不同浓度雷公藤甲素对光老化皮肤成纤维细胞基质金属蛋白酶的影响以维甲酸为标准对照

目的观察基质金属蛋白酶在衰老皮肤真皮成纤维细胞表达中的变化,以及不同浓度雷公藤对其产生的影响,并与标准抗皮肤老化剂芳维甲酸乙酯作比较.方法实验于2004-01/10在泸州医学院组织学与胚胎学实验室完成.经家属同意后取泸州医学院附属医院产科2004-01/06健康新生儿包皮,建立体外培养真皮成纤维细胞光老化模型,用中药雷公藤的有效成分雷公藤甲素与芳维甲酸乙酯进行干扰.按分组接受处理,正常对照组不予干预;阳性对照组长波紫外辐射+8-甲氧补骨脂素;维甲酸实验组长波紫外辐射+8-甲氧补骨脂素+芳维甲酸乙酯;雷公藤甲素实验组长波紫外辐射+8-甲氧补骨脂素+雷公藤甲素.雷公藤甲素实验组又按剂量分为3组雷公藤甲素1(10 mg/L)、雷公藤甲素2(20 mg/L)、雷公藤甲素3(40 mg/L)组.①观察衰老皮肤真皮成纤维细胞的基质金属蛋白酶表达的变化用免疫组织化学技术显示.②成纤维细胞形态变化用光学显微镜观察.结果①阳性对照组基质金属蛋白酶1和基质金属蛋白酶3表达比对照组增强(P＜0.05);维甲酸实验组、雷公藤各组比阳性对照组明显减弱(P＜0.05);维甲酸实验组、雷公藤各组之间无显著差异(P＞0.05).②各组的成纤维细胞的密度没有明显的差异.在8-甲氧补骨脂素与紫外线作用后2 h后,培养成纤维细胞逐渐出现细胞老化的形态学改变.在免疫组织化学染色切片中,可以看到各组成纤维细胞内部基质金属蛋白酶1和基质金属蛋白酶3阳性产物.表达48 h最强,然后逐渐减弱,72 h逐渐消失.结论雷公藤甲素3种浓度组对体外培养的真皮成纤维细胞胞质内基质金属蛋白酶1、基质金属蛋白酶3的表达的抑制作用均与芳维甲酸乙酯的相似,提示3种浓度雷公藤甲素对皮肤光老化均具有与维甲酸相似的预防和治疗作用,即通过调节基质金属蛋白酶的活性而防治皮肤的光老化.     

不同浓度雷公藤甲素对光老化皮肤成纤维细胞基质金属蛋白酶的影响：以维甲酸为标准对照

目的：观察基质金属蛋白酶在衰老皮肤真皮成纤维细胞表达中的变化，以及不同浓度雷公藤对其产生的影响，并与标准抗皮肤老化剂芳维甲酸乙酯作比较。方法：实验于2004-01／10在泸州医学院组织学与胚胎学实验室完成。经家属同意后取泸州医学院附属医院产科2004-01／06健康新生儿包皮，建立体外培养真皮成纤维细胞光老化模型，用中药雷公藤的有效成分雷公藤甲素与芳维甲酸乙酯进行干扰。按分组接受处理。正常对照组不予干预；阳性对照组：长波紫外辐射+8-甲氧补骨脂素；维甲酸实验组：长波紫外辐射+8-甲氧补骨脂素+芳维甲酸乙酯；雷公藤甲素实验组：长波紫外辐射+8-甲氧补骨脂素+雷公藤甲素。雷公藤甲素实验组又按剂量分为3组：雷公藤甲素1(10mg／L)、雷公藤甲素2(20mg／L)、雷公藤甲素3(40mg／L)组。①观察衰老皮肤真皮成纤维细胞的基质金属蛋白酶表达的变化用免疫组织化学技术显示。②成纤维细胞形态变化用光学显微镜观察。结果：①阳性对照组基质金属蛋白酶1和基质金属蛋白酶3表达比对照组增强(P&;lt;0．05)；维甲酸实验组、雷公藤各组比阳性对照组明显减弱(P&;lt;0．05)；维甲酸实验组、雷公藤各组之间无显著差异(P&;gt;0．05)。②各组的成纤维细胞的密度没有明显的差异。在8-甲氧补骨脂素与紫外线作用后2h后，培养成纤维细胞逐渐出现细胞老化的形态学改变。在免疫组织化学染色切片中，可以看到各组成纤维细胞内部基质金属蛋白酶1和基质金属蛋白酶3阳性产物。表达48h最强，然后逐渐减弱，72h逐渐消失。结论：雷公藤甲素3种浓度组对体外培养的真皮成纤维细胞胞质内基质金属蛋白酶1、基质金属蛋白酶3的表达的抑制作用均与芳维甲酸乙酯的相似，提示3种浓度雷公藤甲素对皮肤光老化均具有与维甲酸相似的预防和治疗作用，即通过调节基质金属蛋白酶的活性而防治皮肤的光老化。     

