OBJECTIVE To explore the effect of connexin(Cx)40-formed gap junctional intercellular communication(GJIC)on Photofrin-photodynamic therapy(PDT)phototoxicity in Cx40-transfected He La cells and its potential mechanisms...OBJECTIVE To explore the effect of connexin(Cx)40-formed gap junctional intercellular communication(GJIC)on Photofrin-photodynamic therapy(PDT)phototoxicity in Cx40-transfected He La cells and its potential mechanisms.METHODS He La cell line stably transfected to express Cx40 was seeded at high and low cell density,respectively,to assess in vitro photosensitivity using CCK8 assay.Western blot assay was performed to detect the expression of Cx40.The intracellular ROS and Ca^(2+) concentrations were determined using flow cytometer.4-HNE and ceramide were measured using ELISA assay.RESULTS Cx40-composed GJ formation at high density enhances the phototoxicity of PhotofrinPDT.When the Cx40 is not expressed or Cx40 channels are blocked,the phototoxicity in high-density cultures substantially reduces,indicating that the enhanced PDT phototoxicity at high density is mediated by Cx40-composed GJIC.The GJIC-mediated increase in PDT phototoxicity was associated with ROS and calcium-mediated stress signaling pathways.CONCLUSION The work uniquely presents the ability of Cx40-composed GJIC to enhance the sensitivity of malignant cells to PDT,and indicates that maintenance or increase of Cx40-formed GJIC may be a profitable strategy towards the enhancement of PDT therapeutic efficiency.展开更多
目的研究晚期糖基化终产物（advanced glycation end products，AGEs）对体外培养的人脐静脉内皮细胞缝隙连接蛋白Connexin40基因表达的影响。方法D-葡萄糖孵育牛血清白蛋白制备糖基化牛血清白蛋白作为晚期糖基化终产物。体外常规培养...目的研究晚期糖基化终产物（advanced glycation end products，AGEs）对体外培养的人脐静脉内皮细胞缝隙连接蛋白Connexin40基因表达的影响。方法D-葡萄糖孵育牛血清白蛋白制备糖基化牛血清白蛋白作为晚期糖基化终产物。体外常规培养的人脐静脉内皮细胞分为4组，分别为对照组、100mg／LAGEs干预组、200mg／LAGEs干预组和400mg／LAGEs干预组，干预24h后通过RT—PCR方法检测各组Connexin40基因mRNA的表达有无差异；通过免疫细胞荧光方法检测对照组和200mg／LAGEs干预组之间Connexin40基因蛋白的表达水平有无差异。结果不同浓度（100、200、400mg／L）的AGEs干预24h均能引起人脐静脉内皮细胞Connexin40基因mRNA的表达减少（均P〈0．01）；200mg／L的AGEs干预24h引起人脐静脉内皮细胞Con—nexin40基因的蛋白表达减少（P〈0．01）结论AGEs体外短期干预可引起人脐静脉内皮细胞Connexin40基因的mRNA和蛋白表达水平减少。展开更多
The aim of the study was to assess the distribution of connexin43 (Cx43) and connexin40 (Cx40) in human and bovine ciliary bodies. The effect of the second messengers cAMP and cyclic cGMP on Cx43 protein expression wa...The aim of the study was to assess the distribution of connexin43 (Cx43) and connexin40 (Cx40) in human and bovine ciliary bodies. The effect of the second messengers cAMP and cyclic cGMP on Cx43 protein expression was also investigated. Enucleated human eyes (remnant after corneal transplantation) and bovine eyes were used. Tissue preparations of the anterior segments of the eyes have proceeded for immunohistochemical staining with polyclonal antibodies of Cx43 and Cx40. Isolated ciliary bodies of human and bovine eyes were incubated with cAMP analog 8-Bromo-cAMP or the cGMP analog 8-Bromo-cGMP, the expression of Cx43 protein in the tissues was then assessed by Western blot assay. Both in human and bovine ciliary bodies, strong immunoreactivity of Cx43, but not Cx40, was observed predominantly in the apical cytoplasmic portions of the pigment ciliary epithelial cells and non-pigmented ciliary epithelial cells. In human ciliary body both cAMP and cGMP up-regulated Cx43 expression, while in the bovine ciliary body, cGMP increased Cx43 expression but cAMP decreased it. Cx43 is the major component of human and bovine gap junctions in the ciliary epithelium. The regulation on the Cx43 expression by cAMP and cGMP in human and bovine ciliary bodies suggests the possibly different roles of these signal messengers in the intracellular communication.展开更多
