Objective： To investigate the characteristics of CGI-100- knockdown K562 cells and the effect of CGI-100 RNA interference （RNAi） on matrine-treated K562 cells. Methods： Three oligonucleotides targeting CGI-100 ge...Objective： To investigate the characteristics of CGI-100- knockdown K562 cells and the effect of CGI-100 RNA interference （RNAi） on matrine-treated K562 cells. Methods： Three oligonucleotides targeting CGI-100 gene and a pair of negative control containing the same nucleotide composition with a different sequence were devised and chemically synthesized. The inhibition efficiency of CGI-100 expression by shRNA-CGI-100 in K562 cells was determined using semiquantitative RT-PCR and dot blot hybridization. The effect of CGI-100 RNAi on the growth of K562 cells was examined using MTT assay and cell differentiation was measured by distinct approaches including flow cytometry, benzidine staining and electron microscope. After CGI-100-konckdown K562 cells were incubated with 0.2 mg/ml of matrine or 30 Ixmol/L of hemin for 48 h, the expression levels of Glycophorin A（GPA）（CD235a） and Growth factor independence-lB mRNA（Gfi-IB mRNA） were measured by RT-PCR and the protein levels of GPA, CD14 and CD15 were detected by flow cytometry. Results： The eukaryotic expression vectors of CGI-100 RNAi were successfully constructed. The K562/shRNA-CGI-100 cell line was established in which the inhibition efficiency of CGI-100 gene expression by shRNA-CGI-100 was 54%. CGI-100-knockdown inhibited the proliferation and induced erythroid differentiation in K562 cells. Compared with the control K562 cells, the K562/shRNA-CGI-100 cells showed decreased absorbance value detected by MTT assay, decreased enchromation, increased heterochromation, increased percentage of G0/Gx phase cells, decreased population of S phase cells, decreased PI （proliferation index of cells）, and elevated percentage of benzidine-positive cells. Moreover, the sensitivity of K562/shRNA-CGI-100 cells to either matrine or hemin was enhanced and the sensitivity of these cells to matrine was higher than that to hemin. Compared with the control K562 cells, matrine treatment in K562/shRNA-CGI-100 cells resulted in increased inhibitory rate of proliferation, elevated percentage of be nzidine-positive cells, obviously up-regulated mRNA expressions of GPA and Gfi-IB, and increased mean fluorescence intensity （MFI） of GPA. No CD14 expression was detected and no statistical significance was found for the detected CD15. Finally, the MFI of GPA increased in K562/shRNA-CGI-100 cells treated with hemin and was 1.7 times less than that in cells exposed to matrine. Conclusion： These results suggest that the function of CGI-100 gene is correlated with the deregulated proliferation and the block of erythroid differentiation in K562 cells and may also be involved in matrine-induced erythroid differentiation in K562 ceils.展开更多
Objective To investigate the effect of exogenous S100A13 gene overexpression on the proliferation of human thyroid cancer cell line TT.Methods The recombinant ORF of S100A13 tagged with six histidines at the 5' en...Objective To investigate the effect of exogenous S100A13 gene overexpression on the proliferation of human thyroid cancer cell line TT.Methods The recombinant ORF of S100A13 tagged with six histidines at the 5' end was subcloned into the pcDNA3.2/V5/GW/D-TOPO vector and sequenced.The eukaryotic expression plasmid pcDNA3.2/V5 /GW/D-S100A13 and empty vector pcDNA3.2/V5/GW/D were transfected into TT cells.The positive clones were selected by G418.The expressions of S100A13 mRNA and protein were detected by real time reverse transcription-polymerase chain reaction(RT-PCR) and Western blot.The effect of S100A13 on cell proliferation and cell cycle was evaluated by cell growth curve,MTT colorimetric assay and flow cytometry.Results S100A13 gene tagged with six histidines at the 5 ' end was confirmed to be inserted into the pcDNA3.2/V5/GW/D vector correctly.TT-S100A13-V5 cells,which over-expressed S100A13,were constructed successfully.TT-S100A13-V5 cells grew much faster than TT-V5 and TT cells(P <0.001).The proportions of both S and G2/M phase cells were significantly higher in TT-S100A13-V5 cells than those in TT-V5 and TT cells(P <0.001).Conclusion The eukaryotic expression vector containing human S100A13 gene has been successfully constructed,which highly expresses S100A13 in TT cells.Exogenous S100A13 gene overexpression accelerates TT cell proliferation and drives the cell cycle progression of TT cells from G0/G1 phase to S and G2/M phases.展开更多
Amyloid beta (1-42) peptide is considered responsible for the formation of senile plaques that accumulate in the brain of patients with Alzheimer’s disease (AD). In the past years considerable attention has been focu...Amyloid beta (1-42) peptide is considered responsible for the formation of senile plaques that accumulate in the brain of patients with Alzheimer’s disease (AD). In the past years considerable attention has been focused on identifying new protective substances that prevent or almost retard the appearance of amyloid beta (1-42)-related neurotoxic effects. In this study, human neuroblastoma cells (IMR-32) was used as system model to evaluate the protective role of S100b, a neurotrophic factor and neuronal survival protein, that is highly expressed by reactive astrocytes in close vicinity of beta-amyloid deposits, against amyloid beta (1-42)-dependent toxicity. Our results show that at nanomolar concentrations, S100b protects cells against Aβmediated cytotoxicity, as assessed by MTS vitality test. The protective mechanism seems to be related to the effect on bcl-2 (an anti-apoptotic gene) expression, which is highly down-regulated by amyloid beta (1-42) treatment, while resulted more expressed in the presence of S100b. On the contrary, Bax, a proapoptotic gene, resulted down-regulated by the treatment with S100 compared with the results obtained in the presence of amyloid beta (1-42) peptide. However, at micromolar doses, S100b is toxic for IMR-32 cells and its toxicity adds to that of the Aβpeptide, suggesting that additional molecular mechanisms may be involved in theneurotoxic process.展开更多
