维胺酯对HaCaT细胞增殖和分化的影响

目的 探讨维胺酯对体外培养角质形成细胞(HaCaT细胞)增殖和分化的影响.方法 将浓度为2,5,10,15,20,25,30 μg/mL的维胺酯作用于培养的HaCaT细胞,采用MTT法检测维胺酯对HaCaT细胞体外增殖的影响,流式细胞仪测定细胞周期及凋亡率变化,逆转录-聚合酶链反应(RT-PCR)半定量检测分化标记物角蛋白10及内披蛋白mRNA的表达水平.结果 2 μg/mL维胺酯处理的HaCaT细胞,48 h时表现出对细胞增殖的抑制作用,随着时间延长和药物剂量加大,抗增殖作用愈明显;当药物质量浓度达到30 μg/mL时,48 h和72 h时的抑制率分别为57.67%和82.00%.与对照组相比,经维胺酯作用48 h后,细胞G₁期比例显著增加,S期与G₂期比例则显著下降,并可抑制G₁/G₂期转换,但对细胞凋亡无影响.细胞内披蛋白mRNA表达水平随维胺酯处理浓度增高而上升,药物浓度达30μg/mL时,表达水平由对照组的40.80%增高至156.12%;而角蛋白10 mRNA表达水平则下降,由96.46%降至14.60%.结论 维胺酯具有抑制角质形成细胞增殖及诱导其分化的作用.     

Reconstruction of a human skin equivalent using a spontaneously transformed keratinocyte cell line (HaCaT)

Reconstruction of a skin equivalent using an immortalized human keratinocyte line, HaCaT, was investigated in an attempt to generate an in vitro system representative for human skin. Three different substrates were used to establish air-exposed cultures of HaCaT cells: de-epidermized dermis, collagen gels, and filter inserts. Effects of variations in culture conditions on tissue morphology, on the expression of proliferation-specific and differentiation-specific protein markers, and on lipid profiles were investigated. When grown at the air-liquid interface HaCaT cells initially developed a multilayered epithelium, but during the course of culture marked alterations in tissue architecture were observed. Ultrastructurally, a disordered tissue organization was evident as judged from the presence of rounded cells with abnormally shaped nuclei. Keratins K1 and K10 were irregularly expressed in all cell layers, including stratum basale. Staining of K6/K16 was evident in all cell layers. Locally, basal and suprabasal cells were positive for K4 and additionally expressed K13 and K19. The cornified envelope precursors were expressed only in older cultures (>2 wk after air exposure), except for transglutaminase and small proline rich protein 1, which were irregularly expressed in both early and older cultures. In addition, HaCaT cells showed an impaired capacity to synthesize lipids that are necessary for a proper barrier formation as indicated by the absence of free fatty acids and a very low content and incomplete profile of ceramides. Our data demonstrate that the ultimate steps of terminal differentiation in HaCaT cells do not occur irrespective of the type of substrate or the culture conditions.     

人乳头瘤病毒11型基因组DNA在HaCaT株中的转染

目的 探讨转染与筛选技术获取稳定携带有HPV11基因组DNA的细胞的可能性。方法 对含有pBR322.HPV11质粒的大肠杆菌培养扩增,然后进行质粒的提取与纯化,并用限制性内切酶BamHⅠ切割下HPV11全长基因。低熔点琼脂糖回收后,用T4 DNA连接酶进行线状DNA的自身环化,再与pIK-neo质粒、用Lipofectamine试剂共转染HaCaT细胞。通过G418筛选阳性细胞克隆,将阳性克隆细胞合并与培养扩增后,用FQ-PCR技术检测细胞内HPV11DNA的存在,用巢式RT-PCR技术检测HPV11 E1^E4 mRNA的表达。结果 HPV11型基因组DNA转染至HaCaT细胞后,经G418筛选约2周,培养皿内即可见对G418抵抗的阳性细胞克隆出现,其形态与普通HaCaT细胞相似。用FQ-PCR在筛选后的HaCaT细胞中检测到HPV11DNA的存在,平均病毒DNA载量为（15.9 ± 16.8）拷贝/细胞。传代3次后细胞内病毒DNA无丢失,载量为（23.9 ± 1.1）拷贝/细胞。与未传代细胞相比,二者间差异无统计学意义（t = -0.822,P > 0.05）。巢式RT-PCR扩增产物经琼脂糖凝胶电泳后检测到HPV11 E1^E4 mRNA表达的特异性628 bp条带。结论 HPV11基因组DNA可通过脂质体转染法成功导入HaCaT细胞内,通过筛选可获得阳性细胞,且经3次传代后HPV11DNA仍然存在。     

