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
Objective To evaluate the effects of acute glucose level changes on expression of prepro-orexin, orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R) mRNA in rat hypothalamus tissue and pancreatic islets cells. Metho...Objective To evaluate the effects of acute glucose level changes on expression of prepro-orexin, orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R) mRNA in rat hypothalamus tissue and pancreatic islets cells. Methods Thirty adult male Wistar rats were randomly divided into three equal groups (n = 10). The acute hypoglycemia rat model was induced by a single subcutaneous injection of insulin. Twenty acute hypoglycemia rats were divided into group B and group C. Group B was allowed to eat freely, while group C was food-deprived. Control rats were injected the same volume of saline. The effect of glucose levels (2.8 mmol/L and 8.3 mmol/L) on pancreatic islet cell orexin system was detected in pancreas islet cell cultured in vitro. The expression of prepro-orexin and OXR mRNA was examined in rat hypothalamus tissue and pancreatic islets cell cultured in vitro using reverse transcription-polymerase chain reaction (RT-PCR). Results Expression of orexin mRNA increased about 150% for the food-deprived hypoglycemia rats in comparison with control group (P < 0.01), whereas expression of OX1R mRNA decreased up to 30% (P < 0.01). However, expression of OX2R mRNA was unchanged in comparison with control group. In vitro, after incubation with 2.8 mmol/L glucose for 6 hours, the expression of prepro-orexin mRNA increased 2 times in rat pancreas islet cells in comparison with 8.3 mmol/L glucose group (P < 0.01). But the expression of OX1R mRNA was not sensitive to acute glucose fluctuation.Conclusions Orexin in rat hypothalamus is stimulated by decline in blood glucose and inhibited by signals related to feeding. Moreover, glucose plays a role in modulating the gene expression of prepro-orexin in rat pancreatic islet cells.展开更多
Regenerative medicine, including cell-replacement strategies, may have an important role in the treatment of type 1 diabetes which is associated with decreased islet cell mass. To date, significant progress has been m...Regenerative medicine, including cell-replacement strategies, may have an important role in the treatment of type 1 diabetes which is associated with decreased islet cell mass. To date, significant progress has been made in generating insulin-secreting 13 cells from pluripotent mouse embryonic stem cells （ESCs）.The aim of this study is to explore the potential of regulating the differentiation of ESCs into pancreatic endocrine cells capable of synthesizing the pancreatic hormones including insulin, glucagon, somatostatin and pancreatic polypeptide under proper conditions. Undifferentiated ES cell line was stably transfected with mouse RIP-YFP plasmid construction in serum-free medium using LipofectamineTM 2000 Reagents. We tested pancreatic specific gene expression and characterized these ESC-derived pancreatic endocrine cells. Most of these insulin-secreting cells co-expressed many of the phenotypic markers characteristic of 13 cells such as insulinl, insulin2, Isletl, MafA, insulinoma-associated antigen 1 （IA1） and so on, indicating a similar gene expression pattern to adult islet 13 cells in vivo. Characterization of this population revealed that it consisted predominantly of pancreatic endocrine cells that were able to undergo pancreatic specification under the appropriate conditions. We also demonstrated that zinc supplementation mediated up-regulation of insulin-secreting cells as an effective inducer promoted the development of ESC-derived diabetes therapy. In conclusion, this work not only established an efficient pancreatic differentiation strategy from ESCs to pancreatic endocrine lineage in vitro, but also leaded to the development of new strategies to derive transplantable islet-replacement 13 cells from embryonic stem cells for the future applications of a stem cell based therapy of diabetes.展开更多
文摘利拉鲁肽(liraglutide,Lira)是胰高血糖素样肽-1的类似物,在糖尿病治疗中发挥重要作用,但利拉鲁肽通过改善胰岛β细胞的功能实现治疗糖尿病的具体机制尚未完全阐明。本研究采用高糖(33 mmol/L)诱导胰岛MIN6细胞48 h建立高糖损伤模型,CCK-8检测发现,与对照组相比,高糖组MIN6细胞活力下降(P<0.05),利拉鲁肽作用高糖组细胞活力升高(P<0.05);小鼠胰岛素和ATP含量检测发现,与对照组相比,高糖组胰岛素分泌降低(P<0.01),ATP含量减少(P<0.001),利拉鲁肽作用高糖组胰岛素释放量增加(P<0.05)和细胞内ATP含量增加(P<0.001);采用活体细胞线粒体膜通道孔(MPTP)荧光检测发现,与对照组相比,高糖组绿色荧光强度降低(P<0.001),利拉鲁肽作用高糖组绿色荧光强度增加(P<0.001);DCFH-DA探针联合流式细胞仪检测细胞活性氧簇(ROS)含量发现,与对照组相比,高糖组ROS水平升高(P<0.001),利拉鲁肽作用高糖组ROS水平降低(P<0.01);细胞内丙二醛(MDA)含量、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)以及细胞上清中乳酸脱氢酶(LDH)活性测定发现,与对照组相比,高糖组MDA和LDH水平升高(P<0.05),SOD和CAT水平降低(P<0.01),利拉鲁肽作用高糖组细胞内MDA含量和LDH活性降低(P<0.05),SOD和CAT活性增加(P<0.05);Western印迹检测解偶联蛋白2(uncoupling protein 2,UCP2)的表达发现,与对照组相比,高糖组UCP2表达上调(P<0.01),利拉鲁肽作用高糖组UCP2表达降低(P<0.05)。结果表明,利拉鲁肽对高糖诱导MIN6细胞的线粒体损伤、氧化应激以及胰岛素分泌具有重要作用,其作用机制可能与下调UCP2的表达相关,为利拉鲁肽更好地应用于临床提供了理论依据。
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 Important FinancialIssueof Shi-Wu Programming Key Problem in Liaoning Provinceand Financial Issue for Scientific Research in the Department of Education.
