小檗碱衍生物HB-13对角质形成细胞分泌IFN-γ的影响

目的研究小檗碱衍生物HB一13对人永生化角质形成细胞（HaCaT细胞）分泌γ干扰素（IFN叫）的影响,探讨HB-13的抗炎机制。方法采用四甲基偶氮唑蓝法确定HB-13作用于HaCaT细胞的安全浓度;细胞经P物质刺激后,加入不同浓度的HB-13分别孵育12、24、48h,应用酶联免疫吸附法（ELISA）检测培养上清液中IFN-γ的含量,应用实时荧光定量PCR法检测细胞内IFN-γmRNA表达情况。结果HB-13浓度为2．5、5、10μg／ml对HaCaT细胞毒性作用不显著,并可促进P物质诱导的IFN-γ的分泌。结论促进表皮细胞分泌IFN-γ可能是HB-13在皮肤局部发挥抗炎作用的机制之一。     

Structural alterations provoked by narrow-band ultraviolet B in immortalized keratinocytes: assessment by atomic force microscopy

We applied atomic force microscopy (AFM) to visualize ultrastructural changes of the keratinocyte morphology after narrow-band ultraviolet B (NB-UVB) irradiation. Immortalized human keratinocytes were cultured under standard conditions, irradiated with NB-UVB light at doses ranging from 50 to 800 mJ/cm2 and imaged by AFM mounted on an inverted optical microscope. It was observed, that NB-UVB irradiation provoked dose-dependent alterations of the keratinocyte morphology. While the surface of non-irradiated cells exhibited homogenously distributed crest-like shaped protrusions (height 0.16 +/- 0.05 microm), cells irradiated with a dose of 800 mJ/cm2 in addition showed round shaped protrusions (height 0.14 +/- 0.06 microm) distributed predominantly around the nucleus and bleb-like protrusions irregularly distributed on the cell surface (height 0.95 +/- 0.29 microm). These irradiated cells easily detached from the supporting glass surface, showed impaired contact with adjacent keratinocytes and significantly rearranged their cytoskeleton network. We hypothesize that these structural and functional alterations reflect ongoing apoptosis in UVB treated cells.     

芳维A酸氨丁三醇对HaCaT细胞分化的影响

目的观察芳维A酸氨丁三醇作用于无血清培养的HaCaT细胞后,细胞内分化相关基因TGase1表达的变化,探讨其剂量-时间曲线,并比较芳维A酸氨丁三醇与依曲替酸对HaCaT分化的影响。方法将不同浓度的芳维A酸氨丁三醇和依曲替酸(0.001～1.0μm/L)分别加入无血清培养的HaCaT细胞培养液中,在添加芳维A酸氨丁三醇6h、12h和24h后分别回收总RNA,逆转录PCR(RT-PCR)法检测HaCaT细胞分化的特异性标记基因TGase1 mRNA的表达情况;芳维A酸氨丁三醇和依曲替酸分别在孵育24h后回收总RNA,用RT-PCR法检测TGase1 mRNA的表达,比较这两种药物对HaCaT细胞分化的差异。结果0.001～1.0μm/L浓度的芳维A酸氨丁三醇作用于HaCaT细胞6h、12h和24h后能显著促进HaCaT细胞内分化基因TGase1mRNA表达,且呈剂量和时间依赖性(P<0.05)。芳维A酸氨丁三醇及依曲替酸都可促进TGase1 mRNA表达,且TGase1mRNA的表达呈剂量依赖性,这两种药物的剂量曲线非常相似,其促进TGase1 mRNA表达程度比较,差异无显著性意义(P>0.05)。结论芳维A酸氨丁三醇能显著促进HaCaT细胞内分化基因TGase1 mRNA的表达,且呈时间和剂量依赖性,其促进HaCaT细胞分化的作用与依曲替酸的效果类同。     

降钙素基因相关肽对HaCaT细胞表达血管内皮生长因子的调控

目的 探讨降钙素基因相关肽(CGRP)对角质形成细胞表达和分泌血管内皮生长因子(VEGF)的调控.方法 用实时定量PCR(RT-PCR)方法检测CGRP、CGRP1型受体拮抗剂CGRP8-37、细胞外信号调节激酶ERK1/2特异性的抑制剂PD98059、p38MAPK特异性拮抗剂SB203580对HaCaT角质形成细胞表达VEGFmRNA水平的影响;用酶联免疫吸附方法检测HaCaT细胞分泌至培养液中VEGF蛋白的水平.结果 CGRP时间依赖性地促进HaCaT细胞表达VEGFmRNA和分泌VEGF蛋白;CGRP8-37和PD98059均可明显抑制CGRP刺激的HaCaT细胞表达和分泌VEGF,SB203580不能减弱CGRP刺激的VEGF表达和分泌.结论 CGRP可以上调HaCaT细胞表达和分泌VEGF,CGRP1型受体及其相关的ERK1/2信号通路参与其调控.     