人参皂甙Rg3对乳腺癌细胞(MCF-7)表达金属蛋白酶2、9(MMP-2,MMP-9)的影响

目的为了研究人参皂甙Rg3对乳腺癌细胞(MCF-7)细胞分泌的基质金属蛋白酶(MMP-2,MMP-9)表达的影响。方法免疫组化法测定了用人参皂甙Rg3前后MCF-7细胞基质金属蛋白酶(MMP-2,MMP-9)的表达。结果分析表明人参皂甙Rg3能减少MCF-7细胞基质金属蛋白酶(MMP-2,MMP-9)表达。结论人参皂甙Rg3能抑制基质金属蛋白酶MMP-2、MMP-9的分泌。     

维药买朱尼含药血清影响大鼠关节软骨细胞基质金属蛋白酶1及其组织抑制因子1的表达

背景:基质金属蛋白酶1及其组织抑制因子1的失衡是骨关节炎软骨降解的关键.目的:观察维药买朱尼对白细胞介素1β诱导的大鼠关节软骨细胞基质金属蛋白酶1及其组织抑制因子1表达的影响.方法:从1周龄SD大鼠关节中分离培养原代软骨细胞,鉴定后选用第2代细胞随机分为正常对照组、模型组、买朱尼组.模型组和买朱尼组用10 μg/L的白细胞介素1β干预24 h后,买朱尼组在此基础上加入体积分数10%的买朱尼含药血清,模型组加入正常大鼠血清,继续培养12,24,48 h进行实验.结果与结论:各组细胞培养48 h,RT-PCR检测发现体积分数10%的买朱尼含药血清可上调软骨细胞基质金属蛋白酶组织抑制因子1 mRNA的表达同时抑制基质金属蛋白酶1 mRNA的表达,但此作用在细胞培养的24,36 h时不明显.同时免疫荧光细胞化学染色也显示买朱尼含药血清可促进软骨细胞基质金属蛋白酶组织抑制因子1的表达.说明买朱尼可以调节白细胞介素1β诱导的大鼠关节软骨细胞基质金属蛋白酶1及其组织抑制因子1的表达,且此作用具有时间依赖性.     

自体嗅黏膜移植联合β-七叶皂甙钠干预脊髓损伤大鼠基质金属蛋白酶2、9的表达

背景:脊髓损伤后基质金属蛋白酶2、9的过表达与脊髓组织的继发性损伤有关。目的:观察自体嗅黏膜移植联合β-七叶皂甙钠干预脊髓损伤大鼠脊髓组织内基质金属蛋白酶2、9的表达与动物神经功能的恢复情况。方法:制作半横断脊髓损伤大鼠模型,随机分为4组,分别于腹腔注射β-七叶皂甙钠或生理盐水,以及损伤处植入自体嗅黏膜。结果与结论:干预后7,14d自体嗅黏膜组、β-七叶皂甙钠组、自体嗅黏膜联合β-七叶皂甙钠组BBB评分均高于模型对照组(P<0.05),自体嗅黏膜联合β-七叶皂甙钠组BBB评分高于自体嗅黏膜组、β-七叶皂甙钠组(P<0.05)。干预后1,3,7,14d自体嗅黏膜联合β-七叶皂甙钠组脊髓内基质金属蛋白酶2、9阳性细胞数少于模型对照组、自体嗅黏膜组、β-七叶皂甙钠组(P<0.05)。结果提示自体嗅黏膜移植与β-七叶皂甙钠之间有协同作用,可明显改善神经运动功能恢复情况;此种作用可能与其抑制基质金属蛋白酶2、9过度表达有关。     

