目的探讨长链非编码RNA(lncRNA)-N1LR在脑缺血再灌注损伤后血脑屏障的作用机制。方法原代小鼠脑微血管内皮细胞常规培养,经氧糖剥夺/复糖复氧(OGD/R)处理模拟脑缺血再灌注损伤,实验分对照组、OGD组、lncRNA-N1LR过表达组(OGD处理后转染...目的探讨长链非编码RNA(lncRNA)-N1LR在脑缺血再灌注损伤后血脑屏障的作用机制。方法原代小鼠脑微血管内皮细胞常规培养,经氧糖剥夺/复糖复氧(OGD/R)处理模拟脑缺血再灌注损伤,实验分对照组、OGD组、lncRNA-N1LR过表达组(OGD处理后转染质粒lncRNA-N1LR过表达)、lncRNA-N1LR沉默组(OGD处理后转染质粒lncRNA-N1LR沉默)。采用逆转录聚合酶链反应检测各组中lncRNA-N1LR mRNA、紧密连接蛋白5(claudin-5)及闭合蛋白(occludin)mRNA表达水平;异硫氰酸荧光素-葡聚糖(FITC-dextran)渗透法检测血脑屏障通透性;免疫蛋白印迹法检测claudin-5、occludin蛋白表达。结果与对照组比较,OGD组lncRNA-N1LR mRNA、occludin、claudin-5 mRNA表达水平下降(0.31±0.01 vs 1.00±0.10,0.42±0.03 vs 1.01±0.13,0.38±0.03 vs 1.00±0.15,P<0.05),血脑屏障FITC-dextran通透性明显升高(58.79±3.04 vs 8.87±0.63,P<0.01)。与OGD组比较,lncRNA-N1LR过表达组lncRNA-N1LR mRNA、occludin、claudin-5 mRNA表达水平升高(0.67±0.07 vs 0.31±0.01,0.92±0.02 vs 0.42±0.03,0.70±0.08 vs 0.38±0.03,P<0.05),血脑屏障FITC-dextran通透性降低(41.57±2.43 vs 58.79±3.04,P<0.05)。与OGD组比较,lncRNA-N1LR沉默组lncRNA-N1LR mRNA、occludin、claudin-5 mRNA表达水平降低(0.21±0.02 vs 0.31±0.01,0.31±0.03 vs 0.42±0.03,0.22±0.02 vs 0.38±0.03,P<0.05),血脑屏障FITC-dextran通透性升高(72.34±1.43 vs 58.79±3.04,P<0.05)。结论LncRNA-N1LR上调可能通过降低血脑屏障通透性发挥神经保护作用。展开更多
Leaf rust, caused by Puccinia recondita Rocb. ex Desm. f. sp. tritici Eriks. & Henn, is one of the most important diseases in wheat worldwide. There are more than 40 resistance genes against wheat leaf rust and us...Leaf rust, caused by Puccinia recondita Rocb. ex Desm. f. sp. tritici Eriks. & Henn, is one of the most important diseases in wheat worldwide. There are more than 40 resistance genes against wheat leaf rust and used in wheat breeding. The Lr1 resistance gene is one of them, originates from hexaploid wheat and is present in a number of cultivars. It is a dominant gene located at the distal end of chromosome 5DL of wheat. We are working on isolation of the Lr1 gene using a map based gene cloning approach. Generation of a saturated map around the target gene is the first step of map based gene cloning. Two segregating F 2 populations Thatcher Lr1 ×Thatcher (2814 individual plants) and Thatcher Lr1 ×Frisal (832 plants) are used for fine mapping of the Lr1 gene. Three micro satellite markers (GWM654, GWM269 and GWM272) and four RFLP markers (BCD1421, Psr567, pTAG621 and ABC718) are used to analyze the two mapping populations. The micro satellite marker GWM272 and the RFLP marker ABC718 are tightly linked to Lr1 gene. The two markers are located at 0.1 cM from the Lr1 gene. For physical mapping of the Lr1 gene, genomic BAC and YAC libraries of barley and T.tauschii (D genome) have been screened with the RFLP marker ABC718. Five BAC clones from a genomic library of T.tauschii ,six from a genomic library of barley and one YAC clone from a YAC library of barley were isolated. All ends of BAC and YAC clones have been isolated and analyzed. The ends of BAC and YAC clones from barley could not be used for mapping because they are repetitive or did not hybridize with wheat DNA. Most of BAC ends from T.tauschii BAC clones showed repetitive sequences. Two BAC ends isolated from BAC clone L 1 121K23 (100 kb) are polymorphic and were mapped at the same position as the RFLP marker ABC718. We did not find any recombinants in the 100 kb region around the RFLP marker ABC718.展开更多