基金supported by the National Natural Science Foundation of China(No.30171150)
文摘Objective： To investigate the characteristics of CGI-100- knockdown K562 cells and the effect of CGI-100 RNA interference （RNAi） on matrine-treated K562 cells. Methods： Three oligonucleotides targeting CGI-100 gene and a pair of negative control containing the same nucleotide composition with a different sequence were devised and chemically synthesized. The inhibition efficiency of CGI-100 expression by shRNA-CGI-100 in K562 cells was determined using semiquantitative RT-PCR and dot blot hybridization. The effect of CGI-100 RNAi on the growth of K562 cells was examined using MTT assay and cell differentiation was measured by distinct approaches including flow cytometry, benzidine staining and electron microscope. After CGI-100-konckdown K562 cells were incubated with 0.2 mg/ml of matrine or 30 Ixmol/L of hemin for 48 h, the expression levels of Glycophorin A（GPA）（CD235a） and Growth factor independence-lB mRNA（Gfi-IB mRNA） were measured by RT-PCR and the protein levels of GPA, CD14 and CD15 were detected by flow cytometry. Results： The eukaryotic expression vectors of CGI-100 RNAi were successfully constructed. The K562/shRNA-CGI-100 cell line was established in which the inhibition efficiency of CGI-100 gene expression by shRNA-CGI-100 was 54%. CGI-100-knockdown inhibited the proliferation and induced erythroid differentiation in K562 cells. Compared with the control K562 cells, the K562/shRNA-CGI-100 cells showed decreased absorbance value detected by MTT assay, decreased enchromation, increased heterochromation, increased percentage of G0/Gx phase cells, decreased population of S phase cells, decreased PI （proliferation index of cells）, and elevated percentage of benzidine-positive cells. Moreover, the sensitivity of K562/shRNA-CGI-100 cells to either matrine or hemin was enhanced and the sensitivity of these cells to matrine was higher than that to hemin. Compared with the control K562 cells, matrine treatment in K562/shRNA-CGI-100 cells resulted in increased inhibitory rate of proliferation, elevated percentage of be nzidine-positive cells, obviously up-regulated mRNA expressions of GPA and Gfi-IB, and increased mean fluorescence intensity （MFI） of GPA. No CD14 expression was detected and no statistical significance was found for the detected CD15. Finally, the MFI of GPA increased in K562/shRNA-CGI-100 cells treated with hemin and was 1.7 times less than that in cells exposed to matrine. Conclusion： These results suggest that the function of CGI-100 gene is correlated with the deregulated proliferation and the block of erythroid differentiation in K562 cells and may also be involved in matrine-induced erythroid differentiation in K562 ceils.
基金supported by the Natural Science Fundof Hunan Province(No.06jj5046,No.05jj30039)
文摘Objective To investigate the effect of exogenous S100A13 gene overexpression on the proliferation of human thyroid cancer cell line TT.Methods The recombinant ORF of S100A13 tagged with six histidines at the 5' end was subcloned into the pcDNA3.2/V5/GW/D-TOPO vector and sequenced.The eukaryotic expression plasmid pcDNA3.2/V5 /GW/D-S100A13 and empty vector pcDNA3.2/V5/GW/D were transfected into TT cells.The positive clones were selected by G418.The expressions of S100A13 mRNA and protein were detected by real time reverse transcription-polymerase chain reaction(RT-PCR) and Western blot.The effect of S100A13 on cell proliferation and cell cycle was evaluated by cell growth curve,MTT colorimetric assay and flow cytometry.Results S100A13 gene tagged with six histidines at the 5 ' end was confirmed to be inserted into the pcDNA3.2/V5/GW/D vector correctly.TT-S100A13-V5 cells,which over-expressed S100A13,were constructed successfully.TT-S100A13-V5 cells grew much faster than TT-V5 and TT cells(P <0.001).The proportions of both S and G2/M phase cells were significantly higher in TT-S100A13-V5 cells than those in TT-V5 and TT cells(P <0.001).Conclusion The eukaryotic expression vector containing human S100A13 gene has been successfully constructed,which highly expresses S100A13 in TT cells.Exogenous S100A13 gene overexpression accelerates TT cell proliferation and drives the cell cycle progression of TT cells from G0/G1 phase to S and G2/M phases.
文摘Amyloid beta (1-42) peptide is considered responsible for the formation of senile plaques that accumulate in the brain of patients with Alzheimer’s disease (AD). In the past years considerable attention has been focused on identifying new protective substances that prevent or almost retard the appearance of amyloid beta (1-42)-related neurotoxic effects. In this study, human neuroblastoma cells (IMR-32) was used as system model to evaluate the protective role of S100b, a neurotrophic factor and neuronal survival protein, that is highly expressed by reactive astrocytes in close vicinity of beta-amyloid deposits, against amyloid beta (1-42)-dependent toxicity. Our results show that at nanomolar concentrations, S100b protects cells against Aβmediated cytotoxicity, as assessed by MTS vitality test. The protective mechanism seems to be related to the effect on bcl-2 (an anti-apoptotic gene) expression, which is highly down-regulated by amyloid beta (1-42) treatment, while resulted more expressed in the presence of S100b. On the contrary, Bax, a proapoptotic gene, resulted down-regulated by the treatment with S100 compared with the results obtained in the presence of amyloid beta (1-42) peptide. However, at micromolar doses, S100b is toxic for IMR-32 cells and its toxicity adds to that of the Aβpeptide, suggesting that additional molecular mechanisms may be involved in theneurotoxic process.
