Background: Diabetes mellitus is one of the major health problems for people all over the world today. According to international diabetes federation reports, diabetes affects 382 million people worldwide. Environment...Background: Diabetes mellitus is one of the major health problems for people all over the world today. According to international diabetes federation reports, diabetes affects 382 million people worldwide. Environmental pollutants have deleterious effects on glucose metabolism and cause insulin resistance. We aimed to investigate the effects of the environmental pollutants benzo(a)pyrene, and the therapeutic potential of resveratrol. Methods: 20 μM of benzo(a)pyrene was administered after 48 h of resveratrol (80 μM) application for 24 h in INS-1 (832/13) insulinoma cells. The cells were treated with 20 μM benzo(a)pyrene for 24 hours after 48 hours initial preconditions with 10 μM resveratrol. Oxidative stress status, insulin secretion and apoptosis were analyzed by molecular techniques. Results: Though resveratrol increased the antioxidant status which was decreased by benzo(a)pyrene, interestingly, it increased the oxidative status. Resveratrol increased benzo(a)pyrene-depleted reduced glutathione levels to the control level. The mRNA expression levels of beta-cell functions associated with genes insulin-1, insulin-2 and sirtuin-1 were upregulated by resveratrol. Resveratrol treatment elevated the insulin concentration of culture medium, and the mRNA expression of forkhead box protein-1 gene. Resveratrol upregulated benzo(a)pyrene-downregulated p53 gene expression. On the other hand, benzo(a)pyrene-downregulated mRNA expression of B-cell lymphoma-2 was induced by resveratrol treatment. Conclusion: The data showed that resveratrol could reverse the oxidative alterations, functional impairments and the carcinogenetic effects of benzo(a)pyrene in pancreas beta-cells.展开更多
本文报道一例34岁女性宫颈小细胞神经内分泌癌病例,镜下见弥漫分布的小圆形细胞,胞质少,核深染,核仁不明显;免疫组织化学显示胰岛素瘤相关蛋白1(insulinoma-associated protein 1,INSM1)、CD56等特异性的标志物阳性,病理诊断为宫颈小细...本文报道一例34岁女性宫颈小细胞神经内分泌癌病例,镜下见弥漫分布的小圆形细胞,胞质少,核深染,核仁不明显;免疫组织化学显示胰岛素瘤相关蛋白1(insulinoma-associated protein 1,INSM1)、CD56等特异性的标志物阳性,病理诊断为宫颈小细胞神经内分泌癌,此报道强调了INSM1在诊断小细胞神经内分泌癌中的潜在价值。展开更多
Objective： To investigate the effects of berberine （BBR） and cinnamic acid （CA）, the main active components in Jiaotai Pill （交泰丸, JTP）, on palmitic acid （PA）-induced intracellular tdglyceride （TG） accumu...Objective： To investigate the effects of berberine （BBR） and cinnamic acid （CA）, the main active components in Jiaotai Pill （交泰丸, JTP）, on palmitic acid （PA）-induced intracellular tdglyceride （TG） accumulation in NIT-1 pancreatic 13 cells. Methods： Cells were incubated in culture medium containing PA （0.25 mmol/L） for 24 h. Then treatments with BBR （10 μmol/L）, CA （100 μmol/L） and the combination of BBR and CA （BBR＋CA） were performed respectively. Intracellular lipid accumulation was assessed by Oil Red O staining and TG content was measured by colorimetric assay. The expression of adenosine monophosphate-activated protein kinase （AMPK） protein and its downstream lipogenic and fatty acid oxidation genes, including fatty acid synthase （FAS）, acetyl-coA carboxylase （ACC）, phosphorylation acetyl-coA carboxylase （pACC）, carnitine acyl transferase 1 （CPT-1） and sterol regulating element binding protein lc （SREBP-lc） were determined by Western blot or real time polymerase chain reaction. Results： PA induced an obvious lipid accumulation and a significant increase in intracellular TG content in NIT-1 cells. PA also induced