以小鼠胚胎成纤维细胞为饲养层,收集受孕3.5 d ICR小鼠的囊胚和桑椹胚进行培养,筛选纯化ES细胞集落,使其稳定传代后,对其形态学和生物学性状进行初步鉴定。结果表明,ES细胞有其典型的形态学特征:集落呈鸟巢状,边缘清楚,表面平滑,结构致...以小鼠胚胎成纤维细胞为饲养层,收集受孕3.5 d ICR小鼠的囊胚和桑椹胚进行培养,筛选纯化ES细胞集落,使其稳定传代后,对其形态学和生物学性状进行初步鉴定。结果表明,ES细胞有其典型的形态学特征:集落呈鸟巢状,边缘清楚,表面平滑,结构致密,隆起生长,细胞之间界限不清楚;单个细胞体积小、核大;对ES细胞碱性磷酸酶进行检测,在AKP底物NBT、BCIP作用下,未分化的ES细胞显微镜下为黄褐色,分化的不着色;核型鉴定表明ES细胞具有正常的二倍体核型。展开更多
Induced pluripotent stem cells （iPSCs） resemble embryonic stem cells （ESCs） in morphol- ogy, gene expression and in vitro differentiation, raising new hope for personalized clinical therapy. While many efforts hav...Induced pluripotent stem cells （iPSCs） resemble embryonic stem cells （ESCs） in morphol- ogy, gene expression and in vitro differentiation, raising new hope for personalized clinical therapy. While many efforts have been made to improve reprogramming efficiency, significant problems such as genomic instability of iPSCs need to be addressed before clinical therapy. In this study, we try to figure out the real genomic state of iPSCs and their DNA damage response to ionizing radiation （IR）. We found that iPSC line 3FB4-1 had lower DNA damage repair ability than mouse embryonic fibroblast （MEF） cells, from which 3FB4-11ine was derived. After the introduction of DNA damage by IR, the number of 7-H2AX loci in 3FB4-1 increased modestly compared to a large increase seen in MEF, albeit both significantly （P 〈 0.01）. In addition, whole-genome sequencing analysis showed that after IR, 3FB4-1 possessed more point mutations than MEF and the point mutations spread all over chromosomes. These observations provide evidence that iPSCs are more sensitive to ionizing radiation and their relatively low DNA damage repair capacity may account for their high radiosensitivity. The compromised DNA damage repair capacity of iPSCs should be considered when used in clinical therapy.展开更多
目的构建小鼠胚胎成纤维细胞(MEF)复制性衰老模型,检测衰老溶酶体相关功能改变,为衰老相关溶酶体疾病提供有效的细胞模型。方法通过酶消化法分离提取MEF,体外连续传代培养构建MEF复制性衰老模型,实时定量PCR(qPCR)检测衰老标志物p16和p...目的构建小鼠胚胎成纤维细胞(MEF)复制性衰老模型,检测衰老溶酶体相关功能改变,为衰老相关溶酶体疾病提供有效的细胞模型。方法通过酶消化法分离提取MEF,体外连续传代培养构建MEF复制性衰老模型,实时定量PCR(qPCR)检测衰老标志物p16和p21,衰老相关β⁃半乳糖苷酶(SA⁃β⁃gal)染色方法进一步验证衰老状态;通过溶酶体探针类染料LysoTracker Red DND⁃99探针及LysoSensor Yellow/Blue DND⁃160双激发探针追踪酸性溶酶体定性检测溶酶体酸碱性及定量检测溶酶体pH值;通过偶联自淬BODIPY®染料的牛血清白蛋白(DQ⁃BSA)检测衰老溶酶体的降解能力。结果qPCR和SA⁃β⁃gal染色结果显示,MEF复制性衰老模型构建成功。与年轻P3代MEF相比,衰老P9代MEF溶酶体的酸性明显丧失,差异有统计学意义(P<0.0001),衰老P9代MEF溶酶体的降解能力明显降低(P<0.05)。结论成功构建MEF复制性衰老细胞模型,并证明衰老MEF溶酶体的酸性丧失以及降解能力下降。展开更多
文摘以小鼠胚胎成纤维细胞为饲养层,收集受孕3.5 d ICR小鼠的囊胚和桑椹胚进行培养,筛选纯化ES细胞集落,使其稳定传代后,对其形态学和生物学性状进行初步鉴定。结果表明,ES细胞有其典型的形态学特征:集落呈鸟巢状,边缘清楚,表面平滑,结构致密,隆起生长,细胞之间界限不清楚;单个细胞体积小、核大;对ES细胞碱性磷酸酶进行检测,在AKP底物NBT、BCIP作用下,未分化的ES细胞显微镜下为黄褐色,分化的不着色;核型鉴定表明ES细胞具有正常的二倍体核型。
基金supported by Strategic Priority Research Program of the Chinese Academy of Sciences(Grant No.XDA01040405)National Natural Science Foundation of China(Grant No.91029024)+1 种基金National Basic Research Program of China(973 ProgramGrant No.2013CB911001)
文摘Induced pluripotent stem cells （iPSCs） resemble embryonic stem cells （ESCs） in morphol- ogy, gene expression and in vitro differentiation, raising new hope for personalized clinical therapy. While many efforts have been made to improve reprogramming efficiency, significant problems such as genomic instability of iPSCs need to be addressed before clinical therapy. In this study, we try to figure out the real genomic state of iPSCs and their DNA damage response to ionizing radiation （IR）. We found that iPSC line 3FB4-1 had lower DNA damage repair ability than mouse embryonic fibroblast （MEF） cells, from which 3FB4-11ine was derived. After the introduction of DNA damage by IR, the number of 7-H2AX loci in 3FB4-1 increased modestly compared to a large increase seen in MEF, albeit both significantly （P 〈 0.01）. In addition, whole-genome sequencing analysis showed that after IR, 3FB4-1 possessed more point mutations than MEF and the point mutations spread all over chromosomes. These observations provide evidence that iPSCs are more sensitive to ionizing radiation and their relatively low DNA damage repair capacity may account for their high radiosensitivity. The compromised DNA damage repair capacity of iPSCs should be considered when used in clinical therapy.
文摘目的构建小鼠胚胎成纤维细胞(MEF)复制性衰老模型,检测衰老溶酶体相关功能改变,为衰老相关溶酶体疾病提供有效的细胞模型。方法通过酶消化法分离提取MEF,体外连续传代培养构建MEF复制性衰老模型,实时定量PCR(qPCR)检测衰老标志物p16和p21,衰老相关β⁃半乳糖苷酶(SA⁃β⁃gal)染色方法进一步验证衰老状态;通过溶酶体探针类染料LysoTracker Red DND⁃99探针及LysoSensor Yellow/Blue DND⁃160双激发探针追踪酸性溶酶体定性检测溶酶体酸碱性及定量检测溶酶体pH值;通过偶联自淬BODIPY®染料的牛血清白蛋白(DQ⁃BSA)检测衰老溶酶体的降解能力。结果qPCR和SA⁃β⁃gal染色结果显示,MEF复制性衰老模型构建成功。与年轻P3代MEF相比,衰老P9代MEF溶酶体的酸性明显丧失,差异有统计学意义(P<0.0001),衰老P9代MEF溶酶体的降解能力明显降低(P<0.05)。结论成功构建MEF复制性衰老细胞模型,并证明衰老MEF溶酶体的酸性丧失以及降解能力下降。
