目的观察着色性干皮病C组基因(XPC)缺失对间充质干细胞(MSCs)衰老的影响,并探讨其机制。方法分离培养XPC敲除(XPC-/-)小鼠及野生型(WT)小鼠骨髓MSCs,流式细胞仪检测培养细胞表面标志表达,CCK8法检测细胞增殖情况,茜素红染色以及油红O染...目的观察着色性干皮病C组基因(XPC)缺失对间充质干细胞(MSCs)衰老的影响,并探讨其机制。方法分离培养XPC敲除(XPC-/-)小鼠及野生型(WT)小鼠骨髓MSCs,流式细胞仪检测培养细胞表面标志表达,CCK8法检测细胞增殖情况,茜素红染色以及油红O染色观察成骨分化及成脂分化能力,衰老相关β半乳糖苷酶(SA-β-gal)染色检测细胞衰老情况,实时定量聚合酶链式反应(Real-time PCR)检测衰老相关基因P16、P21 mRNA表达情况。结果分离培养的两组小鼠骨髓来源的MSCs(m BMSCs)均高表达阳性表面抗原标志物CD44、CD29,基本不表达阴性表面抗原标志物CD11b、CD45,但均具有成骨及成脂分化能力。体外培养至20 PD(群体倍增水平)时,XPC-/-m BMSCs中衰老细胞比例(44.41±5.49)%,明显高于WT m BMSCs的(13.17±1.54)%(P<0.01);与WT m BMSCs相比,XPC-/-m BMSCs增殖能力明显下降(4 d时OD值:0.18±0.04 vs 0.36±0.04,P<0.05;5 d时OD值:0.27±0.04 vs 0.56±0.05,P<0.01);XPC-/-m BMSCs中P16 mRNA相对表达量与WT细胞相比无明显差异(P>0.05),而XPC-/-m BMSCs中P21 mRNA相对表达量明显高于WT细胞(3.30±0.23 vs 1.00±0.09,P<0.01);XPC敲除组细胞生长缓慢,出现衰老表型,增殖能力明显下降,SA-β-gal染色阳性率明显高于野生型细胞(P<0.01),衰老相关基因P21表达与野生型相比明显上调(P<0.01)。结论 XPC敲除促进MSCs衰老,其机制可能是由于DNA损伤累积引起的P21/P53信号通路的激活。展开更多
Protein Rad23, a nucleotide excision repair factor, mainly involves in repairing the DNA damage from environment, such as UV light. The function of Rad23 protein involved in DNA damage repair from many environmental f...Protein Rad23, a nucleotide excision repair factor, mainly involves in repairing the DNA damage from environment, such as UV light. The function of Rad23 protein involved in DNA damage repair from many environmental factors has been studied extensively, but it is not clear from ultraviolet irradiation. To further investigate the photo-protective function of Rad23 protein on HeLa cells damaged from UV light irradiation, firstly, HeLa cells were irradiated by UV light and incubated with the fusion protein of pCold-Rad23, then the cell viability and apoptosis rate were detected by MTT and Hoechst33342/Pl fluorescent staining, respectively. The results show that the recombinant Rad23 protein can protect the HeLa cells from UV irradiation, and inhibit the apoptosis of HeLa cell by UV irradiation.展开更多
DNA is constantly exposed to a wide array of genotoxic agents, generating a variety of forms of DNA damage. DNA-protein crosslinks(DPCs)—the covalent linkage of proteins with a DNA strand—are one of the most deleter...DNA is constantly exposed to a wide array of genotoxic agents, generating a variety of forms of DNA damage. DNA-protein crosslinks(DPCs)—the covalent linkage of proteins with a DNA strand—are one of the most deleterious and understudied forms of DNA damage, posing as steric blockades to transcription and replication. If not properly repaired, these lesions can lead to mutations, genomic instability, and cell death. DPCs can be induced endogenously or through environmental carcinogens and chemotherapeutic agents. Endogenously, DPCs are commonly derived through reactions with aldehydes, as well as through trapping of various enzymatic intermediates onto the DNA. Proteolytic cleavage of the protein moiety of a DPC is a general strategy for removing the lesion. This can be accomplished through a DPC-specific protease and and/or proteasome-mediated degradation.Nucleotide excision repair and homologous recombination are each involved in repairing DPCs, with their respective roles likely dependent on the nature and size of the adduct. The Fanconi anemia pathway may also have a role in processing DPC repair intermediates. In this review, we discuss how these lesions are formed, strategies and mechanisms for their removal, and diseases associated with defective DPC repair.展开更多
