【目的】克隆小鼠八聚体结合转录因子1(octamer-binding transcription factor 1,Oct-1)基因序列,探讨八聚体结合转录因子1在小鼠黑素细胞中过表达对毛色主效基因表达的影响及在毛色形成中的作用。【方法】使用实验室冻存的第5代小鼠黑...【目的】克隆小鼠八聚体结合转录因子1(octamer-binding transcription factor 1,Oct-1)基因序列,探讨八聚体结合转录因子1在小鼠黑素细胞中过表达对毛色主效基因表达的影响及在毛色形成中的作用。【方法】使用实验室冻存的第5代小鼠黑素细胞,通过普通PCR方法用引物以小鼠黑素细胞c DNA为模板克隆Oct-1基因c DNA序列,构建小鼠Oct-1克隆载体和真核表达载体;通过KEGG PATHWAY、NCBI、Transfec等软件对获得的序列进行生物信息学分析;在细胞水平通过细胞转染技术过量表达小鼠Oct-1;转染后使用荧光显微镜观察细胞转染效率,采用分光光度计对小鼠黑素细胞中黑色素含量进行测定,并进行Real-time PCR实验检测转染后黑素细胞中毛色主效基因在m RNA水平表达量的变化,Western blot实验检测转染后细胞中MITF、TYR、TYRP-1和TYRP-2蛋白水平的变化。【结果】经测序和拼接最终获得长度为2 313 bp的小鼠Oct-1基因的c DNA序列;成功构建真核表达载体,载体上连有小鼠黑素细胞特异性TYRP-2基因启动子和一个启动报告基因绿色荧光蛋白;通过KEGG PATHWAY分析获得与毛色形成有关的34个候选基因,NCBI查找出34各个基因的启动子,由Transfec启动子分析软件找出Oct-1可以调节的毛色主效基因;细胞转染后,在荧光显微镜下可观察到黑素细胞带有绿色荧光说明转染效率明显;分光光度计检测显示,转染后小鼠黑素细胞中黑色素含量减少(P<0.05);荧光定量检测结果显示,小鼠黑素细胞中Oct-1 m RNA表达量显著增加(P<0.001),表明小鼠Oct-1转染效率显著,MITF m RNA显著降低至0.70倍(P<0.01),TCF m RNA显著降低至0.66倍(P<0.01),Ras、Frizzled、ERK2和TYRP-2 m RNA的表达未见变化,TYR m RNA显著增加至7.69倍(P<0.01),TYRP-1 m RNA升高至3.11倍(P<0.01),αMSH m RNA显著增加至18.49倍(P<0.001),AC m RNA显著增加至6.88倍(P<0.01),c-kit m RNA显著增加至18.75倍(P<0.001),ET1 m RNA增加至1.50倍(P<0.05),ETB-R m RNA显著增加至13.47倍(P<0.001),CAM m RNA增加至1.46倍(P<0.05);蛋白免疫印迹结果显示,小鼠黑素细胞中Oct-1转染组MITF蛋白显著降低至0.67倍(P<0.01),TYR蛋白增加至1.16倍(P<0.05),TYRP-1蛋白升高至1.15倍(P<0.05),TYRP-2蛋白未见变化。【结论】通过PCR和克隆技术及核酸测序技术获得了小鼠Oct-1基因全长2 313 bp的CDS区,经生物信息学分析找出Oct-1作用的毛色主效基因,过表达Oct-1后使黑素细胞中MITF和TCF的表达降低,TYR、TYRP-1、αMSH、AC、c-kit、ET1、ETB-R和CAM的表达增加。证实Oct-1可调节毛色主效基因的表达,参与黑色素合成的调节,改变毛色。展开更多
Objective:To investigate the suitability of citrus-press cakes,by-products of the juice industry as a source for the whitening agents for cosmetic industry.Methods:Ethylacetate extracts of citrus-press cakes(CCE)were ...Objective:To investigate the suitability of citrus-press cakes,by-products of the juice industry as a source for the whitening agents for cosmetic industry.Methods:Ethylacetate extracts of citrus-press cakes(CCE)were examined for their anti-melanogenic potentials in terms of the inhibition of melanin production and mechanisim of melanogenesis by using Western Blot analysis with tyrosinese,tyrosinase-related protein-1(TRP-1),TRP2,and microphthalmia-associated transcription factor(MITF)proteins.To apply the topical agents,citrus-press cakes was investigated the safety in human skin cell line.Finally flavonoid analysis of CCE was also determined by HPLC analysis.Results:Results indicated that CCE were shown to down-regulate melanin content in a dose-dependent pattern.The CCE inhibited tyrosinase,TRP-2,and MITF expressions in a dose-dependent manner.To test the applicability of CCE to human skin,we used MTT assay to assess the cytotoxic effects of CCE on human keratinocyte HaCaT cells.The CCE exhibited low cytotoxicity at 50μg/mL.Characterization of the citrus-press cakes for flavonoid contents using HPLC showed varied quantity of rutin,narirutin,and hesperidin.Conclusions:Considering the anti-melanogenic activity and human safety,CCE is considered as a potential anti-melanogenic agent and may be effective for topical application for treating hyperpigmentation disorders.展开更多
