We explored the appropriate inducing conditions needed to facilitate the differentiation of bone marrow mesenchymal stem cells（BMSCs） into retinal ganglion cells（RGCs）.Math5,a pro-neural basic helixloop-helix（bHL...We explored the appropriate inducing conditions needed to facilitate the differentiation of bone marrow mesenchymal stem cells（BMSCs） into retinal ganglion cells（RGCs）.Math5,a pro-neural basic helixloop-helix（bHLH） gene,was constructed in an adenoviral vector and then infected into the 3rd passage BMSCs.An inverted fluorescence microscope was used to observe the morphological changes of the infected cells.The expressions of Math5,the neuromarkers neuron-specific enolase（NSE）,neurofilament（NF）,Thy1.1,and the RGC-related genes GAP-43 and Brn3b were examined by Western blot and reverse transcription-polymerase chain reaction（RT-PCR）.The results show that cells infected with Math5 adenoviral vector were able to stably express Math5 and presented with a typical morphology of RGCs.Moreover,these cells expressed NSE,NF,Thy1.1,and GAP-43.Under the synergistic induction conditions of retinal conditioned differentiation medium in combination with epidermal growth factor（EGF） and basic fibroblast growth factor（BFGF）,BMSCs infected with Math5 adenoviral vector had a more typical morphology of RGCs,with a greater number of longer axons that connected with each other and formed a net.In addition,the number of NF positive cells was higher,the expression of Brn3b was detected,and the expressions of NSE,NF,and GAP-43 were significantly up-regulated compared to those of them in the control.These results indicate that BMSCs infected with Math5 are able to differentiate into retinal ganglion-like cells.Moreover,Math5 is a stronger activator of the downstream gene Brn3b than the cytokine,which suggests that it is possible to regulate the survival and axon path determination of these differentiated cells.展开更多
A rabbit model of traumatic optic nerve injury, established by occlusion of the optic nerve using a vascular clamp, was used to investigate the effects of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid recep...A rabbit model of traumatic optic nerve injury, established by occlusion of the optic nerve using a vascular clamp, was used to investigate the effects of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist GYKI 52466 on apoptosis of retinal ganglion cells following nerve injury. Hematoxylin-eosin staining and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that retinal ganglion cells gradually decreased with increasing time of optic nerve injury, while GYKI 52466 could inhibit this process. The results demonstrate that following acute optic nerve injury, apoptosis of retinal ganglion cells is a programmed process, which can be inhibited by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist.展开更多
Glaucoma is characterized by the progressive loss of retinal ganglion cells (RGCs),although the pathogenic mechanism remains largely unknown.To study the mechanism and assess RGC degradation,mouse models are often use...Glaucoma is characterized by the progressive loss of retinal ganglion cells (RGCs),although the pathogenic mechanism remains largely unknown.To study the mechanism and assess RGC degradation,mouse models are often used to simulate human glaucoma and specific markers are used to label and quantify RGCs.However,manually counting RGCs is time-consuming and prone to distortion due to subjective bias.Furthermore,semi-automated counting methods can produce significant differences due to different parameters,thereby failing objective evaluation.Here,to improve counting accuracy and efficiency,we developed an automated algorithm based on the improved YOLOv5 model,which uses five channels instead of one,with a squeeze-and-excitation block added.The complete number of RGCs in an intact mouse retina was obtained by dividing the retina into small overlapping areas and counting,and then merging the divided areas using a non-maximum suppression algorithm.The automated quantification results showed very strong correlation (mean Pearson correlation coefficient of 0.993) with manual counting.Importantly,the model achieved an average precision of 0.981.Furthermore,the graphics processing unit (GPU) calculation time for each retina was less than 1 min.The developed software has been uploaded online as a free and convenient tool for studies using mouse models of glaucoma,which should help elucidate disease pathogenesis and potential therapeutics.展开更多
Pituitary adenylate cyclase-activating polypeptide（PACAP） is an endogenous peptide with neuroprotective effects on retinal neurons, but the precise mechanism underlying these effects remains unknown. Considering the...Pituitary adenylate cyclase-activating polypeptide（PACAP） is an endogenous peptide with neuroprotective effects on retinal neurons, but the precise mechanism underlying these effects remains unknown. Considering the abundance of mitochondria in retinal ganglion cells（RGCs）, we postulate that the protective effect of PACAP is associated with the regulation of mitochondrial function. RGC-5 cells were subjected to serum deprivation for 48 hours to induce apoptosis in the presence or absence of 100 nM PACAP. As revealed with the Cell Counting Kit-8 assay, PACAP at different concentrations significantly increased the viability of RGC-5 cells. PACAP also inhibited the excessive generation of reactive oxygen species in RGC-5 cells subjected to serum deprivation. We also showed by flow cytometry that PACAP inhibited serum deprivation-induced apoptosis in RGC-5 cells. The proportions of apoptotic cells and cells with mitochondria depolarization were significantly decreased with PACAP treatment. Western blot assays demonstrated that PACAP increased the levels of Bcl-2 and inhibited the compensatory increase of PAC1. Together, these data indicate protective effects of PACAP against serum deprivation-induced apoptosis in RGCs, and that the mechanism of this action is associated with maintaining mitochondrial function.展开更多