雷公藤多甙对哮喘大鼠肺组织基质金属蛋白酶9及其抑制剂1表达的影响

目的:探讨雷公藤多甙对哮喘大鼠肺组织中细胞外基质相关因子基质金属蛋白酶9及金属蛋白酶组织抑制因子1的表达的影响。方法:①实验于2004-01/09在南京医科大学实验动物中心、南京医科大学生化教研室实验室和病理教研室实验室完成。选用健康雄性Wis-tar大鼠18只。按随机数字表将大鼠分为3组:对照组、哮喘组、药物组,每组6只。②哮喘组和药物组于第1,8天每只腹腔内注射抗原混悬液1mL致敏;第15天开始雾化吸入10g/L鸡卵清蛋白激发哮喘,1次/d,20min/次,连续4周制作哮喘模型。每天激发前30min,药物组预先灌胃雷公藤多甙悬浮液(1.5mL)50mg/(kg·d)。对照组以等量生理盐水行致敏和激发,1次/d,20min/次,连续吸入4周。③采用免疫组化法观察大鼠肺组织中基质金属蛋白酶9、组织金属蛋白酶抑制剂1的蛋白表达,采用反转录聚合酶联反应技术测定3组大鼠肺组织中基质金属蛋白酶9、组织金属蛋白酶抑制剂1mRNA的表达。④多组间均数的比较采用方差分析,组间两两比较采用q检验。结果:大鼠18只均进入结果分析。①大鼠肺组织中基质金属蛋白酶9和组织金属蛋白酶抑制剂1蛋白含量:哮喘组明显高于对照组(q=9.75,7.87,P<0.01);经药物干预后,明显低于哮喘组(q=5.99,3.27,P<0.05),但仍明显高于对照组(q=3.76,4.59,P<0.05)。②大鼠肺组织中基质金属蛋白酶9与组织金属蛋白酶抑制剂1比值:哮喘组>1,明显高于对照组(q=3.87,P<0.05);经药物干预后<1,明显低于对照组(q=3.47,P<0.05),更低于哮喘组(q=7.34,P<0.01)。③大鼠肺组织中基质金属蛋白酶9、组织金属蛋白酶抑制剂1mRNA表达水平:哮喘组明显高于对照组(q=16.71,15.37,P<0.01);经药物干预后显著下降,明显低于哮喘组(q=11.14,7.21,P<0.01),但未完全降至正常,仍明显高于对照组(q=5.57,8.17,P<0.01)。④大鼠肺组织中基质金属蛋白酶9与组织金属蛋白酶抑制剂1mRNA比值:哮喘组>1,明显高于对照组(q=3.45,P<0.05);经药物干预后<1,明显低于对照组(q=3.45,P<.05),更低于哮喘组(q=6.89,P<0.01)。结论:雷公藤多甙可能下调基质金属蛋白酶9的表达,调节基质金属蛋白酶9与组织金属蛋白酶抑制剂1比值的平衡,干预细胞外基质重塑。     