a remarkable decrease in AMPK protein expression and its downstream targets such as pACC and CPT-I. Meanwhile, AMPK downstream lipogenic genes including SREBP-lc mRNA, FAS and ACC protein expressions were increased. Treatments with BBR and BBR＋CA, superior to CA, significantly reversed the above genes changes in NIT-1 pancreatic 13 cells. However, the synergistic effect of BBR and CA on intracellular TG content was not observed in the present study. Conclusion： It can be concluded that in vitro, BBR and BBR＋CA could inhibit PA-induced lipid accumulation by decreasing lipoqenesis and increasin.cl lipid oxidation in NIT-1 pancreatic B cells.展开更多
Objective:To investigate apoptotic effects of berberine,a significant alkaloids component existing in Rhizoma coptidis,and its possible acting mechanism in insulinoma cells.Methods:Different concentrations of berberin...Objective:To investigate apoptotic effects of berberine,a significant alkaloids component existing in Rhizoma coptidis,and its possible acting mechanism in insulinoma cells.Methods:Different concentrations of berberine were used to treat mouse insulinoma(MIN6)cells for various period of time.The viability and apoptosis of the cells were analyzed using methylthiazolyldiphenvl-tetrazolium bromide assay,flow cytometry and enzyme-linked immuno sorbent assay.Changes in the relating pro-and anti-apoptosis proteins were detected by western-blotting.Results:The half-maximal inhibitory concentration(IC50)of berberine was 5.7μmol/L on MIN6 cells viability for 16 h.Berberine caused a 20%reduction(P<0.05)in cell number after only 4-h incubation;which reached 50%after 24 h(P<0.01).Berberine treatment for 16 h significantly increased the level of DNA fragmentation.The flow cytometry showed the apoptotic rate increased 2.9-and 4.6-fold after treating with berberine(5μmol/L)for 8 and 16 h,while 3-and 8.7-fold after 10μmol/L treatment for 8 and 16 h(P<0.01).Berberine treatment dramatically elevated the expression ratio of Bax to Bcl-2.Meanwhile,berberine notably increased the apoptosis-inducing factors and cytochrome C transforming from the mitochondria to the cytoplasm.Apoptotic protease-activating factor 1(Apaf-1)was subsequently activated after cytochrome C release.Furthermore,caspase-3 and poly adenosine diphosphate-ribose polymerase were also activated to trigger apoptosis cascade.Conclusion:High concentration(5 and 10μmol/L)of berberine could induce the apoptosis of MIN6 cells through cytochrome C/Apaf-1/caspase-3 and apoptosis inducing factor(AIF)pathway.展开更多
Background Increased levels of plasma lipopolysaccharide （LPS） have been found in obesity and diabetes patients. This study was to investigate the effect of LPS on pancreatic beta-cell viability and the involvement ...Background Increased levels of plasma lipopolysaccharide （LPS） have been found in obesity and diabetes patients. This study was to investigate the effect of LPS on pancreatic beta-cell viability and the involvement of caspase 3 in NIT-1 cell line. Methods Mouse insulinoma NIT-1 cells were treated with LPS for the indicated time and dose. Cell viability was measured by cell counting kit-8 reagent. Toll-like receptor 4 （TLR4）, caspase 3 and cleaved caspase 3 were detected by Western blotting. Insulin was determined by radioimmunoassay （RIA）. Results LPS promoted NIT-1 cell proliferation at 1 μg/ml, peaked at 72 hours of incubation. A reduction in cleavage of caspase 3 was observed upon LPS treatment. Bay11-7082, a specific inhibitor of nuclear factor （NF）-κB, blunted LPS-induced inhibition of caspase 3 cleavage. Reduction in chronic insulin secretion was observed after treatment with LPS at 1 μg/ml for 48 and 72 hours, not for 24 hours. TLR4 protein was upregulated when NIT-1 cells were treated with LPS at 1 μg/ml for 24 hours. Conclusions LPS promotes early NIT-1 cell proliferation in association with NF-KB-mediated inhibition of caspase 3 cleavage. LPS exerts a time-dependent inhibitory effect on chronic insulin secretion from NIT-1 cells.展开更多