AIM： To investigate the potential of pigment epitheliumderived factor（PEDF） to protect the immortalized rat retinal ganglion cells-5（RGC-5） exposed to Co Cl2-induced chemical hypoxia. METHODS： After being differ...AIM： To investigate the potential of pigment epitheliumderived factor（PEDF） to protect the immortalized rat retinal ganglion cells-5（RGC-5） exposed to Co Cl2-induced chemical hypoxia. METHODS： After being differentiated with staurosporine（SS）, RGC-5 cells were cultured in four conditions： control group cells cultured in Dulbecco ＇s modified eagle medium（DMEM） supplemented with 10% fetal bovine serum, 100 μmol/m L streptomycin and penicillin（named as normal conditions）; hypoxia group cells cultured in DMEM containing 300 μmol/m L Co Cl2; cells in the group protected by PEDF were first pretreated with 100 ng/m L PEDF for 2h and then cultured in the same condition as hypoxia group cells; and PEDF group cells that were cultured in the presence of 100 ng/m L PEDF under normal conditions. The cell viability was assessed by MTT assay, the percentage of apoptotic cells was quantified using Annexin V-FITC apoptosis kit, and intra-cellar reactive oxygen species（ROS） was measured by dichloro-dihydro-fluorescein diacetate（DCFH-DA） probe. The mitochondria-mediated apoptosis was also examined to further study the underlying mechanism of the protective effect of PEDF. The opening of mitochondrial permeability transition pores（m PTPs） and membrane potential（Δψm） were tested as cellular adenosine triphosphate（ATP） level and glutathione（GSH）. Also, the expression and distribution of Cyt C and apoptosis inducing factor（AIF） were observed.RESULTS： SS induced differentiation of RGC-5 cells resulting in elongation of their neurites and establishing contacts between outgrowths. Exposure to 300 μmol/m L Co Cl2 triggered death of 30% of the total cells in cultures within 24 h. At the same time, pretreatment with 100 ng/m L PEDF significantly suppressed the cell death induced by hypoxia（P〈0.05）. The apoptosis induced by treatment of Co Cl2 was that induced cell death accompanied with increasing intracellar ROS and decreasing GSH and ATP level. PEDF pretreatment suppressed these effects（P〈0.05）. Additionally, PEDF treatment inhibited the opening of m PTPs and suppressed decreasing of Δψm in RGC-5 cells, resulting in blocking of the mitochondrial apoptotic pathway.CONCLUSION： Pretreatment of RGC-5 cells with 100 ng/m L PEDF significantly decreases the extent of apoptosis. PEDF inhibits the opening of m PTPs and suppresses decreasing of Δψm. Moreover, PEDF also reduces ROS production and inhibits cellular ATP level＇s reduction. Cyt C and AIF activation in PEDF-pretreated cultures are also reduced. These results demonstrate the potential for PEDF to protect RGCs against hypoxic damage in vitro by preventing mitochondrial dysfunction.展开更多
Objective To observe the change of the neuropeptide pro-protein processing system in the ischemic retina ganglion cell-5(RGC-5) cells,pro-protein convertase-2(PC2),carboxypeptidase-E(CPE) and preproneuropeptide Y(prep...Objective To observe the change of the neuropeptide pro-protein processing system in the ischemic retina ganglion cell-5(RGC-5) cells,pro-protein convertase-2(PC2),carboxypeptidase-E(CPE) and preproneuropeptide Y(preproNPY) protein levels in the ischemic RGC-5 cells and conditioned medium were analyzed. Methods The RGC-5 cell was differentiated in 0.1 μmol/L staurosporine for 24 h and then stressed by different doses of oxygen and glucose deprivation(OGD). The acute or chronic OGD-induced cell death rates w...展开更多
基金Supported by the Postdoctoral Science Foundation of China(No.20110490731)
文摘We explored the appropriate inducing conditions needed to facilitate the differentiation of bone marrow mesenchymal stem cells（BMSCs） into retinal ganglion cells（RGCs）.Math5,a pro-neural basic helixloop-helix（bHLH） gene,was constructed in an adenoviral vector and then infected into the 3rd passage BMSCs.An inverted fluorescence microscope was used to observe the morphological changes of the infected cells.The expressions of Math5,the neuromarkers neuron-specific enolase（NSE）,neurofilament（NF）,Thy1.1,and the RGC-related genes GAP-43 and Brn3b were examined by Western blot and reverse transcription-polymerase chain reaction（RT-PCR）.The results show that cells infected with Math5 adenoviral vector were able to stably express Math5 and presented with a typical morphology of RGCs.Moreover,these cells expressed NSE,NF,Thy1.1,and GAP-43.Under the synergistic induction conditions of retinal conditioned differentiation medium in combination with epidermal growth factor（EGF） and basic fibroblast growth factor（BFGF）,BMSCs infected with Math5 adenoviral vector had a more typical morphology of RGCs,with a greater number of longer axons that connected with each other and formed a net.In addition,the number of NF positive cells was higher,the expression of Brn3b was detected,and the expressions of NSE,NF,and GAP-43 were significantly up-regulated compared to those of them in the control.These results indicate that BMSCs infected with Math5 are able to differentiate into retinal ganglion-like cells.Moreover,Math5 is a stronger activator of the downstream gene Brn3b than the cytokine,which suggests that it is possible to regulate the survival and axon path determination of these differentiated cells.