文摘【目的】克隆小鼠八聚体结合转录因子1(octamer-binding transcription factor 1,Oct-1)基因序列,探讨八聚体结合转录因子1在小鼠黑素细胞中过表达对毛色主效基因表达的影响及在毛色形成中的作用。【方法】使用实验室冻存的第5代小鼠黑素细胞,通过普通PCR方法用引物以小鼠黑素细胞c DNA为模板克隆Oct-1基因c DNA序列,构建小鼠Oct-1克隆载体和真核表达载体;通过KEGG PATHWAY、NCBI、Transfec等软件对获得的序列进行生物信息学分析;在细胞水平通过细胞转染技术过量表达小鼠Oct-1;转染后使用荧光显微镜观察细胞转染效率,采用分光光度计对小鼠黑素细胞中黑色素含量进行测定,并进行Real-time PCR实验检测转染后黑素细胞中毛色主效基因在m RNA水平表达量的变化,Western blot实验检测转染后细胞中MITF、TYR、TYRP-1和TYRP-2蛋白水平的变化。【结果】经测序和拼接最终获得长度为2 313 bp的小鼠Oct-1基因的c DNA序列;成功构建真核表达载体,载体上连有小鼠黑素细胞特异性TYRP-2基因启动子和一个启动报告基因绿色荧光蛋白;通过KEGG PATHWAY分析获得与毛色形成有关的34个候选基因,NCBI查找出34各个基因的启动子,由Transfec启动子分析软件找出Oct-1可以调节的毛色主效基因;细胞转染后,在荧光显微镜下可观察到黑素细胞带有绿色荧光说明转染效率明显;分光光度计检测显示,转染后小鼠黑素细胞中黑色素含量减少(P<0.05);荧光定量检测结果显示,小鼠黑素细胞中Oct-1 m RNA表达量显著增加(P<0.001),表明小鼠Oct-1转染效率显著,MITF m RNA显著降低至0.70倍(P<0.01),TCF m RNA显著降低至0.66倍(P<0.01),Ras、Frizzled、ERK2和TYRP-2 m RNA的表达未见变化,TYR m RNA显著增加至7.69倍(P<0.01),TYRP-1 m RNA升高至3.11倍(P<0.01),αMSH m RNA显著增加至18.49倍(P<0.001),AC m RNA显著增加至6.88倍(P<0.01),c-kit m RNA显著增加至18.75倍(P<0.001),ET1 m RNA增加至1.50倍(P<0.05),ETB-R m RNA显著增加至13.47倍(P<0.001),CAM m RNA增加至1.46倍(P<0.05);蛋白免疫印迹结果显示,小鼠黑素细胞中Oct-1转染组MITF蛋白显著降低至0.67倍(P<0.01),TYR蛋白增加至1.16倍(P<0.05),TYRP-1蛋白升高至1.15倍(P<0.05),TYRP-2蛋白未见变化。【结论】通过PCR和克隆技术及核酸测序技术获得了小鼠Oct-1基因全长2 313 bp的CDS区,经生物信息学分析找出Oct-1作用的毛色主效基因,过表达Oct-1后使黑素细胞中MITF和TCF的表达降低,TYR、TYRP-1、αMSH、AC、c-kit、ET1、ETB-R和CAM的表达增加。证实Oct-1可调节毛色主效基因的表达,参与黑色素合成的调节,改变毛色。
基金Supported by the Next-Generation BioGreen 21 Program,Rural Development Administration,Republic of Korea,Grant No.PJ009583002013
文摘Objective:To investigate the suitability of citrus-press cakes,by-products of the juice industry as a source for the whitening agents for cosmetic industry.Methods:Ethylacetate extracts of citrus-press cakes(CCE)were examined for their anti-melanogenic potentials in terms of the inhibition of melanin production and mechanisim of melanogenesis by using Western Blot analysis with tyrosinese,tyrosinase-related protein-1(TRP-1),TRP2,and microphthalmia-associated transcription factor(MITF)proteins.To apply the topical agents,citrus-press cakes was investigated the safety in human skin cell line.Finally flavonoid analysis of CCE was also determined by HPLC analysis.Results:Results indicated that CCE were shown to down-regulate melanin content in a dose-dependent pattern.The CCE inhibited tyrosinase,TRP-2,and MITF expressions in a dose-dependent manner.To test the applicability of CCE to human skin,we used MTT assay to assess the cytotoxic effects of CCE on human keratinocyte HaCaT cells.The CCE exhibited low cytotoxicity at 50μg/mL.Characterization of the citrus-press cakes for flavonoid contents using HPLC showed varied quantity of rutin,narirutin,and hesperidin.Conclusions:Considering the anti-melanogenic activity and human safety,CCE is considered as a potential anti-melanogenic agent and may be effective for topical application for treating hyperpigmentation disorders.