目的探究慢病毒介导RNAi沉默SGMS2基因的单克隆细胞系构建中最佳感染复数(multiplicity of infection,MOI)及BSD基因筛选抗生素(blasticidin)浓度。方法荧光标记小鼠SGMS2干扰阴性对照慢病毒并按照MOI值0、10、30、60、120(TU number/ce...目的探究慢病毒介导RNAi沉默SGMS2基因的单克隆细胞系构建中最佳感染复数(multiplicity of infection,MOI)及BSD基因筛选抗生素(blasticidin)浓度。方法荧光标记小鼠SGMS2干扰阴性对照慢病毒并按照MOI值0、10、30、60、120(TU number/cell)分别侵染INS-1空白细胞,培养72 h后使用荧光显微镜拍照并计算细胞的荧光比率(%)及死亡率(%),以确定最佳MOI值。小鼠胰岛素瘤INS-1空白细胞中加入0、1、2、3μg/m L blasticidin,第7天时采用MTT法检测细胞的死亡率,以确定细胞抗生素敏感浓度。使用SGMS2干扰阴性对照慢病毒及SGMS2干扰慢病毒(病毒滴度:1×108TU/m L)按照最佳MOI值侵染细胞,并用blasticidin敏感浓度进行阳性细胞筛选,获得混合系细胞。当细胞的荧光率达90%时,进行单克隆稳转细胞系的构建。结果最佳MOI值为60,此时细胞的荧光率达100%,但细胞的死亡率<0.5%,细胞保持原有的形态。当blasticidin敏感浓度为2μg/m L,此时空白细胞失去原有的贴壁性,全部死亡。INS-1-SEMS2细胞第2次检测的Ct值28.21大于第1次检测的Ct值27.58,且siRNA的干扰效率为77.78%,siRNA成功表达,混合稳转细胞系构建成功。成功构建小鼠胰岛素瘤INS-1-SEMS2单克隆细胞系。结论慢病毒介导RNAi沉默基因SGMS2的单克隆细胞系构建成功。展开更多
Purpose Dinutuximab beta is a monoclonal antibody used only in glioblastoma.Streptozotocin is an agent that is particularly toxic to pancreatic beta cells.Dinutuximab beta causes cytotoxicity through natural killer ce...Purpose Dinutuximab beta is a monoclonal antibody used only in glioblastoma.Streptozotocin is an agent that is particularly toxic to pancreatic beta cells.Dinutuximab beta causes cytotoxicity through natural killer cells and neutrophils and shows effects.In this study,cytotoxicity was induced by streptozotocin without natural killer cells and neutrophils.Gaining the ability to show the effect of Dinutuximab beta without a natural killer was the first aim of this study.This will be especially important in cases where the immune system is deficient,such as cancer.The second aim of the study was to investigate the effects of Dinutuximab beta on cell viability and cell death in insulinoma under the conditions created.Methods The effect of Dinutuximab beta in the presence of natural killer cells in vivo was created by the application of Streptozotocin to Beta-cell tumors of the pancreas in vitro.The cell viability was determined with WST-1 assay.Reactive oxygen species were measured by using dichlorofluorescein diacetate as a spectrophotometer.The cells were marked with DAPI to indicate apoptotic markers(nuclear condensation and fragmentation)with the confocal microscope.GLUT2(Glucose transporter 2),IR(Insulin receptor),INS1,and INS2 expression levels were analyzed with q-RT-PCR.Results The cell cytotoxicity was induced by Streptozotocin.The cells proliferated with the administration of Dinutuximab beta alone.The result of Dinutuximab beta administered following Streptozotocin administration resulted in more cell death,increased ROS levels,GLUT2,Ins1,and Ins2 mRNA expression levels,and decreased IR mRNA expression levels.Furthermore,the cells predominantly died via apoptosis showing cytoplasmic condensation and DNA fragmentation.Conclusions The lethal effect of Dinutuximab beta without a natural killer was provided by Streptozotocin in Beta cell tumors of the pancreas.展开更多
基金supported by the Scientific Research Projects Unit of Afyon Kocatepe University(Project No:10.TIP.18/MSc Thesis in Medical Biochemistry)NATO-SPS Program(Project No:MYP-G5266).
