目的研究蛋白激酶A(PKA)在人诱导多能干细胞(iPSCs)向巨核细胞(MKs)分化过程中的调控作用。方法构建PKA过表达质粒,利用慢病毒转染系统将其导入人脐血来源的iPSCs中建立PKA过表达细胞系;采用CRISPR/Cas9基因编辑技术构建PKA基因敲除iPS...目的研究蛋白激酶A(PKA)在人诱导多能干细胞(iPSCs)向巨核细胞(MKs)分化过程中的调控作用。方法构建PKA过表达质粒,利用慢病毒转染系统将其导入人脐血来源的iPSCs中建立PKA过表达细胞系;采用CRISPR/Cas9基因编辑技术构建PKA基因敲除iPS细胞系。通过蛋白水平检测验证两种细胞系中PKA的表达情况。在无血清、无饲养层的“旋转-拟胚体(Spin-EB)”培养体系中,将细胞分为对照组(untreated组)、PKA过表达组(OV组)和PKA敲除组(KO组),在培养至第21天时,收集各组细胞,采用流式细胞术检测巨核系标志物(CD41^(+))和成熟巨核标志物(CD41^(+)CD42a^(+))的表达水平。结果构建PKA过表达和PKA敲除的人iPS细胞系:PKA OV iPSCs和PKA KO iPSCs,并经western-blot验证。在“Spin-EB”分化体系中培养至第21天,PKA OV iPSCs分化生成的CD41^(+)细胞比例及细胞数量无显著差异(P>0.05),CD41^(+)CD42a^(+)细胞比例和数量明显减少(P<0.01;P<0.05)。在PKA KO iPSCs中,分化生成的CD41^(+)细胞比例及数量、CD41^(+)CD42a^(+)细胞比例及数量均显著增加(P<0.001;P<0.001;P<0.01;P<0.01)。结论本研究成功构建了PKA基因修饰iPSCs,并验证PKA在iPSCs分化为MKs中的调控作用,为优化人诱导多能干细胞源血小板生成体系提供了新靶点。PKA过表达抑制了iPSCs向成熟MKs分化,而PKA敲除则显著促进了iPSCs向MKs的分化。展开更多
This study investigated the expression characteristics and clinical significance of gamma-aminobutyric acid A(GABAA)receptor subunit gamma1(GABRG1)in glioma by integrating cancer and normal tissue data from multiple d...This study investigated the expression characteristics and clinical significance of gamma-aminobutyric acid A(GABAA)receptor subunit gamma1(GABRG1)in glioma by integrating cancer and normal tissue data from multiple databases and combining them with experimental evidence.The research revealed that GABRG1 exhibited low expression levels in glioma,and its low expression was closely associated with unfavorable clinical outcomes.Furthermore,a prognostic model with strong decision-making ability was constructed using multivariate Cox regression analysis,incorporating age,isocitric dehydrogenase(IDH)mutation status,O6-methylguanine-DNA methyltransferase(MGMT)methylation status,World Health Organization(WHO)grade,and GABRG1 expression.Additionally,GABRG1 might be associated with the expression of tumor-associated macrophages and was significantly linked to immune regulatory processes.This study provided a new target for immunotherapy in glioma and offered a novel indicator for predicting and evaluating the clinical prognosis of patients.展开更多
文摘目的研究蛋白激酶A(PKA)在人诱导多能干细胞(iPSCs)向巨核细胞(MKs)分化过程中的调控作用。方法构建PKA过表达质粒,利用慢病毒转染系统将其导入人脐血来源的iPSCs中建立PKA过表达细胞系;采用CRISPR/Cas9基因编辑技术构建PKA基因敲除iPS细胞系。通过蛋白水平检测验证两种细胞系中PKA的表达情况。在无血清、无饲养层的“旋转-拟胚体(Spin-EB)”培养体系中,将细胞分为对照组(untreated组)、PKA过表达组(OV组)和PKA敲除组(KO组),在培养至第21天时,收集各组细胞,采用流式细胞术检测巨核系标志物(CD41^(+))和成熟巨核标志物(CD41^(+)CD42a^(+))的表达水平。结果构建PKA过表达和PKA敲除的人iPS细胞系:PKA OV iPSCs和PKA KO iPSCs,并经western-blot验证。在“Spin-EB”分化体系中培养至第21天,PKA OV iPSCs分化生成的CD41^(+)细胞比例及细胞数量无显著差异(P>0.05),CD41^(+)CD42a^(+)细胞比例和数量明显减少(P<0.01;P<0.05)。在PKA KO iPSCs中,分化生成的CD41^(+)细胞比例及数量、CD41^(+)CD42a^(+)细胞比例及数量均显著增加(P<0.001;P<0.001;P<0.01;P<0.01)。结论本研究成功构建了PKA基因修饰iPSCs,并验证PKA在iPSCs分化为MKs中的调控作用,为优化人诱导多能干细胞源血小板生成体系提供了新靶点。PKA过表达抑制了iPSCs向成熟MKs分化,而PKA敲除则显著促进了iPSCs向MKs的分化。
基金Supported by the National Natural Science Foundation of China(82271340,82071368)the Outstanding Youth Funding of Harbin Medical University(HYD2020JQ0016)the Horizontal Subject(86010900)。
文摘This study investigated the expression characteristics and clinical significance of gamma-aminobutyric acid A(GABAA)receptor subunit gamma1(GABRG1)in glioma by integrating cancer and normal tissue data from multiple databases and combining them with experimental evidence.The research revealed that GABRG1 exhibited low expression levels in glioma,and its low expression was closely associated with unfavorable clinical outcomes.Furthermore,a prognostic model with strong decision-making ability was constructed using multivariate Cox regression analysis,incorporating age,isocitric dehydrogenase(IDH)mutation status,O6-methylguanine-DNA methyltransferase(MGMT)methylation status,World Health Organization(WHO)grade,and GABRG1 expression.Additionally,GABRG1 might be associated with the expression of tumor-associated macrophages and was significantly linked to immune regulatory processes.This study provided a new target for immunotherapy in glioma and offered a novel indicator for predicting and evaluating the clinical prognosis of patients.