miR-145对人角质形成细胞系HaCaT细胞增殖、凋亡和细胞周期的影响

目的 研究miR-145对人角质形成细胞系HaCaT细胞增殖、细胞周期及凋亡的调控效应。 方法化学合成miR-145的模拟物,采用瞬时转染的方法过表达miR-145。采用实时PCR方法检测miR-145的表达。MTS方法检测过表达miR-145对HaCaT细胞增殖的影响。流式细胞仪分析过表达miR-145对细胞周期及凋亡的影响。采用荧光素酶实验,实时PCR和Western印迹鉴定NRAS是否为miR-145的靶基因。 结果 与阴性对照（NC）mimics转染组相比,miR-145 mimics转染组miR-145表达水平上调（85.00 ± 1.21）倍,两组差异有统计学意义（t = 115.90,P < 0.001）。转染miR-145 mimics有抑制HaCaT细胞的增殖作用（F = 8.76,P = 0.008）;转染后的时间因素（24、48、72、96 h）对细胞有影响（F = 17.85,P < 0.001）,转染mimics和培养时间之间不存在交互作用（F = 1.21,P = 0.18）。与NC mimics转染组相比,miR-145 mimics转染组的早期凋亡、晚期凋亡细胞比例均明显增加,差异有统计学意义（18.9% ± 4.1%比4.3% ± 1.2%,t = 7.126,P < 0.01;9.3% ± 2.3%比3.6% ± 1.6%,t = 12.38,P < 0.01）。与NC mimics转染组相比,miR-145 mimics转染组HaCaT细胞的G2及S期细胞比例均明显降低,差异均有统计学意义（6.26% ± 1.2%比19.36% ± 3.45%,t =7.610,P = 0.017;7.91% ± 1.3%比18.56% ± 5.23%,t = 7.230,P = 0.019）,而处于G1期的细胞比例升高,差异也有统计学意义（85.83% ± 5.2%比62.08% ± 6.23%,t = 11.78,P = 0.007）。与NC mimics联合psi-CHECK2-NRAS-wild组相比,在293T细胞中共转染miR-145 mimics和psi-CHECK2-NRAS-wild质粒,其荧光素酶值明显下降,miR-145可抑制含NRAS mRNA 3′UTR报告基因的荧光素酶表达（t = 11.09,P = 0.008）;而将NRAS mRNA 3′UTR报告基因上miR-145的结合位点进行突变后,转染miR-145 mimics对含NRAS mRNA 3′UTR报告基因的荧光素酶表达无明显的影响（P > 0.05）。实时PCR和Western印迹结果表明,过表达miR-145 mimics后,NRAS mRNA表达水平未出现明显变化（P > 0.05）,对NRAS蛋白水平的表达有明显的抑制（1.52 ± 0.07比0.20 ± 0.02,t = 28.43,P < 0.01）。 结论 miR-145可能通过NRAS影响HaCaT细胞的周期从而抑制细胞的增殖,同时促进HaCaT细胞的凋亡。     

Interleukin-1β upregulates tissue-type plasminogen activator in a keratinocyte cell line (HaCaT)

