1.贵州医科大学细胞工程生物医药技术国家地方联合工程实验室,贵州省再生医学重点实验室,贵州医科大学 免疫教研室,贵阳 550004; 2.中国医学科学院成体干细胞转化研究重点实验室,组织工程与干细胞实验中心, 贵阳 550004; 3.贵州医科大学附属医院儿科,贵阳 550004
R-33
MO Jing
1.National&Guizhou Joint Engineering Laboratory for Cell Engineering and Biomedicine Technique, Guizhou Province Key Laboratory of Regenerative Medicine, Department of Immunology, Guizhou Medical University, Guiyang 550004, China. 2. Key Laboratory of Adult Stem Cell Translational Research, Center for Tissue Engineering and Stem Cell research, Guiyang 550004LIU Han
1.National&Guizhou Joint Engineering Laboratory for Cell Engineering and Biomedicine Technique, Guizhou Province Key Laboratory of Regenerative Medicine, Department of Immunology, Guizhou Medical University, Guiyang 550004, China. 2. Key Laboratory of Adult Stem Cell Translational Research, Center for Tissue Engineering and Stem Cell research, Guiyang 550004FAN Anran
1.National&Guizhou Joint Engineering Laboratory for Cell Engineering and Biomedicine Technique, Guizhou Province Key Laboratory of Regenerative Medicine, Department of Immunology, Guizhou Medical University, Guiyang 550004, China. 2. Key Laboratory of Adult Stem Cell Translational Research, Center for Tissue Engineering and Stem Cell research, Guiyang 550004ZHANG Peng
1.National&Guizhou Joint Engineering Laboratory for Cell Engineering and Biomedicine Technique, Guizhou Province Key Laboratory of Regenerative Medicine, Department of Immunology, Guizhou Medical University, Guiyang 550004, China. 2. Key Laboratory of Adult Stem Cell Translational Research, Center for Tissue Engineering and Stem Cell research, Guiyang 550004ZHOU Yanhua
1.National&Guizhou Joint Engineering Laboratory for Cell Engineering and Biomedicine Technique, Guizhou Province Key Laboratory of Regenerative Medicine, Department of Immunology, Guizhou Medical University, Guiyang 550004, China. 2. Key Laboratory of Adult Stem Cell Translational Research, Center for Tissue Engineering and Stem Cell research, Guiyang 550004SHU Liping
1.National&Guizhou Joint Engineering Laboratory for Cell Engineering and Biomedicine Technique, Guizhou Province Key Laboratory of Regenerative Medicine, Department of Immunology, Guizhou Medical University, Guiyang 550004, China. 2. Key Laboratory of Adult Stem Cell Translational Research, Center for Tissue Engineering and Stem Cell research, Guiyang 5500041.National&Guizhou Joint Engineering Laboratory for Cell Engineering and Biomedicine Technique, Guizhou Province Key Laboratory of Regenerative Medicine, Department of Immunology, Guizhou Medical University, Guiyang 550004, China. 2. Key Laboratory of Adult Stem Cell Translational Research, Center for Tissue Engineering and Stem Cell research, Guiyang 550004. 3. Department of pediatrics, the affiliated hospital of Guizhou Medical University, Guiyang 550004
目的 构建可诱导 Tet1CD 过表达的稳转细胞系,探究 Tet1 蛋白对细胞增殖、周期及凋亡的影响。 方法 构建重组质粒 pTRE-Tight_Tet1CD_IRES_RFP_RPBSA_M2rtTA_Puro,然后与表达转座酶的“睡美人”质粒通 过脂质体法共同转染到 HEK 293T 细胞,通过嘌呤霉素筛选和多西环素诱导构建诱导表达型的 Tet1CD 稳转细胞系 293T(Tet1CD)。 通过 Western blot 检测 Tet1 蛋白的表达;通过 DNA dot blot 检测细胞基因组中 5hmC 的含量,通过 流式细胞术检测 Tet1CD 蛋白对细胞周期和凋亡的影响;通过细胞计数检测 Tet1CD 蛋白对细胞增殖的影响。结果 成功建立了可诱导 Tet1CD 过表达的 HEK293T 细胞系 293T(Tet1CD);Western blot 结果显示诱导后细胞内 Tet1CD 蛋白表达上升;DNA dot blot 结果显示过表达 Tet1CD 蛋白细胞内 5hmC 含量升高;流式细胞术结果显示, Tet1CD 过表达促进了细胞凋亡(P< 0. 05)及增加 G2 时期细胞比例(P< 0. 05);细胞计数结果显示,Tet1CD 过表达 抑制细胞增殖(P< 0. 0001)。 结论 Tet1 蛋白可能通过促进细胞凋亡及阻滞细胞周期最终抑制细胞增殖。
Objective To explore the effects of Tet1 protein on cell proliferation, the cell cycle and apoptosis, an inducible Tet1CD overexpression cell line was established. Methods A recombinant pTRE-Tight_Tet1CD_IRES_RFP_ RPBSA_ M2rtTA _ Puro plasmid was constructed, then the recombinant plasmid and the “ Sleeping Beauty” plasmid expressing a transposase were cotransfected into HEK293T cells using lipofectamine. The inducible stable 293T (Tet1CD) cell line was selected using puromycin. Tet1 protein expression was detected via western blot, and the 5hmC content in the genome was detected using DNA Dot Blot. The effect of Tet1CD protein on the cell cycle and apoptosis was detected using flow cytometry, and the effect of Tet1CD protein on cell proliferation was detected by cell counting. Results The HEK293T (Tet1CD) cell line with inducible Tet1CD expression was successfully established. Western blot and DNA Dot Blot assays showed increased amounts of Tet1CD proteins and increased 5hmC content in inducible cells. Flow cytometry result demonstrated that Tet1CD expression promoted apoptosis (P< 0. 05) and increased the proportion of cells in G2 phase (P< 0. 05). Cell counts showed that Tet1CD overexpression inhibited cell proliferation (P< 0. 0001). Conclusions Tet1 protein may inhibit cell proliferation by promoting apoptosis and delaying the cell cycle.
莫 晶,杨红兰,刘 含,金 皎,范安然,张 鹏,周艳华,舒莉萍. Tet1 蛋白对 HEK293T 细胞增殖、周期及凋亡的影响[J].中国比较医学杂志,2020,30(6):69~75.
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