 or keratin 10. These genes are expressed in suprabasal epidermal layers, resulting in abnormal keratin-intermediate filament cytoskeleton. We present a male patient with generalized hyperkeratosis involving palms and soles. In lesional skin massive hyperkeratosis and cytolysis in the suprabasal layers of the epidermis were observed. Immunohistochemistry staining for keratin 1 (and keratin 10) showed abnormal clumping in suprabasal keratinocytes. By electron microscopy perinuclear intermediate filament clumps were detected in the keratinocytes. A heterozygous missense mutation, designated L187F, was identified in exon 1 of the keratin 1 gene by direct sequencing. This mutation was not detected in his unaffected parents, indicative of a de novo mutational event. The homologous mutation (L187F, also designated L7F) in basal keratin genes keratin 5 or -14 causes epidermolysis bullosa simplex. The amount of keratin 1-mRNA in the patient's skin was not altered compared to controls.We propose that the severe EHK phenotype observed in our patient results from a dominant negative effect of the L187F mutant Keratin 1 allele exerted on keratin 10, the associated partner-keratin. These findings should be helpful for genetic counseling, prenatal diagnosis and studying molecular structure-function relationship in EHK.     

Keratin 16 expression defines a subset of epithelial cells during skin morphogenesis and the hair cycle

The morphogenesis of skin epithelia and adult hair follicle cycling both require integrated signaling between the epithelium and underlying mesenchyme. Because of their unique regulation, keratin intermediate filaments represent useful markers for the analysis of determination and differentiation processes in complex epithelia, such as the skin. In this study, we analyzed the distribution of mouse type I keratin 16 during skin morphogenesis, in the adult hair cycle, and in challenged epidermis. In mature hair follicles, we find keratin 16 along with its type II keratin partner keratin 6 in the companion layer of the outer root sheath during anagen and in the club hair sheath during catagen and telogen. During embryonic development, the distribution of keratin 16 is uncoupled from its presumed polymerization partner, keratin 6. Keratin 16 initially localizes within early hair germs, but rapidly shifts to a subset of cells at the interface of basal and suprabasal cells above and around the hair germ. The presence of keratin 16 at the transition between mitotically active and differentiating cells is recapitulated in primary keratinocytes cultured in vitro and in phorbol 12-myristate 13-acetate-treated back skin in vivo. We propose that keratin 16 marks cells in an intermediate state of cellular properties in which keratinocytes retain the flexibility required for activities such as cell migration and even mitosis but are resilient enough to provide the structural integrity required of the early suprabasal layers in the context of development, adult hair cycling, and wound repair.     

应用表皮干细胞与去表皮的真皮体外构建组织工程皮肤

目的 用人皮肤表皮干细胞 （ESCs）接种至同种去表皮的真皮上，体外构建组织工程皮肤。方法 用两步酶消化法分离获得角质形成细胞，并用Ⅳ型胶原快速粘附法筛选ESCs并培养；进行细胞形态学观察和用免疫组化法对ESCs进行鉴定；将ESCs接种至去表皮的真皮上浸没培养7 d后转成气-液面培养，4周后取培养的组织工程皮肤切片分别做HE染色和角蛋白的免疫组化染色。结果 培养的ESCs呈铺路石样、克隆状生长；角蛋白K19和β1-整合素的免疫组化染色阳性。培养5周的组织工程皮肤可见有3 ～ 6层细胞，并可见角质层，表皮角蛋白染色阳性。结论 应用人皮肤的ESCs与去表皮的真皮可以体外构建组织工程皮肤。     

Transient expression of a transfected gene in cultured epidermal keratinocytes: implications for future studies

We examine the effect of keratinocyte differentiation upon transient expression of a nonepithelial gene following DNA-mediated transfer. Cultures of primary epidermal keratinocytes were transfected with the reporter gene, chloramphenicol acetyltransferase (CAT). The CAT gene was linked at the 5' end to the long terminal repeat (LTR) regulatory sequences from Rous sarcoma virus, and gene transfer was accomplished by the calcium phosphate coprecipitation method. Transfected cells were fractionated on Ficoll 400 density gradients. The major finding of this study was that the larger, more differentiated cells displayed five- to seven-fold higher levels of CAT activity per cell than the smaller, less differentiated cells. The higher levels of CAT activity did not result from greater uptake of DNA because cells of all gradient fractions contained one to two copies of plasmid DNA per cell. Furthermore, the CAT gene linked to the regulatory sequences from another virus, SV40, gave the same result. We conclude that the CAT gene, when controlled by these viral regulatory sequences, is expressed more efficiently in differentiated keratinocytes. These results have important implications for the interpretation of future studies of gene expression in transfected keratinocytes.