Human keratinocytes synthesize and secrete tissue-type plasminogen activator (tPA). tPA converts the inactive precursor enzyme plasminogen into the trypsin-like proteinase plasmin. tPA is not found in normal epidermis, but in lesional epidermis from patients with a variety of cutaneous diseases, including psoriasis, pemphigus and pemphigoid. The presence of tPA is probably a reaction to the disease process rather than the initiating event in these etiologically and histopathologically diverse lesions. However, the factor(s) that upregulate tPA expression and secretion in keratinocytes have remained largely elusive. We sought to determine whether the inflammatory cytokine interleukin-1β (IL-1β), which is commonly present in diverse epidermal lesions, influences tPA production. Accordingly, we studied the influence of IL-1β on secretion of tPA by cells of the human keratinocyte cell line HaCaT. We found that IL-1β increased tPA secretion in these cells. Given the observation that IL-1β is a common proinflammatory mediator in cutaneous diseases, our findings may explain the increase in tPA in clinically and etiologically diverse inflammatory epidermal lesions. Received: 9 October 1995     

Induction of apoptosis by synthetic ceramide analogues in the human keratinocyte cell line HaCaT

Abstract: In contrast to extracellular, long chain ceramides which comprise a structural component of the epidermal water barrier, intracellular ceramides originating from sphingomyelin hydrolysis have been shown to inhibit proliferation and to induce apoptosis in different cell populations. To further elucidate the possible role of intracellular ceramides in human epidermis, two new cell-permeable ceramide analogues, N -thioacetylsphingosine (C₂-Cer=S) and 4-dodecanoylamino-decan-5-ol (FS-5), were synthesized and tested for their ability to suppress cell growth and to induce apoptosis in immortalized human keratinocytes. It was shown that the well-investigated ceramide analogue N -acetylsphingosine (C₂-Cer=O), as well as the new compound C₂-Cer=S inhibited proliferation of HaCaT cells with half-inhibitory concentrations (IC₅₀) of 20 μg/ml and 10 μg/ml, respectively, whereas FS-5 has been potent with an IC₅₀>40 μg/ml. Overall, all three ceramide analogues induced apoptosis in HaCaT cells as assessed by DNA-fragmentation using ELISA technique and in situ nick end labelling, thereby confirming the importance of ceramide signalling in keratinocytes.     

鳄梨油和橄榄油对HaCaT细胞增殖和分化的影响

目的 探讨天然植物鳄梨油和橄榄油对HaCaT细胞系增殖及分化的影响。方法 将培养的HaCaT细胞分为鳄梨油组、橄榄油组及对照组。应用MTT实验确定鳄梨油和橄榄油对HaCaT细胞的适用剂量。以免疫细胞化学、免疫斑点印迹技术分别检测各组HaCaT细胞的c-myc原癌基因（c-myc）、有丝分裂原激活蛋白激酶（MAPK）、NF-κB、丝聚蛋白、内披蛋白、角蛋白10的表达。结果 对照组c-myc、MAPK、NF-κB免疫化学反应的扫描灰度均值（GSM）分别为101.9 ± 8.9、91.4 ± 5.1、94.3 ± 7.0,鳄梨油组分别为131.4 ± 6.6、136.3 ± 4.5、134.3 ± 5.2,与对照组比较均显著增加（P < 0.05）;橄榄油组分别为121.1 ± 4.5、107.9 ± 7.3、106.4 ± 5.4,与对照组比较均亦显著增加（P < 0.05）;而鳄梨油组与橄榄油组相比,前者各项GSM均高于后者（P < 0.05）。鳄梨油组和橄榄油组丝聚蛋白、内披蛋白、角蛋白10免疫化学反应的GSM均高于对照组（P < 0.05）;而鳄梨油组与橄榄油组相比,后者均高于前者（P < 0.05）。此外,各组各项免疫斑点印迹的GSM与免疫细胞化学的GSM数据基本相符,在鳄梨油组两者呈显著正相关,r = 0.94,P < 0.01;在橄榄油组两者也呈现显著正相关,r = 0.97,P < 0.01。结论 一定浓度鳄梨油和橄榄油,尤其是鳄梨油对HaCaT细胞可呈现显著的促生长、增殖效应;橄榄油和鳄梨油,尤其橄榄油对HaCaT细胞可呈现显著的促分化效应。     

