p53抑制剂PFT-α对阿霉素诱导的人皮肤角质形成细胞凋亡的影响

目的探讨p53抑制剂PFT-α对阿霉素诱导的人皮肤角质形成细胞凋亡的影响。方法用MTT法通过比色分析测定吸光度(OD)值,检测0,4,8,12mg/LPFT-α和阿霉素给药对角质形成细胞增殖的影响,用流式细胞仪检测细胞凋亡。结果阿霉素抑制体外培养的人皮肤角质形成细胞增殖,先给予8,12mg/LPFT-α处理细胞,然后给予阿霉素,与对照组比较,PFT-α升高OD值(P<0.05),降低细胞凋亡率(P<0.05)。结论先行给予适当剂量PFT-α处理人皮肤角质形成细胞,能一定程度减轻阿霉素对人皮肤角质形成细胞的损伤,表现出对阿霉素致凋亡的保护作用。     

齐墩果酸对阿霉素诱导的培养人皮肤角质形成细胞凋亡的影响

目的探讨齐墩果酸对阿霉素诱导的培养人皮肤角质形成细胞凋亡的影响。方法用MTT法通过比色分析测定吸光度(A)值,检测不同剂量齐墩果酸和阿霉素给药对角质形成细胞增殖的影响,用流式细胞仪检测细胞凋亡。结果阿霉素抑制体外培养的人皮肤角质形成细胞增殖,先给予齐墩果酸处理细胞,然后给予阿霉素,齐墩果酸升高A值(P<0.05),降低细胞凋亡率(P<0.05)。结论先行给予适当剂量齐墩果酸处理细胞,能一定程度减轻阿霉素对细胞的损伤,表现出对阿霉素致凋亡的保护作用。     

存活素反义寡核苷酸对角质形成细胞增殖和凋亡的影响

目的探讨存活素反义寡核苷酸对角质形成细胞增殖和凋亡的影响。方法以脂质体介导存活素反义寡核苷酸转染体外培养的角质形成细胞。采用MTT方法观察存活素反义寡核苷酸对角质形成细胞生长曲线的影响;采用RT-PCR方法观察存活素反义寡核苷酸对角质形成细胞存活素表达水平的影响;采用流式细胞仪检测存活素反义寡核苷酸对角质形成细胞细胞周期和凋亡的影响。结果存活素反义寡核苷酸作用于角质形成细胞后,细胞增殖受到明显抑制,存活素mRNA表达水平明显下降,细胞出现明显的凋亡现象。结论存活素反义寡核苷核酸能够抑制角质形成细胞增殖,促进角质形成细胞凋亡。提示存活素可能会成为一个新的治疗银屑病的靶分子。     

反义寡核苷酸阻遏角蛋白14的表达

目的 研究脂质体介导角蛋白K14反义寡核苷酸(ASODN)阻遏人角质形成细胞K14基因和蛋白的表达及抑制体外增殖活性的效果.方法 人表皮角质形成细胞原代培养,3~10代用于实验,利用脂质体将人工合成的正义、反义及错配K14寡核苷酸基因片段导入角质形成细胞,应用流式细胞仪、逆转录聚合酶链反应(RT-PCR)和免疫组化(SABC)方法检测反义寡核苷酸对角质形成细胞的细胞周期、K14基因和蛋白表达的影响.结果 脂质体介导的K14反义寡核苷酸转染角质形成细胞后,能有效地抑制角质形成细胞中K14基因的表达;48h后可阻遏K14蛋白表达;流式细胞仪检测见反义寡核苷酸处理组细胞周期发生明显改变,G₁期细胞百分率上升,S期细胞百分率下降.而正义寡核苷酸1组、错义寡核苷酸组及空白对照组均无此变化.结论 应用反义技术封闭K14基因,可以阻遏角蛋白K14基因和蛋白的表达,并可以抑制体外培养的角质形成细胞的增殖.     

Survivin与皮肤病的研究进展

Survivin是凋亡抑制蛋白家族新成员.其在皮肤恶性肿瘤、癌前期皮肤病、某些良性角质形成细胞过度增生性皮肤病中高表达.Survivin通过抑制细胞凋亡、调节细胞分裂、参与肿瘤血管形成等多重功能参与皮肤疾病的发生.抑癌基因p53通过对抗Survivin蛋白的抗凋亡活性诱导肿瘤细胞凋亡.检测癌组织中Survivin和p53基因的表达可以为肿瘤患者的诊断和判断预后提供参考指标,并且针对Survivin基因及Survivin联合p53基因的靶向治疗将为皮肤癌的治疗开辟新的途径.     