基金The Foundation of Xinjiang Uygur Autonomous Region in China, No. 200821137the National Natural Science Foundation of China, No. 81160153
文摘A rabbit model of traumatic optic nerve injury, established by occlusion of the optic nerve using a vascular clamp, was used to investigate the effects of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist GYKI 52466 on apoptosis of retinal ganglion cells following nerve injury. Hematoxylin-eosin staining and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that retinal ganglion cells gradually decreased with increasing time of optic nerve injury, while GYKI 52466 could inhibit this process. The results demonstrate that following acute optic nerve injury, apoptosis of retinal ganglion cells is a programmed process, which can be inhibited by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist.
基金supported by the National Natural Science Foundation of China (61405028 to J.Z.,81770935 to H.B.Z.)Fundamental Research Funds for the Central Universities (University of Electronic Science and Technology of China)(ZYGX2019J053 to J.Z.)Department of Science and Technology of Sichuan Province,China (2020YJ0445 to H.B.Z.)。
文摘Glaucoma is characterized by the progressive loss of retinal ganglion cells (RGCs),although the pathogenic mechanism remains largely unknown.To study the mechanism and assess RGC degradation,mouse models are often used to simulate human glaucoma and specific markers are used to label and quantify RGCs.However,manually counting RGCs is time-consuming and prone to distortion due to subjective bias.Furthermore,semi-automated counting methods can produce significant differences due to different parameters,thereby failing objective evaluation.Here,to improve counting accuracy and efficiency,we developed an automated algorithm based on the improved YOLOv5 model,which uses five channels instead of one,with a squeeze-and-excitation block added.The complete number of RGCs in an intact mouse retina was obtained by dividing the retina into small overlapping areas and counting,and then merging the divided areas using a non-maximum suppression algorithm.The automated quantification results showed very strong correlation (mean Pearson correlation coefficient of 0.993) with manual counting.Importantly,the model achieved an average precision of 0.981.Furthermore,the graphics processing unit (GPU) calculation time for each retina was less than 1 min.The developed software has been uploaded online as a free and convenient tool for studies using mouse models of glaucoma,which should help elucidate disease pathogenesis and potential therapeutics.
基金supported by grants from the Medical Scientific Research Foundation of Guangdong Province of China,No.A2016271the Natural Science Foundation of Guangdong Province of China,No.2016A030313208the Science and Technology Planning Project of Guangdong Province of China,No.2014A020212393
文摘Pituitary adenylate cyclase-activating polypeptide（PACAP） is an endogenous peptide with neuroprotective effects on retinal neurons, but the precise mechanism underlying these effects remains unknown. Considering the abundance of mitochondria in retinal ganglion cells（RGCs）, we postulate that the protective effect of PACAP is associated with the regulation of mitochondrial function. RGC-5 cells were subjected to serum deprivation for 48 hours to induce apoptosis in the presence or absence of 100 nM PACAP. As revealed with the Cell Counting Kit-8 assay, PACAP at different concentrations significantly increased the viability of RGC-5 cells. PACAP also inhibited the excessive generation of reactive oxygen species in RGC-5 cells subjected to serum deprivation. We also showed by flow cytometry that PACAP inhibited serum deprivation-induced apoptosis in RGC-5 cells. The proportions of apoptotic cells and cells with mitochondria depolarization were significantly decreased with PACAP treatment. Western blot assays demonstrated that PACAP increased the levels of Bcl-2 and inhibited the compensatory increase of PAC1. Together, these data indicate protective effects of PACAP against serum deprivation-induced apoptosis in RGCs, and that the mechanism of this action is associated with maintaining mitochondrial function.