同型半胱氨酸对血管平滑肌细胞基质金属蛋白酶1及金属蛋白酶组织抑制剂1的影响

背景:同型半胱氨酸已被证实是动脉粥样硬化发生的一个独立危险因素。目的:观察同型半胱氨酸对血管平滑肌细胞基质金属蛋白酶1及金属蛋白酶组织抑制剂1的分泌量和活性的影响。设计:自身对照观察。单位:吉林大学再生医学科学研究所病理室。材料:体外培养的鼠龄4～6周,体质量150g左右雄性Wistar大鼠的血管平滑肌细胞。大鼠由吉林大学白求恩医学部实验动物室提供。方法:实验于2001-05/2003-05在吉林大学再生医学科学研究所病理室完成。采用体外培养大鼠血管平滑肌细胞,分成5组,分别加入浓度为0(对照组),0.10,0.25,0.50和1.00mmol/L同型半胱氨酸作用48h,通过酶谱分析同型半胱氨酸对血管平滑肌细胞细胞基质金属蛋白酶1活性的影响,Western blot技术和半定量RT-PCR方法观察细胞基质金属蛋白酶1和金属蛋白酶组织抑制剂1分泌量及其mRNA的表达。主要观察指标:细胞基质金属蛋白酶1的活性,金属蛋白酶1和金属蛋白酶组织抑制剂1分泌量及其mRNA的表达。结果:①金属蛋白酶1和金属蛋白酶组织抑制剂1分泌量:半胱氨酸0.25,0.50和1.00mmol/L组金属蛋白酶1显著低于对照组(P<0.05～0.01);半胱氨酸0.10,0.25,0.50和1.00mmol/L组金属蛋白酶组织抑制剂1均高于对照组(P<0.05～0.01);②基质金属蛋白酶1活性随着半胱氨酸剂量增加而降低,0.50和1.00mmol/L组降低显著(P<0.01);③加入同型半胱氨酸4组金属蛋白酶1mRNA表达显著低于对照组(P<0.01)],半胱氨酸0.25mmol/L组金属蛋白酶组织抑制剂1mRNA高于对照组(P<0.05),0.50和1.00mmol/L组显著高于对照组(P<0.01))。结论:同型半胱氨酸通过抑制细胞基质金属蛋白酶1的分泌,抑制其酶活性,减少胶原降解而使胶原蓄积。提示同型半胱氨酸减少胶原降解可能是动脉粥样硬化的发病机制之一。     

自体嗅黏膜移植联合β-七叶皂甙钠干预脊髓损伤大鼠基质金属蛋白酶2、9的表达

背景：脊髓损伤后基质金属蛋白酶2、9的过表达与脊髓组织的继发性损伤有关。目的：观察自体嗅黏膜移植联合β-七叶皂甙钠干预脊髓损伤大鼠脊髓组织内基质金属蛋白酶2、9的表达与动物神经功能的恢复情况。方法：制作半横断脊髓损伤大鼠模型,随机分为4组,分别于腹腔注射β-七叶皂甙钠或生理盐水,以及损伤处植入自体嗅黏膜。结果与结论：干预后7,14d自体嗅黏膜组、β-七叶皂甙钠组、自体嗅黏膜联合β-七叶皂甙钠组BBB评分均高于模型对照组（P〈0.05）,自体嗅黏膜联合β-七叶皂甙钠组BBB评分高于自体嗅黏膜组、β-七叶皂甙钠组（P〈0.05）。干预后1,3,7,14d自体嗅黏膜联合β-七叶皂甙钠组脊髓内基质金属蛋白酶2、9阳性细胞数少于模型对照组、自体嗅黏膜组、β-七叶皂甙钠组（P〈0.05）。结果提示自体嗅黏膜移植与β-七叶皂甙钠之间有协同作用,可明显改善神经运动功能恢复情况;此种作用可能与其抑制基质金属蛋白酶2、9过度表达有关。     