中波紫外线诱导皮肤角质形成细胞凋亡机制的研究进展

紫外线照射表皮后引起DNA损伤的表皮细胞通过日晒伤细胞(凋亡的角质形成细胞)的形成来清除。p53、Fas、Trp53等基因的表达促进角质形成细胞凋亡,而survivin的表达则抑制角质形成细胞凋亡。紫外线照射后皮肤可产生环丁烷嘧啶二聚体和6-4光产物,这两种光产物可以作为UVB诱导凋亡的起始信号。凋亡在清除DNA损伤的皮肤起着重要作用。在皮肤中通过凋亡清除DNA损伤的细胞,防止日光诱导癌变,而不是依赖于DNA损伤的校正修复。silibinin对中波紫外线引起人HaCaT永生化角质形成细胞凋亡具有双向调控作用。对UVB引起角质形成细胞凋亡的调控药物,值得进一步研究。     

某些中药煎剂对阿霉素抑制的猪毛囊真皮鞘细胞增殖的影响

目的:探讨何首乌、女贞子等中药煎剂对阿霉素抑制的猪毛囊真皮鞘细胞增殖的影响。方法:用MTT法通过比色分析测定吸光度值(OD),检测不同剂量(0、5、10、20)mg/L阿霉素作用24h和10mg/L阿霉素作用不同时间(4、8、12、24、48h)对猪毛囊真皮鞘细胞增殖的影响;用流式细胞仪检测不同剂量的阿霉素作用24h后猪毛囊真皮鞘细胞的凋亡率,从而找出用于以下研究中阿霉素的最适浓度。用MTT法检测不同剂量中药煎剂和10mg/L阿霉素同时给药共同作用24h以及先给予不同剂量中药煎剂作用24h,然后给予10mg/L阿霉素作用24h两种条件下对猪毛囊真皮鞘细胞增殖的影响。结果:阿霉素抑制体外培养的猪毛囊真皮鞘细胞增殖,其抑制效应随阿霉素浓度的增大和作用时间的延长而增强;细胞凋亡率以阿霉素浓度为10mg/L时最高。中药煎剂和阿霉素10mg/L同时给药条件下,前者不能有效升高OD值(P>0.05);如果先给予中药煎剂处理,后给10mg/L阿霉素,则中等剂量的中药煎剂即能有效升高OD值(P<0.001)。结论:阿霉素抑制体外培养的猪毛囊真皮鞘细胞增殖,先给予适当剂量的中药煎剂处理细胞,能在一定程度上减轻阿霉素对细胞的损伤。     

凉血活血胶囊对皮肤角质形成细胞凋亡的研究

目的 观察凉血活血胶囊对体外角质形成细胞株凋亡的影响,初步探讨其治疗银屑病的机制。方法 以TUNEL法检测银屑病患者皮损细胞凋亡、PCNA检测增殖细胞核抗原,并以碘化丙啶法和膜联蛋白V法在流式细胞仪上检测凉血活血胶囊对体外角质形成细胞凋亡作用的影响。结果 银屑病皮损中基底层和棘细胞中下层角质形成细胞增殖和凋亡较正常皮肤均有所增加。凉血活血胶囊可诱导培养的角质形成细胞凋亡,在1、5、10 mg/mL时分别为24.67±5.07、50.33±10.04、66.20±6.91,随着浓度增加,细胞凋亡率增加,与正常对照组比较差异有显著性,与试验对照复方青黛胶囊组比较差异无显著性。结论 银屑病皮损中角质形成细胞增殖和凋亡发生紊乱,两者在较高水平保持相对平衡。凉血活血胶囊可以诱导细胞凋亡,从而达到治疗银屑病的目的。     

rhFGF-7对体外培养人皮肤角质形成细胞增殖的影响

目的建立一种人皮肤角质形成细胞分离和培养的方法,研究重组人成纤维细胞生长因子7(rhFGF-7)对人皮肤角质形成细胞增殖的影响。方法取环切术后包皮,分别用胰酶和dispaseⅡ酶消化法分离表皮,用无血清培养基进行无滋养层培养,采用免疫细胞化学方法对培养的细胞进行鉴定,并用细胞计数法测定细胞的生长曲线,MTT法检测rhFGF-7对人皮肤角质形成细胞增殖的影响。结果与胰酶消化分离表皮相比,dispaseⅡ酶消化获得的表皮,其细胞贴壁率高,增殖速度快,且成纤维细胞污染少;荧光显微镜下细胞胞浆呈黄绿色,为角蛋白阳性染色;培养的角质形成细胞可持续增殖至第8代,10ng/mL的rhFGF-7促角质形成细胞增殖作用最显著。结论所建立的人皮肤角质形成细胞分离和培养方法,可获得高纯度的人皮肤角质形成细胞,rhFGF-7可促进体外培养人皮肤角质形成细胞的增殖。     

