DNA damage response （DDR） in different cell cycle status of human peripheral blood lymphocytes （PBLs） and the role of H2AX in DDR were investigated. The PBLs were stimulated into cell cycle with phytohemagglutinin...DNA damage response （DDR） in different cell cycle status of human peripheral blood lymphocytes （PBLs） and the role of H2AX in DDR were investigated. The PBLs were stimulated into cell cycle with phytohemagglutinin （PHA）. The apoptotic ratio and the phosphorylation H2AX （S139） were flow cytometrically measured in resting and proliferating PBLs after treatment with camptothecin （CPT） or X-ray. The expressions of γH2AX, Bcl-2, caspase-3 and caspase-9 were detected by Western blotting. DDR in 293T cells was detected after H2AX was silenced by RNAi method. Our results showed that DNA double strand breaks （DSBs） were both induced in quiescent and proliferating PBLs after CPT or X-ray treatment. The phosphorylation of H2AX and apoptosis were more sensitive in proliferating PBLs compared with quiescent lymphocytes （P0.05）. The expression levels of anti-apoptotic proteins Bcl-2 were reduced and cleaved caspase-3 and caspase-9 were increased. No significant changes were observed in CPT-induced apoptosis in 293T cells between H2AX knocking down group and controls. It was concluded that proliferating PBLs were more vulnerable to DNA damage compared to non-stimulated lymphocytes and had higher apoptosis rates. γH2AX may only serve as a marker of DNA damage but exert no effect on apoptosis regulation.展开更多
Objective:To establish a method of genomic DNA extraction from whole blood using Fe3O4/Au composite particles as a carrier. Methods: Two crucial conditions (sodium chloride concentration and amount of the magnetic par...Objective:To establish a method of genomic DNA extraction from whole blood using Fe3O4/Au composite particles as a carrier. Methods: Two crucial conditions (sodium chloride concentration and amount of the magnetic particles) were optimized and 8 different human whole blood samples were used to purify genomic DNA under the optimal condition. Then agarose gel electrophoresis and polymerase chain reaction (PCR) were performed. Results: The optimal binding condition was 1.5 mol/L NaCl/10% PEG, and the optimal amount of Fe3O4/Au composite particles was 600 μg. The yields of the genomic DNA from 100 μl of different whole blood samples were 2-5 μg, and the ratio of A260/A280 was in the range of 1.70-1.90. The size of genomic DNA was about 23 kb and the PCR was valid. Conclusion: The purification system using Fe3O4/Au composite microparticles has advantages in high yield, high purity, ease of operating, time saving and avoiding centrifugation. The purified sample was found to function satisfactorily in PCR amplification.展开更多
To investigate the distribution of mitochondria ONA 4 977 bp deletion, a common deletion （CD）, in normal populations of Chinese, human peripheral blood samples from sixty healthy donors were collected, and levels of...To investigate the distribution of mitochondria ONA 4 977 bp deletion, a common deletion （CD）, in normal populations of Chinese, human peripheral blood samples from sixty healthy donors were collected, and levels of the CD in genomic DNA from the samples were detected using real-time PCR. The results showed that the CD was found in 27 health donors, with its positive rate being 45% （27/60）. The CD ratio was between 0 and 0.000308%, and not affected by age and gender in sixty healthy donors. Our studies indicate that the CD ratio is low, and do not show the age-dependent accumulation and any gender difference in peripheral whole blood from the normal Chinese population.展开更多
基金supported by grants from Program 973 from Ministry of ScienceTechnology of China (Nos. 2004CB518705, 2009CB5218702)the National Natural Sciences Foundation of China (Nos. 30872472, 30800569)
文摘DNA damage response （DDR） in different cell cycle status of human peripheral blood lymphocytes （PBLs） and the role of H2AX in DDR were investigated. The PBLs were stimulated into cell cycle with phytohemagglutinin （PHA）. The apoptotic ratio and the phosphorylation H2AX （S139） were flow cytometrically measured in resting and proliferating PBLs after treatment with camptothecin （CPT） or X-ray. The expressions of γH2AX, Bcl-2, caspase-3 and caspase-9 were detected by Western blotting. DDR in 293T cells was detected after H2AX was silenced by RNAi method. Our results showed that DNA double strand breaks （DSBs） were both induced in quiescent and proliferating PBLs after CPT or X-ray treatment. The phosphorylation of H2AX and apoptosis were more sensitive in proliferating PBLs compared with quiescent lymphocytes （P0.05）. The expression levels of anti-apoptotic proteins Bcl-2 were reduced and cleaved caspase-3 and caspase-9 were increased. No significant changes were observed in CPT-induced apoptosis in 293T cells between H2AX knocking down group and controls. It was concluded that proliferating PBLs were more vulnerable to DNA damage compared to non-stimulated lymphocytes and had higher apoptosis rates. γH2AX may only serve as a marker of DNA damage but exert no effect on apoptosis regulation.
基金Supported by the National High Technology Research and Development Program of China (2006AA020705)
文摘Objective:To establish a method of genomic DNA extraction from whole blood using Fe3O4/Au composite particles as a carrier. Methods: Two crucial conditions (sodium chloride concentration and amount of the magnetic particles) were optimized and 8 different human whole blood samples were used to purify genomic DNA under the optimal condition. Then agarose gel electrophoresis and polymerase chain reaction (PCR) were performed. Results: The optimal binding condition was 1.5 mol/L NaCl/10% PEG, and the optimal amount of Fe3O4/Au composite particles was 600 μg. The yields of the genomic DNA from 100 μl of different whole blood samples were 2-5 μg, and the ratio of A260/A280 was in the range of 1.70-1.90. The size of genomic DNA was about 23 kb and the PCR was valid. Conclusion: The purification system using Fe3O4/Au composite microparticles has advantages in high yield, high purity, ease of operating, time saving and avoiding centrifugation. The purified sample was found to function satisfactorily in PCR amplification.
基金supported by grants from the building project for the National Key Clinical Special Department of China(No.2011-17)the Medical Science and Technology Foundation of Henan Province(No.201204123)the Priority Academic Program Development of Jiangsu Higher Education Institutions
文摘To investigate the distribution of mitochondria ONA 4 977 bp deletion, a common deletion （CD）, in normal populations of Chinese, human peripheral blood samples from sixty healthy donors were collected, and levels of the CD in genomic DNA from the samples were detected using real-time PCR. The results showed that the CD was found in 27 health donors, with its positive rate being 45% （27/60）. The CD ratio was between 0 and 0.000308%, and not affected by age and gender in sixty healthy donors. Our studies indicate that the CD ratio is low, and do not show the age-dependent accumulation and any gender difference in peripheral whole blood from the normal Chinese population.