曲伏前列腺素对体外培养人眼睫状肌细胞基质金属蛋白酶2的作用

背景:曲伏前列腺素可通过增加眼睫状肌细胞间间隙,使葡萄膜巩膜房水流出通道流出阻力下降,进而降低眼压,这一作用途径是否通过增强睫状肌基质金属蛋白酶的表达而完成?目的:观察曲伏前列腺素干预人眼睫状肌细胞基质金属蛋白酶2表达活性的作用。设计:观察对比分析。单位:中山大学附属第二医院眼科。材料:实验于2005-08/2006-04在中山大学附属眼科中心完成。供体摘自1例死亡1h内无眼疾青年尸体单侧眼球(均经其家属同意),取自中山眼科医院,供体排除眼部疾患。兔抗人基质金属蛋白酶2多克隆抗体(武汉博士德生物工程有限公司),曲伏前列腺素(美国ALCON公司,批号:86610F,0.004%溶液)。方法:实验干预:在人睫状肌细胞无牛血清培养基中加入1μmol/L曲伏前列腺素为实验组,同时以无药物干预的细胞为对照组,实验组于加药后6,12,24h收集细胞进行观察。实验评估:采用RT-PCR和ELISA法分别检测各组人睫状肌细胞基质金属蛋白酶2在基因和蛋白水平的表达;采用Zymography技术分别检测各组细胞基质金属蛋白酶2的活性。主要观察指标:人眼睫状体细胞内基质金属蛋白酶2 mRNA的表达,细胞外液基质金属蛋白酶2蛋白表达以及基质金属蛋白酶2活性。结果:①基质金属蛋白酶2 mRNA的表达:实验组加药后6,12,24h基质金属蛋白酶2 mRNA相对表达量呈逐渐升高趋势(F=236.959,P<0.01)。②基质金属蛋白酶2蛋白表达:实验组加药后6,12,24h基质金属蛋白酶2表达随曲伏前列腺素作用时间延长逐渐升高(F=38.110,P<0.01)。③基质金属蛋白酶2活性:Zymography技术检测实验组加药后6,12,24h基质金属蛋白酶2活性随曲伏前列腺素作用时间延长而逐渐增强(F=74.348,P<0.01)。结论:体外培养的人睫状肌细胞接受曲伏前列腺素作用后,基质金属蛋白酶2表达随药物作用时间延长逐渐增加,活性逐渐增强。     

血管内皮生长因子对佐剂性关节炎滑膜细胞基质金属蛋白酶3及基质金属蛋白酶9表达的影响

目的:观察血管内皮生长因子对佐剂性关节炎滑膜细胞质金属蛋白酶3及质金属蛋白酶9表达的影响,并探讨其意义。方法:实验于2006-02/12在桂林医学院实验中心完成。①实验材料:清洁级8周龄雄性Wistar大鼠6只,血管内皮生长因子为Peprotech EC LTD公司产品,基质金属蛋白酶3(扩增369bp)上游引物、下游引物,基质金属蛋白酶9(扩增405bp)上游引物、下游引物均购自晶美公司。②实验干预:先用弗氏完全佐剂造Wistar大鼠模型;造模20d后取Wistar大鼠右后足滑膜细胞进行原代培养。③实验分组:实验分为血管内皮生长因子组:取P2代细胞接种于6孔培养板,分别加入终浓度为5,25,50μg/L血管内皮生长因子;对照组:不加血管内皮生长因子。④实验评估:取病理切片观察滑膜细胞形态学改变;采用半定量反转录聚合酶链反应检测Wistar大鼠佐剂性关节炎滑膜细胞基质金属蛋白酶3及基质金属蛋白酶9的m-RNA表达。结果:①培养细胞的形态学观察:原代培养14d滑膜细胞从组织块边缘逸出,21d密集生长开始传代;传代细胞48h可明显分辨出树突样细胞、巨噬细胞样细胞和成纤维细胞样细胞;传至21代,细胞生长及特性稳定。②病理切片:滑膜组织有中性粒细胞、单核细胞、淋巴细胞浸润,滑膜细胞增生、排列紊乱,纤维素渗出,胶原纤维沉着,纤维素样坏死,呈滑膜炎表现。③基质金属蛋白酶3、基质金属蛋白酶9的mRNA表达:对照组基质金属蛋白酶3mRNA表达相对灰度值与血管内皮生长因子终浓度5,25,50μg/L组比较,差异有显著性意义(0.32±0.03,0.77±0.06,1.12±0.12,1.59±0.02,P<0.05);对照组基质金属蛋白酶9mRNA表达相对灰度值与血管内皮生长因子终浓度5,25,50μg/L组比较,差异有显著性意义(0.47±0.07,0.50±0.10,0.91±0.10,1.31±0.06,P<0.05);基质金属蛋白酶3和基质金属蛋白酶9mRNA表达随血管内皮生长因子浓度的加大表达增加。结论:血管内皮生长因子以剂量递增的方式对体外培养的佐剂性关节炎滑膜细胞基质金属蛋白酶3及基质金属蛋白酶9的表达有促进作用。     