文摘目的探讨长链非编码RNA(lncRNA)-N1LR在脑缺血再灌注损伤后血脑屏障的作用机制。方法原代小鼠脑微血管内皮细胞常规培养,经氧糖剥夺/复糖复氧(OGD/R)处理模拟脑缺血再灌注损伤,实验分对照组、OGD组、lncRNA-N1LR过表达组(OGD处理后转染质粒lncRNA-N1LR过表达)、lncRNA-N1LR沉默组(OGD处理后转染质粒lncRNA-N1LR沉默)。采用逆转录聚合酶链反应检测各组中lncRNA-N1LR mRNA、紧密连接蛋白5(claudin-5)及闭合蛋白(occludin)mRNA表达水平;异硫氰酸荧光素-葡聚糖(FITC-dextran)渗透法检测血脑屏障通透性;免疫蛋白印迹法检测claudin-5、occludin蛋白表达。结果与对照组比较,OGD组lncRNA-N1LR mRNA、occludin、claudin-5 mRNA表达水平下降(0.31±0.01 vs 1.00±0.10,0.42±0.03 vs 1.01±0.13,0.38±0.03 vs 1.00±0.15,P<0.05),血脑屏障FITC-dextran通透性明显升高(58.79±3.04 vs 8.87±0.63,P<0.01)。与OGD组比较,lncRNA-N1LR过表达组lncRNA-N1LR mRNA、occludin、claudin-5 mRNA表达水平升高(0.67±0.07 vs 0.31±0.01,0.92±0.02 vs 0.42±0.03,0.70±0.08 vs 0.38±0.03,P<0.05),血脑屏障FITC-dextran通透性降低(41.57±2.43 vs 58.79±3.04,P<0.05)。与OGD组比较,lncRNA-N1LR沉默组lncRNA-N1LR mRNA、occludin、claudin-5 mRNA表达水平降低(0.21±0.02 vs 0.31±0.01,0.31±0.03 vs 0.42±0.03,0.22±0.02 vs 0.38±0.03,P<0.05),血脑屏障FITC-dextran通透性升高(72.34±1.43 vs 58.79±3.04,P<0.05)。结论LncRNA-N1LR上调可能通过降低血脑屏障通透性发挥神经保护作用。
文摘Leaf rust, caused by Puccinia recondita Rocb. ex Desm. f. sp. tritici Eriks. & Henn, is one of the most important diseases in wheat worldwide. There are more than 40 resistance genes against wheat leaf rust and used in wheat breeding. The Lr1 resistance gene is one of them, originates from hexaploid wheat and is present in a number of cultivars. It is a dominant gene located at the distal end of chromosome 5DL of wheat. We are working on isolation of the Lr1 gene using a map based gene cloning approach. Generation of a saturated map around the target gene is the first step of map based gene cloning. Two segregating F 2 populations Thatcher Lr1 ×Thatcher (2814 individual plants) and Thatcher Lr1 ×Frisal (832 plants) are used for fine mapping of the Lr1 gene. Three micro satellite markers (GWM654, GWM269 and GWM272) and four RFLP markers (BCD1421, Psr567, pTAG621 and ABC718) are used to analyze the two mapping populations. The micro satellite marker GWM272 and the RFLP marker ABC718 are tightly linked to Lr1 gene. The two markers are located at 0.1 cM from the Lr1 gene. For physical mapping of the Lr1 gene, genomic BAC and YAC libraries of barley and T.tauschii (D genome) have been screened with the RFLP marker ABC718. Five BAC clones from a genomic library of T.tauschii ,six from a genomic library of barley and one YAC clone from a YAC library of barley were isolated. All ends of BAC and YAC clones have been isolated and analyzed. The ends of BAC and YAC clones from barley could not be used for mapping because they are repetitive or did not hybridize with wheat DNA. Most of BAC ends from T.tauschii BAC clones showed repetitive sequences. Two BAC ends isolated from BAC clone L 1 121K23 (100 kb) are polymorphic and were mapped at the same position as the RFLP marker ABC718. We did not find any recombinants in the 100 kb region around the RFLP marker ABC718.