皮肤科学基础

20042639　角蛋白K14反义寡核苷酸对角质形成细胞增殖的影响/陈玉欣(四军大西京医院全军皮肤性病中心)…//临床皮肤科杂志.-2004,33(5).-263～265采用脂质体将人工合成的正义、反义及错配寡核苷酸基因片段导入体外培养的角质形成细胞,应用细胞生长抑制实验、透射电镜和流式细胞仪分别检测反义寡核苷酸对角质形成细胞的增殖、超微结构和细胞增殖周期的影响。结果显示,脂质体介导的K14反义寡核苷酸对角质形成细胞的增殖活性有明显抑制作用;电镜下见角质形成细胞中角蛋白合成明显减少;流式细胞仪检测见细胞周期发生明显改变,G1期细胞百分率上…     

Surface glycoproteins of cultured human keratinocytes from normal and uninvolved psoriatic epidermis

Surface glycoproteins of cultured human keratinocytes from normal skin and uninvolved psoriatic epidermis, isolated by the suction blister method, were studied by two different methods. Cells were cultured on collagen-coated culture dishes and showed a fibrillar keratin-specific staining by immunofluorescence. Surface labelling experiments using the neuraminidase/galactose oxidase/sodium borohydride method (which labels the penultimate galactose moieties of glycoproteins) revealed one major glycoprotein with M_r 53 kD (kilodaltons) both in normal keratinocytes and in keratinocytes from uninvolved psoriatic skin. The periodate/sodium borohydride method (which labels the terminal sialic acids in glycoproteins) by contrast revealed three major glycoproteins, with M_r 53 kD to 63 kD, in normal keratinocytes but only a single major glycoprotein, with M_r 53 kD, in keratinocytes from uninvolved psoriatic skin. Treatment of cultured keratinocytes with etretinate appeared to restore the normal pattern of surface glycoproteins in uninvolved psoriatic keratinocytes.     

Mutation spectra of epidermal p53 clones adjacent to basal cell carcinoma and squamous cell carcinoma

Foci of normal keratinocytes overexpressing p53 protein are frequently found in normal human skin. Such epidermal p53 clones are common in chronically sun-exposed skin and have been suggested to play a role in skin cancer development. In the present study, we have analyzed the prevalence of p53 mutations in epidermal p53 clones from normal skin surrounding basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Using laser-assisted microdissection, 37 epidermal p53 clones adjacent to BCC (21) and SCC (16) were collected. Genetic analysis was performed using a multiplex/nested polymerase chain reaction followed by direct DNA sequencing of p53 exons 2-11. In total, 21 of 37 analyzed p53 clones consisted of p53-mutated keratinocytes. The identified mutations were located in p53 exons 4-8, corresponding to the sequence-specific DNA-binding domain. All mutations were missense, and 78% displayed a typical ultraviolet signature. The frequency of p53 mutations was similar in skin adjacent to BCC compared to SCC. The presented data confirm and extend previous knowledge on the genetic background of epidermal p53 clones. The mutation spectra found in epidermal p53 clones resemble that of non-melanoma skin cancer. Approximately, 40% of the epidermal p53 clones lacked an underlying p53 mutation, suggesting that other genetic events in genes up- or downstream of the p53 gene can generate foci of normal keratinocytes overexpressing p53 protein.     

Enhanced expression of p16 in seborrhoeic keratosis; a lesion of accumulated senescent epidermal cells in G1 arrest

BACKGROUND: Seborrhoeic keratosis (SK) is a common skin disease associated with skin ageing and photoageing, but only limited studies have been performed on SK and the senescence of keratinocytes. OBJECTIVES: We sought to clarify the genetic basis of SK and the senescence of keratinocytes. METHODS: Expression of p16, cyclins A, D and E, p21, p53, retinoblastoma (Rb) gene product and telomerase-associated protein 1 (TP1) in SK was examined by immunohistochemistry. DNA fragmentation in SK was detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling method. We cultured keratinocytes from SK lesions and non-lesional epidermis and examined expression of p16, observed morphology of the cultured cells by light and electron microscopy and measured survival time. RESULTS: p16, a cyclin-dependent kinase inhibitor, was expressed in all cells from SK lesions, whereas normal keratinocytes expressed p16 only in the granular cells. Other factors such as cyclins A, D and E, p21, p53, Rb gene product, and TP1, were not expressed in SK cells. These results suggest that p16 expression is a marker of SK and that p16 has a role in the pathogenesis of SK. DNA fragmentation was not detected in four of five SK tissue samples; one of the SK tissue samples showed DNA fragmentation only in the superficial upper layer of an SK lesion, suggesting that apoptosis was inhibited in SK cells. In contrast, normal epidermis showed DNA fragmentation in the granular and squamous layers. Immunohistochemical examination of cultured SK cells also revealed the presence of p16. A greater number of SK cells survived after 3 weeks of culture in comparison with normal keratinocytes. Features of senescence, such as a balloon-like appearance after lengthy culture and increased amounts of tonofilaments in cytoplasm, were observed in SK cells in culture. CONCLUSIONS: These results suggest that SK is a benign neoplasm where keratinocytes in a senescent condition and G1 arrest are accumulated.     