High density of beta2-adrenoceptors in a human keratinocyte cell line with complete epidermal differentiation capacity (HaCaT)

Summary A non-tumorigenic keratinocyte cell line with complete epidermal differentiation capacity (HaCaT) was used in radioligand binding experiments to determine the number of beta-adrenoceptors. Intact cells were saturated with ³H-labelled (–)CGP-12177 (CGP), a hydrophilic non-selective beta-adrenergic antagonist as radioligand. In order to investigate the beta-adrenergic subtype selectivity, displacement experiments were performed with different antagonists and agonists. Binding of CGP to keratinocytes has been shown to be reversible and saturable and to have high affintiy (B _max=114.0±8.8 fmol/10⁷ cells with 6866 receptors/cell, K _D=0.095±0.017 nmol/l; n=11). Betaadrenergic antagonists inhibited binding yielding monophasic displacement curves. IC₅₀-values (nmol/l) were: propranolol (non-selective) 1.68; CGP-12177 (non-selective) 1.08; ICI 118,551 (beta₂-selective) 2.92; bisoprolol (beta₁-selective) 1230; and CGP-20712 (beta₁-selective) 24980. Agonists displaced CGP in the order isoprenaline> adrenaline>noradrenaline. We conclude that HaCaT cells express a high density of beta₂-adrenoceptors providing a good model system to study adrenergic receptor mechanisms under reproducible experimental conditions in keratinocytes.Part of this work was presented at the Meeting of the European Society of Dermatological Research (ESDR), Turin, June 9–12, 1990     

稳定表达人乳头瘤病毒16E7蛋白HaCaT细胞株的建立

目的建立稳定表达人乳头瘤病毒(HPV)16E7蛋白的HaCaT细胞株.方法利用PCR及酶切技术扩增出CaSki细胞中HPV16E7基因并定向克隆到真核细胞表达载体pcDNA3.1(+)中,构建真核表达质粒pcDNA3.1(+)-HPV16E7.将重组质粒转染入HaCaT细胞,经G418筛选稳定表达HPV16E7蛋白的细胞株并予以鉴定.结果经酶切、测序鉴定构建pcDNA3.1(+)-HPV16E7成功.RT-PCR扩增产物经琼脂糖凝胶电泳后检测到HPV16E7mRNA表达的特异性297 bp条带;Western印迹可检测到E7蛋白稳定表达.结论成功构建出稳定表达HPV16E7蛋白的HaCaT细胞株.     

稳定表达人乳头瘤病毒16E7蛋白HaCaT细胞株的建立

目的 建立稳定表达人乳头瘤病毒（HPV）16E7蛋白的HaCaT细胞株。方法 利用PCR及酶切技术扩增出CaSki细胞中HPV16E7基因并定向克隆到真核细胞表达载体pcDNA3.1（+）中,构建真核表达质粒pcDNA3.1（+）-HPV16E7。将重组质粒转染入HaCaT细胞,经G418筛选稳定表达HPV16E7蛋白的细胞株并予以鉴定。结果 经酶切、测序鉴定构建pcDNA3.1（+）-HPV16E7成功。RT-PCR扩增产物经琼脂糖凝胶电泳后检测到HPV16E7mRNA表达的特异性297 bp条带;Western印迹可检测到E7蛋白稳定表达。结论 成功构建出稳定表达HPV16E7蛋白的HaCaT细胞株。     

稳定表达人乳头瘤病毒16E7蛋白HaCaT细胞株的建立

目的建立稳定表达人乳头瘤病毒(HPV)16E7蛋白的HaCaT细胞株.方法利用PCR及酶切技术扩增出CaSki细胞中HPV16E7基因并定向克隆到真核细胞表达载体pcDNA3.1(+)中,构建真核表达质粒pcDNA3.1(+)-HPV16E7.将重组质粒转染入HaCaT细胞,经G418筛选稳定表达HPV16E7蛋白的细胞株并予以鉴定.结果经酶切、测序鉴定构建pcDNA3.1(+)-HPV16E7成功.RT-PCR扩增产物经琼脂糖凝胶电泳后检测到HPV16E7mRNA表达的特异性297 bp条带;Western印迹可检测到E7蛋白稳定表达.结论成功构建出稳定表达HPV16E7蛋白的HaCaT细胞株.     