The expression of keratinocyte growth factor receptor (FGFR2-IIIb) correlates with the high proliferative rate of HaCaT keratinocytes

Keratinocyte growth factor receptor (KGFR = FGFR2-IIIb) is a tyrosine kinase receptor expressed by keratinocytes, which mediates the effects of fibroblast growth factors (FGF). There are contradictory data in the literature regarding the role of FGFR2-IIIb during the proliferation/differentiation programme of keratinocytes. In this study, we aimed to investigate whether overexpression of FGFR2-IIIb may have a role in the regulation of keratinocyte proliferation. We analysed the expression of FGFR2-IIIb in an in vitro HaCaT model system representing different stages of proliferation and differentiation of keratinocytes. Real-time RT-PCR and Western blot analyses demonstrated a correlation between FGFR2-IIIb mRNA and protein expression and the proportion of cells in S/G2/M phase in synchronized HaCaT keratinocytes and thus with proliferation activity (r = 0.96). After treatment with the antipsoriatic drug, dithranol, FGFR2-IIIb is downregulated dose dependently both at mRNA and protein levels. Moreover, when the rate of proliferation is decreased by the lack of cell attachment to the culturing surface, FGFR2-IIIb mRNA (P = 0.0315) and protein expressions were also reduced (P = 0.0242), while a differentiation marker, keratin 10, mRNA (P = 0.0003) and protein levels (P = 0.001) were increased (r = -0.92). Based on our results we conclude that FGFR2-IIIb expression in HaCaT keratinocytes corresponds with the proliferative activation of the cells and is not related to the differentiation programme.     

HaCaT细胞蛋白质组双向电泳图谱的建立

背景与目的： 建立HaCaT细胞蛋白质组双向电泳图谱。 材料与方法： 用含10％ 胎牛血清的RPMI1640培养液培养HaCaT细胞,胰酶消化后收集细胞,加入细胞裂解液使细胞充分裂解,离心后取上清液进行第一向第电聚焦电泳和平衡,再进行第二向十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和考马斯亮蓝染色及图像扫描与分析。 结果： 通过对HaCaT细胞蛋白提取液进行双向电泳,在 17 cm pH 3-10 L IPG胶条上可分离到(700±23)个重复性及分辨率均较好的蛋白位点,蛋白斑点的匹配率为72.2％。 结论： HaCaT细胞蛋白质组双向电泳图谱的建立,为其蛋白质组学的进一步研究奠定了基础。     

芦荟大黄素对人永生化角质形成细胞增殖和凋亡作用研究

目的研究芦荟大黄素（AE）对人永生化角质形成细胞（HaCaT）的生长抑制效应和诱导细胞凋亡能力,探讨其治疗银屑病的可能机制。方法四甲基偶氮唑蓝（MTT）法检测芦荟大黄素对HaCaT细胞增殖的抑制作用,倒置显微镜观察细胞形态学变化,流式细胞术检测细胞周期变化及凋亡。结果芦荟大黄素质量浓度在20～80μg／mL范围内可抑制HaCaT细胞增殖且呈剂量依赖性;镜下观察到细胞稀疏,生长减慢,细胞形态拉长,细胞被阻滞于S期而凋亡。结论芦荟大黄素能抑制HaCaT细胞的增殖、诱导HaCaT细胞的凋亡。可用于治疗银屑病。     

姜黄素对低氧诱导的HaCaT细胞增殖以及HIF-1α蛋白的影响

目的 研究姜黄素对HaCaT细胞增殖的影响以及对HaCaT细胞在缺氧培养条件下细胞中低氧诱导因子(HIF)-1α蛋白表达的影响,探讨姜黄素对银屑病的作用机制.方法 用不同浓度的姜黄素作用于HaCaT细胞,观察其对细胞增殖的影响;以不同浓度的姜黄素作用于缺氧培养的HaCaT细胞后,免疫组化检测其对HIF-1α蛋白表达的影响.结果 姜黄素对HaCaT细胞的增殖具有明显的抑制作用,作用于缺氧培养的Ha-CaT细胞后其HIF-1α蛋白表达减弱.结论 姜黄素可抑制HaCaT细胞增殖及缺氧培养的HaCaT细胞HIF-1α蛋白的表达.     

无机砷化合物对HaCaT细胞毒性作用

目的探讨无机砷化合物对人表皮角质细胞(HaCaT)毒性与氧化应激和砷代谢关系。方法用四甲基偶氮唑盐法测定细胞生存率;检测砷化物对丙二醛(MDA)含量、超氧化物歧化酶(SOD)活力影响;流式细胞仪检测细胞内活性氧(ROS);原子荧光分析方法检测细胞内外不同形态砷含量。结果200.0 μmol/L As₂O₃(iAs^Ⅲ)、50.0~500.0 μmol/L Na₂HAsO₄·7H₂O(iAs^Ⅴ)能够明显降低HaCaT细胞生存率,生存率分别为(54.8±5.9)%、(85.4±9.5)%、(70.0±4.0)%、(58.8±6.0)%,在低剂量时,2种砷化物表现为促进HaCaT细胞增殖(P<0.05);iAs^Ⅲ组随剂量增加MDA含量梯度增加,iAs^Ⅴ组MDA含量先增加后降低呈倒U形;细胞内ROS含量iAs^Ⅲ组> iAs^Ⅴ组;细胞外iAs^Ⅲ、iAs^Ⅴ含量均高于细胞内,细胞内砷甲基化率表现为5.0 mol/L iAs^Ⅲ组(87.3%)>50.0 mol/L iAs^Ⅲ组(61.8%),5.0 mol/L iAs^Ⅴ组(40.0%)>50.0 mol/L iAs^Ⅴ组(10.5%),且iAs^Ⅲ组高于iAs^Ⅴ组。结论无机砷化物对HaCaT 细胞毒性影响与氧化应激和砷代谢水平有关。