文摘Objective To evaluate the effects of acute glucose level changes on expression of prepro-orexin, orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R) mRNA in rat hypothalamus tissue and pancreatic islets cells. Methods Thirty adult male Wistar rats were randomly divided into three equal groups (n = 10). The acute hypoglycemia rat model was induced by a single subcutaneous injection of insulin. Twenty acute hypoglycemia rats were divided into group B and group C. Group B was allowed to eat freely, while group C was food-deprived. Control rats were injected the same volume of saline. The effect of glucose levels (2.8 mmol/L and 8.3 mmol/L) on pancreatic islet cell orexin system was detected in pancreas islet cell cultured in vitro. The expression of prepro-orexin and OXR mRNA was examined in rat hypothalamus tissue and pancreatic islets cell cultured in vitro using reverse transcription-polymerase chain reaction (RT-PCR). Results Expression of orexin mRNA increased about 150% for the food-deprived hypoglycemia rats in comparison with control group (P < 0.01), whereas expression of OX1R mRNA decreased up to 30% (P < 0.01). However, expression of OX2R mRNA was unchanged in comparison with control group. In vitro, after incubation with 2.8 mmol/L glucose for 6 hours, the expression of prepro-orexin mRNA increased 2 times in rat pancreas islet cells in comparison with 8.3 mmol/L glucose group (P < 0.01). But the expression of OX1R mRNA was not sensitive to acute glucose fluctuation.Conclusions Orexin in rat hypothalamus is stimulated by decline in blood glucose and inhibited by signals related to feeding. Moreover, glucose plays a role in modulating the gene expression of prepro-orexin in rat pancreatic islet cells.
文摘Regenerative medicine, including cell-replacement strategies, may have an important role in the treatment of type 1 diabetes which is associated with decreased islet cell mass. To date, significant progress has been made in generating insulin-secreting 13 cells from pluripotent mouse embryonic stem cells （ESCs）.The aim of this study is to explore the potential of regulating the differentiation of ESCs into pancreatic endocrine cells capable of synthesizing the pancreatic hormones including insulin, glucagon, somatostatin and pancreatic polypeptide under proper conditions. Undifferentiated ES cell line was stably transfected with mouse RIP-YFP plasmid construction in serum-free medium using LipofectamineTM 2000 Reagents. We tested pancreatic specific gene expression and characterized these ESC-derived pancreatic endocrine cells. Most of these insulin-secreting cells co-expressed many of the phenotypic markers characteristic of 13 cells such as insulinl, insulin2, Isletl, MafA, insulinoma-associated antigen 1 （IA1） and so on, indicating a similar gene expression pattern to adult islet 13 cells in vivo. Characterization of this population revealed that it consisted predominantly of pancreatic endocrine cells that were able to undergo pancreatic specification under the appropriate conditions. We also demonstrated that zinc supplementation mediated up-regulation of insulin-secreting cells as an effective inducer promoted the development of ESC-derived diabetes therapy. In conclusion, this work not only established an efficient pancreatic differentiation strategy from ESCs to pancreatic endocrine lineage in vitro, but also leaded to the development of new strategies to derive transplantable islet-replacement 13 cells from embryonic stem cells for the future applications of a stem cell based therapy of diabetes.