基金supported by National Natural Science Foundation of China(81402946)Initializing Fund of Xuzhou Medical University of China(D2014017 and D2014010)Natural Science Research Grant of Higher Education of Jiangsu Province of China(14KJD310002)
文摘OBJECTIVE To explore the effect of connexin(Cx)40-formed gap junctional intercellular communication(GJIC)on Photofrin-photodynamic therapy(PDT)phototoxicity in Cx40-transfected He La cells and its potential mechanisms.METHODS He La cell line stably transfected to express Cx40 was seeded at high and low cell density,respectively,to assess in vitro photosensitivity using CCK8 assay.Western blot assay was performed to detect the expression of Cx40.The intracellular ROS and Ca^(2+) concentrations were determined using flow cytometer.4-HNE and ceramide were measured using ELISA assay.RESULTS Cx40-composed GJ formation at high density enhances the phototoxicity of PhotofrinPDT.When the Cx40 is not expressed or Cx40 channels are blocked,the phototoxicity in high-density cultures substantially reduces,indicating that the enhanced PDT phototoxicity at high density is mediated by Cx40-composed GJIC.The GJIC-mediated increase in PDT phototoxicity was associated with ROS and calcium-mediated stress signaling pathways.CONCLUSION The work uniquely presents the ability of Cx40-composed GJIC to enhance the sensitivity of malignant cells to PDT,and indicates that maintenance or increase of Cx40-formed GJIC may be a profitable strategy towards the enhancement of PDT therapeutic efficiency.
文摘目的研究晚期糖基化终产物（advanced glycation end products，AGEs）对体外培养的人脐静脉内皮细胞缝隙连接蛋白Connexin40基因表达的影响。方法D-葡萄糖孵育牛血清白蛋白制备糖基化牛血清白蛋白作为晚期糖基化终产物。体外常规培养的人脐静脉内皮细胞分为4组，分别为对照组、100mg／LAGEs干预组、200mg／LAGEs干预组和400mg／LAGEs干预组，干预24h后通过RT—PCR方法检测各组Connexin40基因mRNA的表达有无差异；通过免疫细胞荧光方法检测对照组和200mg／LAGEs干预组之间Connexin40基因蛋白的表达水平有无差异。结果不同浓度（100、200、400mg／L）的AGEs干预24h均能引起人脐静脉内皮细胞Connexin40基因mRNA的表达减少（均P〈0．01）；200mg／L的AGEs干预24h引起人脐静脉内皮细胞Con—nexin40基因的蛋白表达减少（P〈0．01）结论AGEs体外短期干预可引起人脐静脉内皮细胞Connexin40基因的mRNA和蛋白表达水平减少。
文摘The aim of the study was to assess the distribution of connexin43 (Cx43) and connexin40 (Cx40) in human and bovine ciliary bodies. The effect of the second messengers cAMP and cyclic cGMP on Cx43 protein expression was also investigated. Enucleated human eyes (remnant after corneal transplantation) and bovine eyes were used. Tissue preparations of the anterior segments of the eyes have proceeded for immunohistochemical staining with polyclonal antibodies of Cx43 and Cx40. Isolated ciliary bodies of human and bovine eyes were incubated with cAMP analog 8-Bromo-cAMP or the cGMP analog 8-Bromo-cGMP, the expression of Cx43 protein in the tissues was then assessed by Western blot assay. Both in human and bovine ciliary bodies, strong immunoreactivity of Cx43, but not Cx40, was observed predominantly in the apical cytoplasmic portions of the pigment ciliary epithelial cells and non-pigmented ciliary epithelial cells. In human ciliary body both cAMP and cGMP up-regulated Cx43 expression, while in the bovine ciliary body, cGMP increased Cx43 expression but cAMP decreased it. Cx43 is the major component of human and bovine gap junctions in the ciliary epithelium. The regulation on the Cx43 expression by cAMP and cGMP in human and bovine ciliary bodies suggests the possibly different roles of these signal messengers in the intracellular communication.
