目的:研究脑痰清(Nao Tan Qing,NTQ)对阿尔茨海默病(Alzheimer’s disease,AD)小鼠中神经干细胞增殖的影响,并探究其分子机制。方法:将五转家族性阿尔茨海默病模型小鼠(transgenic mice with five familial Alzheimer’s disease,5×...目的:研究脑痰清(Nao Tan Qing,NTQ)对阿尔茨海默病(Alzheimer’s disease,AD)小鼠中神经干细胞增殖的影响,并探究其分子机制。方法:将五转家族性阿尔茨海默病模型小鼠(transgenic mice with five familial Alzheimer’s disease,5×FAD小鼠)随机分为两组:AD组、AD+NTQ组,分别使用去离子水或NTQ灌胃处理;利用免疫荧光染色、实时荧光定量PCR、蛋白质印迹法等检测海马区神经干细胞增殖情况;体外分离培养C57/BL6J小鼠胚胎神经干细胞,分别使用PBS、NTQ处理细胞,利用CCK-8法检测细胞增殖情况,TUNEL检测细胞凋亡,使用免疫荧光染色检测Y染色体性别决定区(sex-determing region of Y chromosome,SRY)盒转录因子2(SRY-box transcription factor 2,SOX2)阳性、5-溴脱氧尿嘧啶核苷(5-bromo-2-deoxy uridine,BrdU)阳性及双皮质素(doublecortin,DCX)阳性细胞,通过实时荧光定量PCR及蛋白质印迹法检测SOX2、DCX表达。利用实时荧光定量PCR及蛋白质印迹法检测cyclin D1、p27/Kip1及GATA2的表达情况;使用cyclin D1-细胞周期依赖性蛋白激酶(cyclin-depen-dent kinase,CDK)抑制剂体外处理神经干细胞后,通过实时荧光定量PCR及蛋白质印迹法检测SOX2表达水平。结果:与AD组相比,AD+NTQ组小鼠海马区SOX2+细胞数量增多、SOX2 mRNA及蛋白水平显著增加;NTQ处理神经干细胞后,神经球直径显著增加,BrdU+、SOX2+及DCX+细胞数目增加,SOX2、DCX mRNA水平增加,SOX2蛋白水平显著增加。AD+NTQ组小鼠海马区GATA2及下游分子p27/Kip1表达下降,对cyclin D1的抑制作用减弱,使细胞发生增殖。添加cyclin D1-CDK抑制剂可减弱NTQ所引发的SOX2、DCX表达量增加。结论:NTQ通过调控GATA2-p27/Kip1-cyclin D1信号通路,维持神经干细胞增殖,改善AD小鼠认知障碍。展开更多
The advent of gene editing represents one of the most transformative breakthroughs in life science,making genome manipulation more accessible than ever before.While traditional CRISPR/Cas-based gene editing,which invo...The advent of gene editing represents one of the most transformative breakthroughs in life science,making genome manipulation more accessible than ever before.While traditional CRISPR/Cas-based gene editing,which involves double-strand DNA breaks(DSBs),excels at gene disruption,it is less effective for accurate gene modification.The limitation arises because DSBs are primarily repaired via non-homologous end joining(NHEJ),which tends to introduce indels at the break site.While homology directed repair(HDR)can achieve precise editing when a donor DNA template is provided,the reliance on DSBs often results in unintended genome damage.HDR is restricted to specific cell cycle phases,limiting its application.Currently,gene editing has evolved to unprecedented levels of precision without relying on DSB and HDR.The development of innovative systems,such as base editing,prime editing,and CRISPR-associated transposases(CASTs),now allow for precise editing ranging from single nucleotides to large DNA fragments.Base editors(BEs)enable the direct conversion of one nucleotide to another,and prime editors(PEs)further expand gene editing capabilities by allowing for the insertion,deletion,or alteration of small DNA fragments.The CAST system,a recent innovation,allows for the precise insertion of large DNA fragments at specific genomic locations.In recent years,the optimization of these precise gene editing tools has led to significant improvements in editing efficiency,specificity,and versatility,with advancements such as the creation of base editors for nucleotide transversions,enhanced prime editing systems for more efficient and precise modifications,and refined CAST systems for targeted large DNA insertions,expanding the range of applications for these tools.Concurrently,these advances are complemented by significant improvements in in vivo delivery methods,which have paved the way for therapeutic application of precise gene editing tools.Effective delivery systems are critical for the success of gene therapies,and recent developments in both viral and non-viral vectors have improved the efficiency and safety of gene editing.For instance,adeno-associated viruses(AAVs)are widely used due to their high transfection efficiency and low immunogenicity,though challenges such as limited cargo capacity and potential for immune responses remain.Non-viral delivery systems,including lipid nanoparticles(LNPs),offer an alternative with lower immunogenicity and higher payload capacity,although their transfection efficiency can be lower.The therapeutic potential of these precise gene editing technologies is vast,particularly in treating genetic disorders.Preclinical studies have demonstrated the effectiveness of base editing in correcting genetic mutations responsible for diseases such as cardiomyopathy,liver disease,and hereditary hearing loss.These technologies promise to treat symptoms and potentially cure the underlying genetic causes of these conditions.Meanwhile,challenges remain,such as optimizing the safety and specificity of gene editing tools,improving delivery systems,and overcoming off-target effects,all of which are critical for their successful application in clinical settings.In summary,the continuous evolution of precise gene editing technologies,combined with advancements in delivery systems,is driving the field toward new therapeutic applications that can potentially transform the treatment of genetic disorders by targeting their root causes.展开更多