尘螨变应原rDer p1诱导HaCaT细胞表达胸腺基质淋巴生成素的研究

【摘要】 目的 探讨尘螨变应原rDer p1在体外对角质形成细胞表达胸腺基质淋巴生成素（TSLP）的影响。 方法 体外培养人角质形成细胞株HaCaT细胞,予以0.1、1和10 mg/L尘螨变应原rDer p1与蛋白酶活化受体2（PAR2）特异性激动剂SLGIKV（500 μmol/L）和拮抗剂VKGILS（500 μmol/L）共培养,以无血清培养基为空白对照。用ELISA法和荧光定量PCR法检测各实验组和对照组TSLP蛋白及mRNA表达水平;通过激光共聚焦显微镜检测细胞内钙流变化分析rDer p1诱导HaCaT细胞产生TSLP和PAR2受体活化的关系。 结果 1 mg/L和10 mg/L rDer p1组培养12 h上清中TSLP水平分别为（155.5 ± 5.9） ng/L和（228.8 ± 28.7） ng/L,显著高于空白对照组（54.3 ± 13.9 ng/L,P < 0.01）,TSLP表达水平随rDer p1浓度增加而升高。SLGIKV组TSLP表达［（166.2 ± 8.8） ng/L］也显著高于空白对照组（P < 0.01）。10 mg/L rDer p1组和SLGIKV组TSLP mRNA相对表达量在8 h最高,分别为（3.28 ± 0.27）倍和（2.15 ± 0.26）倍,与4 h和24 h相比差异有统计学意义（P < 0.01）。SLGIKV可引起HaCaT细胞钙内流增加;10 mg/L rDer p1也可引起HaCaT细胞钙内流增加,经过PAR2受体特异性阻断剂VKGILS（500 μmol/L）处理后再给予rDer p1刺激,细胞内钙内流峰值明显降低。 结论 尘螨变应原rDer p1能够通过部分活化HaCaT细胞表面的PAR2受体诱导产生促炎细胞因子TSLP。     

Characterization of the ultraviolet B and X-ray response of primary cultured epidermal cells from patients with disseminated superficial actinic porokeratosis

BACKGROUND: Disseminated superficial actinic porokeratosis (DSAP) is the most common porokeratosis and is characterized by multiple keratotic lesions which tend to occur at sun-exposed sites. A mild hypersensitivity to X-rays has been reported for DSAP-derived fibroblasts and frequent over-expression of p53 has been found in lesional epidermis. OBJECTIVES: In order to clarify whether genome maintenance mechanisms might be compromised in this disease the following approaches were undertaken: (i) primary cultured keratinocytes and fibroblasts from DSAP patients were characterized for ultraviolet (UV) B and X-ray response; (ii) 15 lesions were studied for p53 mutations, and (iii) the differentiation status of DSAP-derived keratinocytes was evaluated. METHODS: Primary cultures of keratinocytes and fibroblasts were established from lesional and nonlesional skin biopsies of two subjects with DSAP. p53 mutations were analysed by DNA sequencing of the conserved region of the TP53 gene. Differentiation was evaluated both in stratified epithelial sheets from confluent keratinocyte cultures and in organotypic skin cultures. RESULTS: The cytotoxic and apoptotic response to UVB or X-irradiation was similar in DSAP-derived keratinocytes and fibroblasts when compared with normal cells. Two of 15 lesions examined presented p53 mutations located at nondipyrimidine sites. A strikingly decreased expression of filaggrin was observed both in reconstructed epidermis and in reconstructed skin. CONCLUSIONS: The UVB and X-ray response of DSAP-derived keratinocytes and fibroblasts indicates that the actinic character of this skin pathology is not due to radiation hypersensitivity. In agreement with this finding, mutations in the p53 gene, which are often associated with UV-related skin carcinogenesis, were rarely detected in DSAP lesions and were not UV-specific. Reconstructed epidermis and reconstructed skin models successfully reproduced the main features of this genodermatosis, showing that DSAP-derived keratinocytes bear an inherent defect in the terminal differentiation programme.