文摘Background: Diabetes mellitus is one of the major health problems for people all over the world today. According to international diabetes federation reports, diabetes affects 382 million people worldwide. Environmental pollutants have deleterious effects on glucose metabolism and cause insulin resistance. We aimed to investigate the effects of the environmental pollutants benzo(a)pyrene, and the therapeutic potential of resveratrol. Methods: 20 μM of benzo(a)pyrene was administered after 48 h of resveratrol (80 μM) application for 24 h in INS-1 (832/13) insulinoma cells. The cells were treated with 20 μM benzo(a)pyrene for 24 hours after 48 hours initial preconditions with 10 μM resveratrol. Oxidative stress status, insulin secretion and apoptosis were analyzed by molecular techniques. Results: Though resveratrol increased the antioxidant status which was decreased by benzo(a)pyrene, interestingly, it increased the oxidative status. Resveratrol increased benzo(a)pyrene-depleted reduced glutathione levels to the control level. The mRNA expression levels of beta-cell functions associated with genes insulin-1, insulin-2 and sirtuin-1 were upregulated by resveratrol. Resveratrol treatment elevated the insulin concentration of culture medium, and the mRNA expression of forkhead box protein-1 gene. Resveratrol upregulated benzo(a)pyrene-downregulated p53 gene expression. On the other hand, benzo(a)pyrene-downregulated mRNA expression of B-cell lymphoma-2 was induced by resveratrol treatment. Conclusion: The data showed that resveratrol could reverse the oxidative alterations, functional impairments and the carcinogenetic effects of benzo(a)pyrene in pancreas beta-cells.
文摘本文报道一例34岁女性宫颈小细胞神经内分泌癌病例,镜下见弥漫分布的小圆形细胞,胞质少,核深染,核仁不明显;免疫组织化学显示胰岛素瘤相关蛋白1(insulinoma-associated protein 1,INSM1)、CD56等特异性的标志物阳性,病理诊断为宫颈小细胞神经内分泌癌,此报道强调了INSM1在诊断小细胞神经内分泌癌中的潜在价值。
基金Supported by the National Natural Science Foundation of China(No.81373871 and 81273683)
文摘Objective： To investigate the effects of berberine （BBR） and cinnamic acid （CA）, the main active components in Jiaotai Pill （交泰丸, JTP）, on palmitic acid （PA）-induced intracellular tdglyceride （TG） accumulation in NIT-1 pancreatic 13 cells. Methods： Cells were incubated in culture medium containing PA （0.25 mmol/L） for 24 h. Then treatments with BBR （10 μmol/L）, CA （100 μmol/L） and the combination of BBR and CA （BBR＋CA） were performed respectively. Intracellular lipid accumulation was assessed by Oil Red O staining and TG content was measured by colorimetric assay. The expression of adenosine monophosphate-activated protein kinase （AMPK） protein and its downstream lipogenic and fatty acid oxidation genes, including fatty acid synthase （FAS）, acetyl-coA carboxylase （ACC）, phosphorylation acetyl-coA carboxylase （pACC）, carnitine acyl transferase 1 （CPT-1） and sterol regulating element binding protein lc （SREBP-lc） were determined by Western blot or real time polymerase chain reaction. Results： PA induced an obvious lipid accumulation and a significant increase in intracellular TG content in NIT-1 cells. PA also induced a remarkable decrease in AMPK protein expression and its downstream targets such as pACC and CPT-I. Meanwhile, AMPK downstream lipogenic genes including SREBP-lc mRNA, FAS and ACC protein expressions were increased. Treatments with BBR and BBR＋CA, superior to CA, significantly reversed the above genes changes in NIT-1 pancreatic 13 cells. However, the synergistic effect of BBR and CA on intracellular TG content was not observed in the present study. Conclusion： It can be concluded that in vitro, BBR and BBR＋CA could inhibit PA-induced lipid accumulation by decreasing lipoqenesis and increasin.cl lipid oxidation in NIT-1 pancreatic B cells.
