MTT法和水溶性四氮唑(WST-1)法检测中波紫外线照射HaCaT细胞后活力的比较

目的:观察中波紫外线照射后HaCaT细胞活力的变化并探讨水溶性四氮唑(WST-1)法检测的适用性.方法:将HaCaT细胞分为对照组(假照射组)、300J/m2、600J/m2、900J/m2紫外线照射后24h组(每组6个样本).600J/m2紫外线照射后4h组、8h组、1 2h组、24h组、48h组、72h组(每组6个样本).血球计数板计算HaCaT细胞数量.四甲基偶氮唑盐(MTT)法和水溶性四氮唑(WST-1)法活性测定HaCaT细胞活性.结果:MTT法显示300J/m2、600J/m2、900J/m2紫外线照射HaCaT细胞24h后光密度(OD)值明显较0J/m2组低;WST-1法各组OD值明显比0J/m2组低.MTT法显示600J/m2紫外线照射HaCaT细胞8h、12h、24h、48h、72h后OD值明显比0J/m2组低.WST-1法显示600J/m2紫外线照射HaCaT细胞4h后OD值较0J/m2组低;600J/m2紫外线照射HaCaT细胞8h、12h、24h、48h、72h后OD值明显比0J/m2组低.WST-1法的OD值与细胞计数、MT法OD值呈正相关.结论:随着紫外线剂量的加大和照射后时间延长,HaCaT细胞数量减少,细胞的活力下降,WST-1代谢活性亦降低,呈时间和剂量依赖性.HaCaT细胞可对WST-1进行代谢,适用于该细胞增殖研究.     

Site-directed Mutagenesis (Y52E) of POLH Affects Its Ability to Bypass Ultraviolet-induced DNA Lesions in HaCaT Cells

DNA polymerase eta (Pol η) is one of several Y family translesion synthesis (TLS) polymerases in humans and plays an important role in maintaining genome stability after ultraviolet (UV) irradiation, as it carries out error-free TLS at sites of UV-induced lesions. We performed site-directed mutagenesis of human polymerase η gene (Y52E), confirmed by sequencing, then cloned wild-type mutant and POLH genes into the eukaryotic vector pEGFP-N1. After transfecting wild-type and mutant plasmids into HaCaT keratinocytes, we tested for UV-induced cis-syn cyclobutane pyrimidine dimer (CPDs) DNA lesions, and analysed cellular viability by MTT cell proliferation assay. The results showed that CPD levels decreased with empty vector control (EVC), wild-type POLH and Y52E-POLH over 48 hours post-UV irradiation with 0.1 mW/cm2 UVB for 15 minutes (p = 0.025). The rate in CPD reduction of mutant POLH was less than in wild-type POLH. Cell viabilities of all three groups increased over 48 hours after UV irradiation, with the increased rate in the wild-type being higher than for mutant protein (p = 0.046). We conclude that Y52E POLH has reduced capacity to bypass UV-induced DNA lesions in HaCaT cells.     

蜈蚣败毒饮对HaCaT细胞Keratin6 mRNA表达的影响

目的:研究蜈蚣败毒饮对角质形成(Ha Ca T)细胞角蛋白Keratin6 m RNA表达的影响。方法:36只Wistar大鼠随机均分为空白对照(等容生理盐水)组、甲氨蝶呤(0.22 g/100 g)组、复方青黛胶囊(0.26 g/100 g)组与蜈蚣败毒饮高、中、低剂量(11.88、5.94、2.96 g/100 g)组,ig给药,每天2次,连续3 d。末次给药后取腹主动脉血,各组血清作用于相应的Ha Ca T细胞培养24 h。通过实时荧光酶链聚合反应(RT-PCR)法测定Ha Ca T细胞Keratin6 m RNA的表达。结果:与空白对照组比较,各用药组Ha Ca T细胞Keratin6 m RNA表达减弱,蜈蚣败毒饮在减弱Ha Ca T细胞Keratin6 m RNA表达方面呈现出非剂量依赖关系。结论:蜈蚣败毒饮可能通过抑制Keratin6 m RNA的表达,从而降低了Ha Ca T细胞增殖来发挥抗银屑病作用。     