稳定表达人乳头瘤病毒16E7蛋白HaCaT细胞株的建立

目的建立稳定表达人乳头瘤病毒(HPV)16E7蛋白的HaCaT细胞株.方法利用PCR及酶切技术扩增出CaSki细胞中HPV16E7基因并定向克隆到真核细胞表达载体pcDNA3.1(+)中,构建真核表达质粒pcDNA3.1(+)-HPV16E7.将重组质粒转染入HaCaT细胞,经G418筛选稳定表达HPV16E7蛋白的细胞株并予以鉴定.结果经酶切、测序鉴定构建pcDNA3.1(+)-HPV16E7成功.RT-PCR扩增产物经琼脂糖凝胶电泳后检测到HPV16E7mRNA表达的特异性297 bp条带;Western印迹可检测到E7蛋白稳定表达.结论成功构建出稳定表达HPV16E7蛋白的HaCaT细胞株.     

Differentiation of the HaCaT keratinocyte cell line: modulation by adrenomedullin

BACKGROUND: Adrenomedullin (AM) is a multifunctional peptide produced by a wide variety of cells, including keratinocytes. We, and others, have demonstrated that AM has a role as a growth regulatory factor of the skin and contributes as an antimicrobial agent in the integument's protective barrier. It is not known whether AM has a role in differentiating keratinocytes. OBJECTIVES: To study the role of AM in keratinocyte differentiation, modulating the effects of calcium and in addition, to assess whether differentiated keratinocytes are still capable of initiating an inflammatory response. METHODS: HaCaT cells were differentiated using CaCl2. Expression of transglutaminase type 1 (TG1) and E2F1 genes was used to monitor differentiation. AM secretion was measured by enzyme-linked immunosorbent assay (ELISA). NF-kappaB activity and interleukin (IL)-6 secretion in the cells were assessed after exposure to calcium and AM by electrophoretic mobility shift assay and ELISA, respectively. RESULTS: Secretion of AM by the keratinocyte cell line HaCaT was found to be increased during 1 mmol L(-1) CaCl2-induced cell differentiation but not 0.1 mmol L(-1) CaCl2. All treatments showed low levels of the cell proliferation marker, E2F1. Over time, cells incubated in the presence of 0.1 mmol L(-1) or 1 mmol L(-1) of CaCl2 showed an increase in TG1 expression, a marker of early differentiation. The addition of AM showed a decrease in TG1 expression when combined with 0.1 mmol L(-1) CaCl2, but not with 1 mmol L(-1) CaCl2. In addition, cells kept in 0.1 mmol L(-1) CaCl2 showed translocation of NF-kappaB after 48 h and 72 h of incubation, which was abolished when AM was added to the cells. Treatment with 1 mmol L(-1) CaCl2 led to earlier translocation of NF-kappaB at 24 h after treatment and addition of AM did not abolish the effect of 1 mmol L(-1) CaCl2 on NF-kappaB activation. Cells incubated in 0.1 mmol L(-1) CaCl2 showed increased secretion of IL-6 over time, consistent with NF-kappaB activation. The addition of AM to cells incubated with 0.1 mmol L(-1) CaCl2 showed a rapid decrease in IL-6 secretion after only 6 h. However, 1 mmol L(-1) CaCl2 did not induce secretion of IL-6 and the addition of AM did not affect the result. CONCLUSIONS: Our data indicate that AM can reverse calcium-induced differentiation when 0.1 mmol L(-1) CaCl2 is used but not 1 mmol L(-1) CaCl2. Cells differentiated with 0.1 mmol L(-1) CaCl2 are still capable of generating an inflammatory response, showing signs of late NF-kappaB activation and IL-6 secretion that can be inhibited by AM. However, cells differentiated with 1 mmol L(-1) CaCl2 lose their ability to secrete IL-6 but not AM, which could be acting as an antimicrobial peptide.     