Clinical‐instrumental and morphological evaluation of the effect of autologous dermal fibroblasts administration

Basic molecular mechanisms, associated with the main cell population of the dermis – fibroblasts – are the basis of skin aging. The number of functionally active fibroblasts in the skin and their biosynthetic activity decreases with age, thus enhancement of their cell density with synthetically active cells is accepted as a one of the most effective methods. The objective of the present study was to evaluate the safety and effectiveness of intradermal administration of autologous dermal fibroblasts in a year after treatment of 17 patients, aged 45–65 years. Results obtained with modern instrumental skin diagnostic methods (vacuum cutometry, optical profilometry, VISIA photometric analysis, etc.) demonstrate the safety and clinical effectiveness of dermal autofibroblast therapy: after transplantation, cultured autofibroblasts keep their biosynthetic activity and produce extracellular matrix for at least 12 months. As a result, remodelling of the dermis microstructures is observed, accompanied by a progressive increase of collagen content and thickness of the dermis (up to 62.5 ±6.7% in 12 months). This is clinically expressed by increase of skin elasticity (24.0 ±4.3% in periorbital area) and thickness of the skin, and by decrease in the number and depth of wrinkles (46 ±7% by the end of observation period). Copyright © 2014 John Wiley & Sons, Ltd.     

人参皂苷Rd干预体外培养肝储脂细胞胶原的表达

背景:肝储脂细胞产生胶原是导致肝硬化的直接原因,调控肝储脂细胞产生胶原的能力即可以防治肝硬化。目的:观察人参皂苷Rd对体外培养肝储脂细胞Ⅰ,Ⅲ型胶原表达的影响。方法:体外培养肝储脂细胞,随机分为对照组和实验组,实验组用100mg/L人参皂苷Rd进行培养干扰,用免疫组织化学、免疫荧光技术检测Ⅰ,Ⅲ型胶原基因的表达。结果与结论:实验组比对照组,Ⅰ,Ⅲ型胶原阳性产物吸光度和面积比值明显降低(P<0.05),说明人参皂苷Rd对体外培养肝储脂细胞Ⅰ,Ⅲ型胶原的表达有明显调节作用,影响肝储脂细胞产生胶原纤维。     

人参皂甙Rd对高原大鼠运动疲劳后学习记忆及海马CA1区超微结构的影响

目的:观察人参皂甙Rd对高原运动疲劳后大鼠学习记忆能力及海马CA1区超微结构改变的影响。方法:健康成年Wistar大鼠24只,由兰州用汽车直接引入青海省可可西里高原,随机分为对照(A)组、生理盐水重度疲劳(B)组及人参皂甙重度疲劳(C)组各8只;A组正常饲养,不运动;采用跑台运动方式建立重度疲劳模型,B、C组分别腹腔给予生理盐水(2mg/kg)或人参皂甙Rd(2mg/kg);Morris水迷宫测试大鼠学习记忆能力,HE染色观察大鼠海马组织形态学改变,透射电子显微镜观察海马CA1区神经元超微结构的改变。结果:与A组相比,B组大鼠逃避潜伏期显著延长(P0.01),海马CA1区超微结构发生病理性改变,C组大鼠逃避潜伏期较B组缩短(P0.05),海马CA1区超微结构病理性改变减轻。结论:人参皂甙Rd可缓解高原环境重度疲劳导致的大鼠学习记忆能力减退及海马神经元损伤。     

Studies on skin aging. Preparation and properties of fucose-rich oligo- and polysaccharides. Effect on fibroblast proliferation and survival.