文摘目的:研究脑痰清(Nao Tan Qing,NTQ)对阿尔茨海默病(Alzheimer’s disease,AD)小鼠中神经干细胞增殖的影响,并探究其分子机制。方法:将五转家族性阿尔茨海默病模型小鼠(transgenic mice with five familial Alzheimer’s disease,5×FAD小鼠)随机分为两组:AD组、AD+NTQ组,分别使用去离子水或NTQ灌胃处理;利用免疫荧光染色、实时荧光定量PCR、蛋白质印迹法等检测海马区神经干细胞增殖情况;体外分离培养C57/BL6J小鼠胚胎神经干细胞,分别使用PBS、NTQ处理细胞,利用CCK-8法检测细胞增殖情况,TUNEL检测细胞凋亡,使用免疫荧光染色检测Y染色体性别决定区(sex-determing region of Y chromosome,SRY)盒转录因子2(SRY-box transcription factor 2,SOX2)阳性、5-溴脱氧尿嘧啶核苷(5-bromo-2-deoxy uridine,BrdU)阳性及双皮质素(doublecortin,DCX)阳性细胞,通过实时荧光定量PCR及蛋白质印迹法检测SOX2、DCX表达。利用实时荧光定量PCR及蛋白质印迹法检测cyclin D1、p27/Kip1及GATA2的表达情况;使用cyclin D1-细胞周期依赖性蛋白激酶(cyclin-depen-dent kinase,CDK)抑制剂体外处理神经干细胞后,通过实时荧光定量PCR及蛋白质印迹法检测SOX2表达水平。结果:与AD组相比,AD+NTQ组小鼠海马区SOX2+细胞数量增多、SOX2 mRNA及蛋白水平显著增加;NTQ处理神经干细胞后,神经球直径显著增加,BrdU+、SOX2+及DCX+细胞数目增加,SOX2、DCX mRNA水平增加,SOX2蛋白水平显著增加。AD+NTQ组小鼠海马区GATA2及下游分子p27/Kip1表达下降,对cyclin D1的抑制作用减弱,使细胞发生增殖。添加cyclin D1-CDK抑制剂可减弱NTQ所引发的SOX2、DCX表达量增加。结论:NTQ通过调控GATA2-p27/Kip1-cyclin D1信号通路,维持神经干细胞增殖,改善AD小鼠认知障碍。
文摘The advent of gene editing represents one of the most transformative breakthroughs in life science,making genome manipulation more accessible than ever before.While traditional CRISPR/Cas-based gene editing,which involves double-strand DNA breaks(DSBs),excels at gene disruption,it is less effective for accurate gene modification.The limitation arises because DSBs are primarily repaired via non-homologous end joining(NHEJ),which tends to introduce indels at the break site.While homology directed repair(HDR)can achieve precise editing when a donor DNA template is provided,the reliance on DSBs often results in unintended genome damage.HDR is restricted to specific cell cycle phases,limiting its application.Currently,gene editing has evolved to unprecedented levels of precision without relying on DSB and HDR.The development of innovative systems,such as base editing,prime editing,and CRISPR-associated transposases(CASTs),now allow for precise editing ranging from single nucleotides to large DNA fragments.Base editors(BEs)enable the direct conversion of one nucleotide to another,and prime editors(PEs)further expand gene editing capabilities by allowing for the insertion,deletion,or alteration of small DNA fragments.The CAST system,a recent innovation,allows for the precise insertion of large DNA fragments at specific genomic locations.In recent years,the optimization of these precise gene editing tools has led to significant improvements in editing efficiency,specificity,and versatility,with advancements such as the creation of base editors for nucleotide transversions,enhanced prime editing systems for more efficient and precise modifications,and refined CAST systems for targeted large DNA insertions,expanding the range of applications for these tools.Concurrently,these advances are complemented by significant improvements in in vivo delivery methods,which have paved the way for therapeutic application of precise gene editing tools.Effective delivery systems are critical for the success of gene therapies,and recent developments in both viral and non-viral vectors have improved the efficiency and safety of gene editing.For instance,adeno-associated viruses(AAVs)are widely used due to their high transfection efficiency and low immunogenicity,though challenges such as limited cargo capacity and potential for immune responses remain.Non-viral delivery systems,including lipid nanoparticles(LNPs),offer an alternative with lower immunogenicity and higher payload capacity,although their transfection efficiency can be lower.The therapeutic potential of these precise gene editing technologies is vast,particularly in treating genetic disorders.Preclinical studies have demonstrated the effectiveness of base editing in correcting genetic mutations responsible for diseases such as cardiomyopathy,liver disease,and hereditary hearing loss.These technologies promise to treat symptoms and potentially cure the underlying genetic causes of these conditions.Meanwhile,challenges remain,such as optimizing the safety and specificity of gene editing tools,improving delivery systems,and overcoming off-target effects,all of which are critical for their successful application in clinical settings.In summary,the continuous evolution of precise gene editing technologies,combined with advancements in delivery systems,is driving the field toward new therapeutic applications that can potentially transform the treatment of genetic disorders by targeting their root causes.