基金Supported by the National Natural Science Foundation of China(No.NSFC81274041 and No.NSFC81273995)the International Cooperation Projects of Ministry of Education(No.2011DFA30920)the Key Drug Development Program of Ministry of Science an Technology(No.20122X09103201-005)
文摘Objective:To investigate apoptotic effects of berberine,a significant alkaloids component existing in Rhizoma coptidis,and its possible acting mechanism in insulinoma cells.Methods:Different concentrations of berberine were used to treat mouse insulinoma(MIN6)cells for various period of time.The viability and apoptosis of the cells were analyzed using methylthiazolyldiphenvl-tetrazolium bromide assay,flow cytometry and enzyme-linked immuno sorbent assay.Changes in the relating pro-and anti-apoptosis proteins were detected by western-blotting.Results:The half-maximal inhibitory concentration(IC50)of berberine was 5.7μmol/L on MIN6 cells viability for 16 h.Berberine caused a 20%reduction(P<0.05)in cell number after only 4-h incubation;which reached 50%after 24 h(P<0.01).Berberine treatment for 16 h significantly increased the level of DNA fragmentation.The flow cytometry showed the apoptotic rate increased 2.9-and 4.6-fold after treating with berberine(5μmol/L)for 8 and 16 h,while 3-and 8.7-fold after 10μmol/L treatment for 8 and 16 h(P<0.01).Berberine treatment dramatically elevated the expression ratio of Bax to Bcl-2.Meanwhile,berberine notably increased the apoptosis-inducing factors and cytochrome C transforming from the mitochondria to the cytoplasm.Apoptotic protease-activating factor 1(Apaf-1)was subsequently activated after cytochrome C release.Furthermore,caspase-3 and poly adenosine diphosphate-ribose polymerase were also activated to trigger apoptosis cascade.Conclusion:High concentration(5 and 10μmol/L)of berberine could induce the apoptosis of MIN6 cells through cytochrome C/Apaf-1/caspase-3 and apoptosis inducing factor(AIF)pathway.
基金This work was supported by grants from the National Natural Science Foundation of China （No. 30671974 and 81070598）. The authors declare no conflict of interest.
文摘Background Increased levels of plasma lipopolysaccharide （LPS） have been found in obesity and diabetes patients. This study was to investigate the effect of LPS on pancreatic beta-cell viability and the involvement of caspase 3 in NIT-1 cell line. Methods Mouse insulinoma NIT-1 cells were treated with LPS for the indicated time and dose. Cell viability was measured by cell counting kit-8 reagent. Toll-like receptor 4 （TLR4）, caspase 3 and cleaved caspase 3 were detected by Western blotting. Insulin was determined by radioimmunoassay （RIA）. Results LPS promoted NIT-1 cell proliferation at 1 μg/ml, peaked at 72 hours of incubation. A reduction in cleavage of caspase 3 was observed upon LPS treatment. Bay11-7082, a specific inhibitor of nuclear factor （NF）-κB, blunted LPS-induced inhibition of caspase 3 cleavage. Reduction in chronic insulin secretion was observed after treatment with LPS at 1 μg/ml for 48 and 72 hours, not for 24 hours. TLR4 protein was upregulated when NIT-1 cells were treated with LPS at 1 μg/ml for 24 hours. Conclusions LPS promotes early NIT-1 cell proliferation in association with NF-KB-mediated inhibition of caspase 3 cleavage. LPS exerts a time-dependent inhibitory effect on chronic insulin secretion from NIT-1 cells.