Skin aging represents an important chapter of connective tissue aging and concerns an organ of vital importance. Here we describe the preparation as well as the biological properties of fucose-rich oligo- and polysaccharides (FROPs), composed of polymers of a trisaccharide containing galactose, acetyl galacturonic acid and fucose, from the original high molecular weight bacterial polysaccharide (Fucogel), Solabia, France). Using endoglycosidases, oligo- and polysaccharides were prepared and characterized by physical and chemical procedures. The non-reducing end-groups comprise equal amounts of galactose and fucose. The here-described biological properties are: stimulation of cell proliferation of cultured human skin fibroblasts, protection of cells against ascorbate-induced cytotoxicity due to the release of reactive oxygen species (ROS). Properties elsewhere described concern the inhibition of matrix metallo-proteinases 2 and 9 (MMP-2 and MMP-9), their expression and activation. Using fluorescein isothiocyanate (FITC)-labeled polysaccharides, their interaction with cell membranes and also their penetration and accumulation in cells, especially in the cell nucleus could be demonstrated, probably via cell-membrane receptor-mediated mechanisms. We describe some of the symptoms of skin aging and show, that the here-described polysaccharide preparations are susceptible to slow down some of the cellular and molecular mechanisms involved, partly by the mediation of the above-mentioned receptors, partly by acting directly on the regulation of gene expression.     

Engineering a human skin equivalent to study dermis remodelling and epidermis senescence in vitro after UVA exposure

Utra Violet type A (UVA) exposure strongly affects the ageing of human skin by modifying both epidermis and dermis and their cross talk as well. The possibility to get a deep understanding in vitro of such crucial mechanism would have a huge impact in the development of antiageing compounds. Here, we present a full thickness model of human skin equivalent formed by a millimeter‐sized dermis completely composed of fibroblasts embedded in their own extracellular matrix. We show that such endogenous nature of the dermis compartment allows the replication of the complexity of the mutual interactions occurring between cellular and extracellular components of the skin under UVA exposure: (a) oxidative stress formation in the whole tissue (dermis and epidermis); (b) senescence of germinative layer of epidermal tissue in terms of p63, ki67, and activated caspase‐3 regulation; (c) modification of the collagenous network architecture in the dermis compartment. By using this human skin model, it is possible to study a widely shared assumptions not yet proved in vitro such the effect of UVA on the self‐renewal capability of skin stem cells.     

Characterization of chitosan–gelatin scaffolds for dermal tissue engineering

Porous scaffolds for dermal tissue engineering were fabricated by freeze‐drying a mixture of chitosan and gelatin (CG) solutions. Different crosslinking agents including glutaraldehyde, 1‐(3‐dimethylaminopropyl)‐3‐ethyl‐carbodimide hydrochloride (EDC), and genipin were used to crosslink the scaffolds and improve their biostability. The porous structure and mechanical properties were determined for the scaffolds. The proliferation of human fibroblasts in the scaffolds was analyzed. It was found that EDC crosslinked scaffolds had the greatest amount of cells after four days. EDC crosslinked CG scaffolds had tensile modulus in a dry state and compressive modulus in a wet state similar to commercial collagen wound dressing. They also showed appropriate pore size, high water absorption, and good dimensional stability during cell culture. When human fibroblasts were seeded on acellular porcine dermis (APD), acellular human dermis (AHD), and CG scaffolds for 3D cell culture, they were well‐distributed in the centre of the CG scaffolds but stayed only on the superficial layer of APD or AHD after seven days. A gelatin‐based bioglue was applied to the CG scaffolds where the keratinocytes were seeded to mimic epidermal structure. After 14 days, the bioglue degraded and keratinocytes grew to form monolayers on the scaffolds. This study showed that CG scaffolds crosslinked by EDC and seeded with human fibroblasts could serve as dermal constructs, while the bioglue coating seeded with keratinocytes could serve as an epidermal construct. Such a combination could help regenerate skin with integrated dermal and epidermal layers and a have potential use in tissue‐engineered skin. Copyright © 2011 John Wiley & Sons, Ltd.     

