紫外线照射对HaCaT细胞IL-12和IFN-γ受体表达的影响

目的：观察紫外线（UV）照射对HaCaT细胞起源的IL-12、IFN-γ的影响，为进一步阐述光敏性皮炎的发病机制提供依据。方法：以UVA和UVB照射人永生化表皮细胞（HaCaT打细胞），应用免疫组化SP法观察UV照射后HaCaT细胞表面IL-12、IFN-γ受体的表达情况，并以阳性细胞百分比半定量IL-12、IFN-γ在HaCaT细胞中的表达；同时应用ELISA方法检测细胞培养液中IL-12、IFN-γ的含量。结果：静息状态下，HaCaT细胞低水平表达IL-12R、IFN-γR，上清液中IL-12、IFN-γ呈低水平表达或检测不出；UV照射HaCaT细胞后12h，细胞表面IL-12R、IFN-γR及上清液中的IL-12、IFN-γ与照射前无明显差异。结论：紫外线照射后角质形成细胞产生细胞因子致T细胞亚群失衡与光敏性皮炎的相关性，有待进一步探讨。     

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。     

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。     

马钱子组分对人HaCaT细胞毒性作用的比较

目的研究马钱子生物碱及其不同组分对人永生化表皮细胞系Ha Ca T细胞的毒性作用。方法体外培养Ha Ca T细胞,加入相同质量浓度的马钱子碱、士的宁、马钱子碱与士的宁的混合物,和马钱子总生物碱作用于细胞24 h后,采用MTT比色法测定不同生物碱的细胞增殖抑制作用;流式细胞仪检测对Ha Ca T细胞的凋亡,比较马钱子生物碱不同组分对Ha Ca T细胞的毒性作用。结果士的宁对Ha Ca T细胞的抑制率和细胞凋亡率大于马钱子碱;马钱子碱和士的宁混合物组对Ha Ca T细胞的抑制率和细胞凋亡率大于马钱子总生物碱。结论马钱子生物碱组分均对Ha Ca T细胞存在细胞毒作用,呈剂量依赖关系,但马钱子碱的细胞毒性明显小于士的宁。     

五味子提取工艺优化及乙醇提取物的皮肤细胞抗氧化作用研究

目的 探究五味子提取物对人皮肤细胞氧化损伤的保护作用,开发其在皮肤抗氧化方面的应用。方法 分别采用热水蒸煮和水蒸气蒸馏的方法进行脱色和除味;另外,采用叔丁基过氧化氢（tBHP）诱导的HaCaT细胞氧化应激模型评价五味子提取物对人皮肤细胞氧化损伤的保护作用,采用CCK-8试剂盒检测细胞存活率,AnnexinV-FITC/PI双标记流式细胞仪检测细胞凋亡率,2,7-二氯荧光黄双乙酸盐法检测细胞内活性氧的水平。结果 采用优化后的提取工艺得到了气味变淡且颜色明显变浅的五味子提取物;用北五味子提取物预处理HaCaT细胞6 h后,可有效降低tBHP引起的细胞凋亡,提高细胞存活率,降低细胞内ROS水平。结论 优化后的乙醇提取工艺显著改善了五味子提取物的颜色和气味,并保持了提取物的抗氧化活性,因而有望成为一种应对皮肤氧化损伤的外用保护剂。     

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.     

黄芩素对UVB诱导HaCaT细胞光老化及相关p38信号通路的影响

目的:研究黄芩素对中波紫外线(UVB)诱导人皮肤角质形成细胞(HaCaT)光老化及相关p38丝裂原活化蛋白激酶(p38MAPK)信号通路的影响。方法:选择1×10~(-11),1×10~(-10),1×10~(-9),1×10~(-8),1×10~(-7),1×10~(-6),1×10~(-5),1×10~(-4),1×10~(-3)mol·L~(-1)9种浓度黄芩素作用于HaCaT细胞,噻唑蓝(MTT)法筛选出最佳有效浓度。经预实验筛选出辐射强度为0.5 mW·cm~(-2)的UVB,辐射时间为5 min建立光老化模型,筛选出最佳有效浓度作用于光老化模型,另设空白组MTT法检测各组细胞活性。超氧化物歧化酶(SOD),谷胱甘肽过氧化物酶(GSH-Px),过氧化氢酶(CAT)及丙二醛(MDA)试剂盒检测SOD,GSH-Px,CAT活性及MDA含量。实时定量荧光定量聚合酶连式反应(RT-PCR)及蛋白免疫印迹法(Western blot)分别检测各组细胞中mRNA及蛋白表达。结果:筛选出1×10~(-7),1×10~(-6),1×10~(-5)mol·L~(-1)黄芩素为最佳有效浓度;与空白组比较,UVB组对HaCaT细胞无明显的增殖作用。与UVB组比较,1×10~(-7),1×10~(-6),1×10~(-5)mol·L~(-1)黄芩素组对光老化模型无明显增殖作用。与空白组比较,UVB组SOD,GSH-Px及CAT活性明显的降低,MDA含量显著升高(P0.01);与UVB组比较,1×10~(-7),1×10~(-6),1×10~(-5)mol·L~(-1)黄芩素组SOD,GSH,CAT活性明显升高,MDA含量明显降低(P0.05,P0.01)。与空白组比较,UVB组肿瘤坏死因子-α(TNF-α)mRNA及TNF-α,磷酸化p38(p-p38)蛋白表达显著升高(P0.01);与UVB组比较,1×10~(-7),1×10~(-6),1×10~(-5)mol·L~(-1)黄芩素组TNF-αmRNA及TNF-α,p-p38蛋白表达明显降低(P0.05,P0.01)。结论:黄芩素对UVB诱导HaCaT细胞光老化保护作用主要是通过提高氧化酶活性以及阻断p38MAPK信号通路,抑制炎症因子产生。     

Flicking technique for microencapsulation of cells in calcium alginate leading to the microtissue formation

Microbeads have wide applications in biomedical engineering field that include drug delivery, encapsulation of biomolecules, tissue padding and tissue regeneration. In this paper, we report a simple, yet efficient, flicking technique to produce microcapsules of calcium alginate at a narrow distribution of size. The system consists of an infusion pump and a customised flicker that taps the syringe needle for dispersing microcapsules of sodium alginate that polymerised in the calcium chloride solution. The flow rate of the syringe pump and the velocity of the flicker were studied to achieve a well controlled and tunable size distribution of microbeads ranging from 200 to 400?μm. At a flow rate of 4?μl/min and flicking rate of 80?rpm, a narrow size distribution of microbeads were produced. Via this technique, HaCaT cells were encapsulated in calcium alginate microbeads that grown into microtissues with a size ranging from 100 to 300?μm after two weeks of culture. These microtissues could be potentially useful for pharmacological application.