血清含量对HaCaT细胞生长特性及迁移能力的影响

[目的] 考察不同血清含量以及培养不同时间对人永生化角质形成细胞(HaCaT)细胞的生长及迁移特性的影响,为HaCaT细胞划痕实验条件优化提供实验数据支撑。[方法] 培养HaCaT细胞,按10%血清浓度全培基(全培基阳性对照组)、2%低浓度血清培基(低血清培基组)及无血清培基(对照组)分组,通过进行细胞划痕实验,CCK-8细胞活力检测、Brdu细胞增殖实验,分析HaCaT生长特性。[结果] 在24 h内,低血清培基组HaCaT细胞与全培基组相比,细胞增殖能力无明显差异,细胞活力有增高的趋势。细胞孵育48 h后,全培基组细胞增殖能力及活力明显升高。24~48 h,全培基组细胞增殖能力及细胞活力的提升速度明显高于低血清培基组。[结论] 培养基中血清含量对HaCaT细胞生长特性及迁移能力具有重要影响,培养24 h内低血清培养条件有利于划痕实验的观察与测定。     

In vitro wound healing activity of 1-hydroxy-5,7-dimethoxy-2-naphthalene-carboxaldehyde (HDNC) and other isolates of Aegle marmelos L.: Enhances keratinocytes motility via Wnt/β-catenin and RAS-ERK pathways

Wound healing is a complex process in which injured skin and tissues repaired by interaction of a complex cascade of cellular events that generates resurfacing, reconstitution and restoration of the tensile strength of injured skin. It follows β-catenin, extracellular signal regulated kinase (ERK) and Akt signaling pathways. Aegle marmelos L., generally known as bael is found to act as anti-inflammatory, antioxidant and anti-ulcer agent. Furthermore, studies have demonstrated that this Indian traditional medicinal plant, A. marmelos flower extract (AMF) was used for wound injury. Henceforth, the current study was investigated to ascertain the effect of its active constituents in vitro wound healing with mechanism involve in migration of cells and activation of β-catenin in keratinocytes, inhibition of PGE₂ in macrophages and production of collagen in fibroblasts. We have taken full thickness wound of rats and applied AMF for 2 weeks. Cutaneous wound healing activity was performed using HaCaT keratinocytes, Hs68 dermal fibroblasts and RAW264.7 macrophages to determine cell viability, nitric oxide production, collagen expression, cell migration and β-catenin activation. Results shows that AMF treated rats demonstrated reduced wound size and epithelisation was improved, involved in keratinocytes migration by regulation of Akt signaling, beta-catenin and extracellular signal-regulated kinase (ERK) pathways. AMF and its active constituent’s increased mRNA expression, inhibited nitric oxide, PGE₂ release, mRNA expression of mediators in RAW 264.7 macrophages and enhances the motility of HaCaT keratinocytes in vitro wound healing of rats.     

NCSTN基因沉默对HaCaT细胞增殖分化的影响

【摘要】 目的 检测NCSTN基因稳定沉默的人永生化角质形成细胞（HaCaT）增殖活性及相关分化蛋白的改变,初步探索反常性痤疮发病的可能机制。方法 构建慢病毒介导shRNA沉默NCSTN基因的HaCaT细胞模型（shRNA组）,转染空载慢病毒的HaCaT细胞作为阴性对照组,实时定量PCR和Western印迹法检测NCSTN基因的沉默效率。CCK8法检测HaCaT细胞增殖活性,实时定量PCR和Western印迹法检测HaCaT细胞角蛋白（CK1、CK5、CK7、CK10、CK14、CK16、CK17、CK18、CK19、CK20）及其他分化分子（内披蛋白、兜甲蛋白）mRNA和蛋白的表达。两组计量资料的比较采用两独立样本t检验。结果 shRNA组NCSTN mRNA及蛋白表达（0.42 ± 0.19、0.30 ± 0.07）均显著低于阴性对照组（1.00 ± 0.34、1.00 ± 0.26;t = 5.196、2.637,P < 0.001、< 0.05）,基因沉默效率达70%。与阴性对照组相比,shRNA组HaCaT细胞明显增殖活跃,CK16、CK19蛋白表达显著下调（t = 3.787、3.817,P < 0.01、< 0.05）,终末分化分子内披蛋白表达明显降低（t = 2.904,P < 0.05）。结论 稳定沉默NCSTN基因表达会引起HaCaT细胞异常增殖分化,为后续探究NCSTN基因突变引起反常性痤疮提供新思路。