文摘目的探究慢病毒介导RNAi沉默SGMS2基因的单克隆细胞系构建中最佳感染复数(multiplicity of infection,MOI)及BSD基因筛选抗生素(blasticidin)浓度。方法荧光标记小鼠SGMS2干扰阴性对照慢病毒并按照MOI值0、10、30、60、120(TU number/cell)分别侵染INS-1空白细胞,培养72 h后使用荧光显微镜拍照并计算细胞的荧光比率(%)及死亡率(%),以确定最佳MOI值。小鼠胰岛素瘤INS-1空白细胞中加入0、1、2、3μg/m L blasticidin,第7天时采用MTT法检测细胞的死亡率,以确定细胞抗生素敏感浓度。使用SGMS2干扰阴性对照慢病毒及SGMS2干扰慢病毒(病毒滴度:1×108TU/m L)按照最佳MOI值侵染细胞,并用blasticidin敏感浓度进行阳性细胞筛选,获得混合系细胞。当细胞的荧光率达90%时,进行单克隆稳转细胞系的构建。结果最佳MOI值为60,此时细胞的荧光率达100%,但细胞的死亡率<0.5%,细胞保持原有的形态。当blasticidin敏感浓度为2μg/m L,此时空白细胞失去原有的贴壁性,全部死亡。INS-1-SEMS2细胞第2次检测的Ct值28.21大于第1次检测的Ct值27.58,且siRNA的干扰效率为77.78%,siRNA成功表达,混合稳转细胞系构建成功。成功构建小鼠胰岛素瘤INS-1-SEMS2单克隆细胞系。结论慢病毒介导RNAi沉默基因SGMS2的单克隆细胞系构建成功。
基金supported by the Scientific Research Project Coordination Unit of Istanbul University.Project No:34960.
文摘Purpose Dinutuximab beta is a monoclonal antibody used only in glioblastoma.Streptozotocin is an agent that is particularly toxic to pancreatic beta cells.Dinutuximab beta causes cytotoxicity through natural killer cells and neutrophils and shows effects.In this study,cytotoxicity was induced by streptozotocin without natural killer cells and neutrophils.Gaining the ability to show the effect of Dinutuximab beta without a natural killer was the first aim of this study.This will be especially important in cases where the immune system is deficient,such as cancer.The second aim of the study was to investigate the effects of Dinutuximab beta on cell viability and cell death in insulinoma under the conditions created.Methods The effect of Dinutuximab beta in the presence of natural killer cells in vivo was created by the application of Streptozotocin to Beta-cell tumors of the pancreas in vitro.The cell viability was determined with WST-1 assay.Reactive oxygen species were measured by using dichlorofluorescein diacetate as a spectrophotometer.The cells were marked with DAPI to indicate apoptotic markers(nuclear condensation and fragmentation)with the confocal microscope.GLUT2(Glucose transporter 2),IR(Insulin receptor),INS1,and INS2 expression levels were analyzed with q-RT-PCR.Results The cell cytotoxicity was induced by Streptozotocin.The cells proliferated with the administration of Dinutuximab beta alone.The result of Dinutuximab beta administered following Streptozotocin administration resulted in more cell death,increased ROS levels,GLUT2,Ins1,and Ins2 mRNA expression levels,and decreased IR mRNA expression levels.Furthermore,the cells predominantly died via apoptosis showing cytoplasmic condensation and DNA fragmentation.Conclusions The lethal effect of Dinutuximab beta without a natural killer was provided by Streptozotocin in Beta cell tumors of the pancreas.