长波紫外线照射皮肤成纤维细胞表皮生长因子受体的表达

背景:长波紫外线与人体皮肤老化关系密切,主要作用于表皮和真皮全层。目的:观察长波紫外线照射对体外培养的皮肤成纤维细胞的表皮生长因子受体表达的影响。方法:体外培养人成纤维细胞,将细胞分为对照组、长波紫外线照射5,10,20J/cm2组。照射24h后,分别用实时PCR和Western blot检测表皮生长因子受体mRNA和蛋白的表达情况。结果与结论:体外培养的皮肤成纤维细胞随着长波紫外线照射剂量的增加,表皮生长因子受体的mRNA与蛋白水平均显著增加,提示长波紫外线可以诱导成纤维细胞里的表皮生长因子的表达。     

APP17肽通过抑制细胞内ROS保护紫外线照射后人皮肤成纤维细胞(英文)

Objective Ultraviolet light (UV) is known to cause photoaging of skin.UV irradiation can damage proliferation capacity and induce collagenase in fibroblasts in the dermis.Many researchers have explored the potential photo-protective agents;however,no ideal agent has been widely accepted.Amyloid precursor protein 17-mer peptide (APP17-mer peptide),an active peptide segment,has been reported to be responsible for the trophic effect in clonal CNS neuronal line,fibroblast cell line and HaCat cells.The aim of this study was to explore the effects of APP17-mer peptide on cultured fibroblasts after ultraviolet irradiation.Methods Human skin fibroblasts were cultured in DMEM medium with or without APP17-mer peptide (concentrations ranging from 20μmol/L,40 μmol/L,to 80μmol/L).The cultured fibroblasts were exposed to a single UV irradiation,and the proliferation activity of fibroblasts was detected by a MTT assay.The expression of matrix metalloproteinase-1 (MMP-1) mRNA was analyzed quantitatively following real-time RT-PCR.The generation of intracellular reactive oxygen species (ROS) was measured with fluorescent quantitation method.Results A single exposure to UV irradiation depressed proliferation activity of fibroblasts compared with sham-irradiated control (P<0.05).40μmol/L and 80μmol/L APP17-mer peptide increased the cellular proliferation activity in UV irradiated and unirradiated fibroblasts (P<0.05),however,20μmol/L did not show such protective effects (P>0.05).A single exposure of fibroblasts to UV irradiation resulted in 1.78 fold up-regulation of MMP-1 mRNA compared with unirradiated sample (P<0.05),and 40μmol/L and 80μmol/L APP17-mer peptide decreased the expression of MMP-1 mRNA (P<0.05 and P<0.01,respectively).UV irradiation increased generation of ROS in cultured fibroblasts (P<0.05).40μmol/L APP17-mer peptide inhibited the generation of ROS in irradiated fibroblasts.Conclusions APP17-mer peptide can enhance proliferation activity of fibroblasts after exposure to UV irradiation;it can also inhibit MMP-1 mRNA expression and ROS generation induced by UV irradiation.Inhibition of ROS generation after UV irradiation might be involved in the protective mechanism of APP17 peptide on proliferation activity and collagenase induction in UV-irradiated fibroblasts.     

放射性皮肤溃疡中端粒酶逆转录晦表达与癌变及难愈合的机制

INTRODUCTIONAlotofreseachessofarhaveconfirmedthattelomeraseactivityisbecomingincreasinglyregardedasahallmarkofcarcinogenesis．Wepreviouslyreportedthatradiation－inducedchronichumanskinulcer，oneoftheprecancerouslesionswasshowinghighertelom－eraseactivity，althoughweakerthanthatofmalignancy[１－３]inad－ditiontotheoverexpressionsofc－erbB－２，EGFR，MDM２andP５３proteins．ThelimitationofPCR－basedtelomericrepeatamplificationprotocol（TRAP）isthatthemethodcanonlybeuseddetectingtheacti…