人角质形成细胞株HaCaT细胞表皮生长因子受体内化和下调的研究

目的 了解表皮生长因子受体（EGFR）信号转导与负反馈调节的分子生物学机制。方法 采用免疫沉淀、免疫印迹、间接免疫荧光结合激光扫描共聚焦显微镜等技术，研究HaCaT和CHOwt细胞株中多种配体诱导的EGFR内化和下调情况。结果 免疫印迹检测表明，表皮生长因子（EGF）与肝素结合的EGF（HB-EGF）可引起EGFR总量的快速下调，转化生长因子α（TGFα）及Heregulin则未明显促进受体的降解。EGF、HB-EGF和TGFα处理HaCaT细胞较长时间后活化型受体数量亦不同程度减少。间接免疫荧光染色显示，未处理细胞的EGFR主要分布在胞膜，胞质亦见少量分布。经10 min EGF处理后EGFR聚集成斑状结构（HaCaT细胞明显），并形成内吞体（CHOwt细胞明显），而此时EGFR总量尚未发生明显改变。经过4 h EGF处理后，HaCaT和CHOwt细胞内EGFR信号明显减弱，说明此时受体已经下调。结论 不同配体对HaCaT细胞的EGFR总量及活化型受体的下调作用不同。HaCaT和CHOwt细胞EGFR内化和下调的信号转导机制可能存在差异。     

硒代蛋氨酸对中波紫外线致HaCaT细胞氧化损伤的保护作用

目的 研究硒代蛋氨酸对中波紫外线（UVB）致HaCaT细胞氧化损伤的影响及其可能机制。方法 培养HaCaT细胞,分为4组：①正常对照组,不做任何处理;②硒代蛋氨酸组：分别加入1、10、50、100、200 nmol/L和1 μmol/L 硒代蛋氨酸预孵育24 h;③UVB组：30、60、90 mJ/cm2 UVB照射;④硒代蛋氨酸 + UVB组：不同浓度硒代蛋氨酸预孵育24 h后进行不同剂量UVB照射。采用噻唑蓝（MTT）法检测细胞增殖活性,流式细胞仪检测细胞凋亡率,比色法检测超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）活性及丙二醛（MDA）水平。采用析因设计方差分析、单因素方差分析对数据进行统计学分析,多重比较采用LSD法。 结果 析因设计方差分析结果显示,UVB照射对细胞增殖活性有抑制作用（F = 128.04,P < 0.05）,且随UVB强度增加,细胞增殖活性逐渐下降,组间差异有统计学意义（P < 0.05）;硒代蛋氨酸预孵育对细胞增殖活性也有影响（F = 5.95,P < 0.05）,其中10 nmol/L ~ 1 μmol/L硒代蛋氨酸 + UVB组与UVB组比较,细胞增殖活性显著升高（P < 0.05）;UVB照射和硒代蛋氨酸对细胞增殖活性无明显交互作用（F = 1.65,P > 0.05）。30 mJ/cm2 UVB 照射后,UVB组凋亡率（31.9% ± 2.67%）较正常对照组（4.1% ± 0.67%）显著升高（P < 0.05）;而10、50、100、200 nmol/L和1 μmol/L硒代蛋氨酸 + 30 mJ/cm2 UVB组凋亡率［依次为：（21.9 ± 3.72）%、（17.2 ± 1.67）%、（4.6 ± 0.85）%、（7.5 ± 1.86）%、（13.5 ± 1.95）%］均较30 mJ/cm2 UVB组凋亡率显著下降（P < 0.05）。30 mJ/cm2 UVB组与正常对照组相比,SOD和GSH-Px活性降低,MDA含量升高（P < 0.05）;10 nmol/L ~ 1 μmol/L硒代蛋氨酸 + 30 mJ/cm2 UVB组与30 mJ/cm2 UVB组比较,SOD和GSH-Px活性增高,MDA含量下降（P < 0.05）。 结论 硒代蛋氨酸可以减轻UVB诱导HaCaT细胞氧化损伤,其机制与增强抗氧化酶活性、减少氧自由基有关。     