基金Supported by National Natural Science Foundation of China(No.81100665)
文摘AIM： To investigate the potential of pigment epitheliumderived factor（PEDF） to protect the immortalized rat retinal ganglion cells-5（RGC-5） exposed to Co Cl2-induced chemical hypoxia. METHODS： After being differentiated with staurosporine（SS）, RGC-5 cells were cultured in four conditions： control group cells cultured in Dulbecco ＇s modified eagle medium（DMEM） supplemented with 10% fetal bovine serum, 100 μmol/m L streptomycin and penicillin（named as normal conditions）; hypoxia group cells cultured in DMEM containing 300 μmol/m L Co Cl2; cells in the group protected by PEDF were first pretreated with 100 ng/m L PEDF for 2h and then cultured in the same condition as hypoxia group cells; and PEDF group cells that were cultured in the presence of 100 ng/m L PEDF under normal conditions. The cell viability was assessed by MTT assay, the percentage of apoptotic cells was quantified using Annexin V-FITC apoptosis kit, and intra-cellar reactive oxygen species（ROS） was measured by dichloro-dihydro-fluorescein diacetate（DCFH-DA） probe. The mitochondria-mediated apoptosis was also examined to further study the underlying mechanism of the protective effect of PEDF. The opening of mitochondrial permeability transition pores（m PTPs） and membrane potential（Δψm） were tested as cellular adenosine triphosphate（ATP） level and glutathione（GSH）. Also, the expression and distribution of Cyt C and apoptosis inducing factor（AIF） were observed.RESULTS： SS induced differentiation of RGC-5 cells resulting in elongation of their neurites and establishing contacts between outgrowths. Exposure to 300 μmol/m L Co Cl2 triggered death of 30% of the total cells in cultures within 24 h. At the same time, pretreatment with 100 ng/m L PEDF significantly suppressed the cell death induced by hypoxia（P〈0.05）. The apoptosis induced by treatment of Co Cl2 was that induced cell death accompanied with increasing intracellar ROS and decreasing GSH and ATP level. PEDF pretreatment suppressed these effects（P〈0.05）. Additionally, PEDF treatment inhibited the opening of m PTPs and suppressed decreasing of Δψm in RGC-5 cells, resulting in blocking of the mitochondrial apoptotic pathway.CONCLUSION： Pretreatment of RGC-5 cells with 100 ng/m L PEDF significantly decreases the extent of apoptosis. PEDF inhibits the opening of m PTPs and suppresses decreasing of Δψm. Moreover, PEDF also reduces ROS production and inhibits cellular ATP level＇s reduction. Cyt C and AIF activation in PEDF-pretreated cultures are also reduced. These results demonstrate the potential for PEDF to protect RGCs against hypoxic damage in vitro by preventing mitochondrial dysfunction.
文摘目的研究核酶PARP-1在光诱导的视网膜神经节细胞(RGCs)凋亡中的作用。方法应用1000 Lux光诱导视网膜神经节细胞-5(RGC-5)光损伤模型;利用MTT、APOPercentageTM实验及原位TUNEL确定光诱导RGC-5细胞死亡的模式;通过半定量RT-PCR和Western blot方法评估光对RGC-5细胞中核酶PARP-1表达的影响;探讨PARP-1抑制剂尼克酰胺和NU1025对光损伤中RGC-5细胞的保护作用。结果1000 Lux光以时间依赖的方式降低体外培养的RGC-5细胞活性,光暴露4d明显诱导RGC-5细胞凋亡。RGC-5细胞光损伤导致核酶PARP-1转录和表达的上调(F=224.59,P<0.01,n=12,ANOVA,Dunnett t test),PARP-1抑制剂尼克酰胺和NU1025显著提高细胞活性(F=312.87,P<0.01,n=12,ANOVA,Dunnett t test)。结论1000 Lux光可以诱导体外培养的RGC-5细胞凋亡,核酶PARP-1在该细胞凋亡分子机制中起重要作用。
基金supported by Guangdong Pharmaceutical University Grant (No. 2005SMK22) and Key-Teacher Training Grant.
文摘Objective To observe the change of the neuropeptide pro-protein processing system in the ischemic retina ganglion cell-5(RGC-5) cells,pro-protein convertase-2(PC2),carboxypeptidase-E(CPE) and preproneuropeptide Y(preproNPY) protein levels in the ischemic RGC-5 cells and conditioned medium were analyzed. Methods The RGC-5 cell was differentiated in 0.1 μmol/L staurosporine for 24 h and then stressed by different doses of oxygen and glucose deprivation(OGD). The acute or chronic OGD-induced cell death rates w...