文摘目的观察着色性干皮病C组基因(XPC)缺失对间充质干细胞(MSCs)衰老的影响,并探讨其机制。方法分离培养XPC敲除(XPC-/-)小鼠及野生型(WT)小鼠骨髓MSCs,流式细胞仪检测培养细胞表面标志表达,CCK8法检测细胞增殖情况,茜素红染色以及油红O染色观察成骨分化及成脂分化能力,衰老相关β半乳糖苷酶(SA-β-gal)染色检测细胞衰老情况,实时定量聚合酶链式反应(Real-time PCR)检测衰老相关基因P16、P21 mRNA表达情况。结果分离培养的两组小鼠骨髓来源的MSCs(m BMSCs)均高表达阳性表面抗原标志物CD44、CD29,基本不表达阴性表面抗原标志物CD11b、CD45,但均具有成骨及成脂分化能力。体外培养至20 PD(群体倍增水平)时,XPC-/-m BMSCs中衰老细胞比例(44.41±5.49)%,明显高于WT m BMSCs的(13.17±1.54)%(P<0.01);与WT m BMSCs相比,XPC-/-m BMSCs增殖能力明显下降(4 d时OD值:0.18±0.04 vs 0.36±0.04,P<0.05;5 d时OD值:0.27±0.04 vs 0.56±0.05,P<0.01);XPC-/-m BMSCs中P16 mRNA相对表达量与WT细胞相比无明显差异(P>0.05),而XPC-/-m BMSCs中P21 mRNA相对表达量明显高于WT细胞(3.30±0.23 vs 1.00±0.09,P<0.01);XPC敲除组细胞生长缓慢,出现衰老表型,增殖能力明显下降,SA-β-gal染色阳性率明显高于野生型细胞(P<0.01),衰老相关基因P21表达与野生型相比明显上调(P<0.01)。结论 XPC敲除促进MSCs衰老,其机制可能是由于DNA损伤累积引起的P21/P53信号通路的激活。
基金Project(31171176)supported by the National Natural Science Foundation of China
文摘Protein Rad23, a nucleotide excision repair factor, mainly involves in repairing the DNA damage from environment, such as UV light. The function of Rad23 protein involved in DNA damage repair from many environmental factors has been studied extensively, but it is not clear from ultraviolet irradiation. To further investigate the photo-protective function of Rad23 protein on HeLa cells damaged from UV light irradiation, firstly, HeLa cells were irradiated by UV light and incubated with the fusion protein of pCold-Rad23, then the cell viability and apoptosis rate were detected by MTT and Hoechst33342/Pl fluorescent staining, respectively. The results show that the recombinant Rad23 protein can protect the HeLa cells from UV irradiation, and inhibit the apoptosis of HeLa cell by UV irradiation.
基金supported by the National Institutes of Health (CA179441, CA193124-Project 3 to Lei Li)
文摘DNA is constantly exposed to a wide array of genotoxic agents, generating a variety of forms of DNA damage. DNA-protein crosslinks(DPCs)—the covalent linkage of proteins with a DNA strand—are one of the most deleterious and understudied forms of DNA damage, posing as steric blockades to transcription and replication. If not properly repaired, these lesions can lead to mutations, genomic instability, and cell death. DPCs can be induced endogenously or through environmental carcinogens and chemotherapeutic agents. Endogenously, DPCs are commonly derived through reactions with aldehydes, as well as through trapping of various enzymatic intermediates onto the DNA. Proteolytic cleavage of the protein moiety of a DPC is a general strategy for removing the lesion. This can be accomplished through a DPC-specific protease and and/or proteasome-mediated degradation.Nucleotide excision repair and homologous recombination are each involved in repairing DPCs, with their respective roles likely dependent on the nature and size of the adduct. The Fanconi anemia pathway may also have a role in processing DPC repair intermediates. In this review, we discuss how these lesions are formed, strategies and mechanisms for their removal, and diseases associated with defective DPC repair.