空气细颗粒物PM2.5对HaCaT细胞增殖、细胞周期及凋亡的影响

目的 探讨空气细颗粒物PM2.5对HaCaT细胞增殖、细胞周期及凋亡的影响。方法 收集北京市2015—2016年采暖季雾霾天气PM2.5,制备成混悬液。将HaCaT细胞分为空白组（仅用细胞培养基）、对照组（不加PM2.5,其他处理同实验组）和实验组［100 ~ 400 mg/L（细胞形态学观察和增殖实验用50 ~ 800 mg/L）PM2.5混悬液处理］处理24 h后,倒置显微镜下观察细胞形态变化;CCK8法检测细胞存活率;流式细胞仪检测细胞周期分布及细胞凋亡;Western 印迹法检测细胞周期蛋白A2（cyclin A2）及细胞周期蛋白依赖性激酶1（CDK1）的表达水平。结果 随着PM2.5浓度升高,HaCaT细胞形态逐渐发生改变,细胞数目逐渐减少。与对照组（100 ± 4.95）%相比,50 mg/L PM2.5组HaCaT细胞存活率无明显变化（P＞0.05）,100、200、400、800 mg/L PM2.5组细胞存活率［分别为（91.77 ± 2.04）%、（80.01 ± 1.57）%、（57.80 ± 1.56）%、（21.98 ± 0.86）%］均显著下降（P＜0.05）。流式细胞仪检测显示,与对照组相比,PM2.5组（100、200、400 mg/L）S期细胞比例逐渐增高,G2/M期细胞比例逐渐降低,差异均有统计学意义（P＜0.05）。Western印迹法显示,100、200、400 mg/L PM2.5组cyclin A2、CDK1蛋白表达均较对照组有所降低,尤其以200 mg/L组降低最明显,差异均有统计学意义（P＜0.05）。100、200、400 mg/L PM2.5组细胞总凋亡率分别为（9.98 ± 0.21）%、（12.56 ± 0.74）%、（16.74 ± 1.48）%,与对照组（6.24 ± 0.17）%相比,差异均有统计学意义（P＜0.05）。结论 PM2.5可能通过下调cyclin A2、CDK1表达水平诱导HaCaT细胞发生S期阻滞,从而抑制细胞增殖,促进HaCaT细胞凋亡。     

Ultraviolet A irradiation inhibits thymus and activation-regulated chemokine (TARC/CCL17) production by a human keratinocyte HaCaT cell line

Ultraviolet A (UVA) irradiation modulates the immunological functions of skin. We examined the effect of UVA irradiation on the basal and the IFN-gamma-and TNF-alpha-stimulation-induced production of thymus-and activation-regulated chemokine (TARC/CCL17) using HaCaT cells. UVA irradiation inhibited the basal levels of both TARC mRNA expression and TARC protein production. UVA irradiation also significantly inhibited TARC mRNA expression and TARC protein secretion that were induced by co-stimulation with IFN-y and TNF-alpha. A time course study showed that: the significant suppression of TARC mRNA expression was detected 8 hours after irradiation and continued for 36 hours; the strongest inhibition of TARC protein secretion occurred in the first 8 hours after UVA irradiation and continued for 36 hours. Our data provide the first evidence that UVA inhibits TARC mRNA expression and TARC protein production by keratinocytes in a dose-dependent manner. These results may suggest an explanation for the UV-induced therapeutic effect.