Objective To study the differences and similarities of the antisense drugs with different structures on the biological functions of HL60 cells. Methods Cytotoxic effects were measured by cell viability assay. The e...Objective To study the differences and similarities of the antisense drugs with different structures on the biological functions of HL60 cells. Methods Cytotoxic effects were measured by cell viability assay. The expression levels of protein were assayed by immunofluorescence using fluoresce isothiocyanate label. The morphological changes in apoptotic cells were observed. Flow cytometric analysis of DNA fragmentation was also performed. Results Antisense peptide nucleic acid (PNA) targeting the coding region of the Bcl-2 mRNA could effectively inhibit the growth of HL60 cells, down-regulate the synthesis of Bcl-2 protein and induce apoptosis. After HL60 cells were treated with 10 μmol/L Bcl-2 antisense PNA or antisense oligonucleotide for 72 h respectively, apoptotic rates of HL60 cells were 17.80±1.53 and 13.17±1.12, respectively( P <0.05). Conclusion Antisense PNA targeting the coding region of Bcl-2 mRNA may have stronger antisense effects than the antisense oligonucleotides and could induce apoptosis of HL60 cells.展开更多
Objective To optimize experimental parameters for the photosensitization of 5-aminolevulinic acid (ALA) in promyelocytic leukemia cell HL60 and compare them with normal human peripheral blood mononuclear cell (PBMC). ...Objective To optimize experimental parameters for the photosensitization of 5-aminolevulinic acid (ALA) in promyelocytic leukemia cell HL60 and compare them with normal human peripheral blood mononuclear cell (PBMC). Methods ALA incubation time, wavelength applied to irradiate, concentration of ALA incubated, irradiation fluence may modulate the effect of 5-aminolevulinic acid based Photodynamic Therapy (ALA-PDT).The high-pressure mercury lamps of 400W served as light source, the interference filter of 410nm, 432nm, 545nm, 577nm were used to select the specific wavelength. Fluorescence microscope was used to detect the fluorescence intensity and location of protoporphyrin IX (PpIX) endogenously produced by ALA. MTT assay was used to measure the survival of cell. Flow cytometry with ANNEXIN V FITC kit (contains annexin V FITC, binding buffer and PI) was used to detect the mode of cell death. Results ① 1mmol/L ALA incubated 1×105/mL HL60 cell line for 4 hours, the maximum fluorescence of ALA induced PpIX was detected in cytomembrane. ② Irradiated with 410nm for 14.4J/cm2 can result in the minimum survivability of HL60 cell. ③ The main mode of HL60 cell death caused by ALA-PDT is necrosis. Conclusion ALA for 1mmol/L, 4 hours for dark incubation time, 410nm for irradiation wavelength, 14.4J/cm2 for irradiation fluence were the optimal parameters to selectively eliminate promyelocytic leukemia cell HL60 by ALA based PDT. The photosensitization of ALA based PDT caused the necrosis of HL60 cell, so it could be used for inactivation of certain leukemia cells.展开更多
Objective: To clone the gene associated with apoptosis induced by an inhibitor of polyamine biosynthesis, a-difluoromethylornithime(DFMO). Methods: The differential sufbtraction sereening was used for gene cloning f...Objective: To clone the gene associated with apoptosis induced by an inhibitor of polyamine biosynthesis, a-difluoromethylornithime(DFMO). Methods: The differential sufbtraction sereening was used for gene cloning from cDNA library of HL60 cells treated by DFMO. Northern blot,morphological observation, FCM assays and ladder map of DNA electrophoresis were performed. Results: The transfectiong gene expression and activity of inducing apoptosis in the cell transfected from recombinant plasmid containing the cloned fragment df4 wasproved. Conclusion: It is suggest that df4 gene cloned in the study coul be a gene regulating apoptosis of HL60 cells.展开更多
To explore the effect of NF κB on bcl x gene transcription in extended drug resistance leukemia cell line HL 60/E6, drug resistant subline HL 60/E6 was derived by intermittently exposing HL 60 cells to 6 ng/ml ...To explore the effect of NF κB on bcl x gene transcription in extended drug resistance leukemia cell line HL 60/E6, drug resistant subline HL 60/E6 was derived by intermittently exposing HL 60 cells to 6 ng/ml epirubicin. Indirect immunofluorescence was used to demonstrate the location of NF κB RelA in HL 60/E6 cells. FCM analysis and RT PCR were used to detect the efficiency of liposome mediated ODN transfection and the change of bcl x L mRNA levels after 5 μmol/L phosphorothioate (PS) derivatized antisense (AS) oligodeoxynucleotide (ODN) directed to RelA was transferred into HL 60/E6 cells. The results showed that RelA remained persistently active and located at the nuclei of HL 60/E6 cells,but in the cytoplasm of HL 60 cells, the efficiency of liposome mediated ODN transfection was significantly higher than that of null ODN ( P <0.01 in 4 h, 6 h, 12 h, 24 h). Exposure of HL 60/E6 cells to 5 μmol/L AS PS ODN directed to RelA led to a maximal 40 % decline of bcl x L mRNA levels within 8 h. The inhibition rate of bcl x L mRNA was (15±1.79) %, (28±2.34) %, (40±3.47) %, (20±1.54) % in 4 h, 6 h, 8 h, 15 h, respectively, but it was less than 15 % in control group. It was concluded that NF κB was involved in regulating bcl x transcription. It was suggested that NF κB was an important factor for drug resistance in leukemia cells.展开更多
Aim To study the effect of 8 chloroadenosine (8 CA)on undifferentiatied HL 60 cell line. Methods The IC 50 of cancer cell proliferation was determined using a microculture plate reader at 570 nm (MTT) and 54...Aim To study the effect of 8 chloroadenosine (8 CA)on undifferentiatied HL 60 cell line. Methods The IC 50 of cancer cell proliferation was determined using a microculture plate reader at 570 nm (MTT) and 540 nm (SRB). Morphology of HL 60 cells was observed under a scanning electron microscope and a transmission electron microscope. The differentiation of HL 60 cells was examined by nitro blue tetrazolium reduction (NBT) and acid phosphatase assay. The cycle of HL 60 cells was analyzed by flow cytometry. Results 8 CA inhibited proliferation of eight human cancer cell lines. The IC 50 ranked in the following order: KB (0 05 μmol·L -1 ) < HL 60 (0 25 μmol·L -1 ) < Bel 7402 (0 56 μmol·L -1 ) < MCF 7 (0 65 μmol·L -1 ) < HCT (0 79 μmol·L -1 ) < HeLa (0 89 μmol·L -1 ) < BGC 823 (1 149 μmol·L -1 ) < PG (2 50 μmol·L -1 ). The scanning and transmission electron microscopy showed that the microvilli of HL 60 cell surface shortened, and the shape of HL 60 cells nuclei changed to kidney shaped, horse shoe shaped and bilob ated after treatment with 8 CA. Meanwhile, 8 CA promoted NBT reduction and increased activity of acid phosphatase in HL 60 cells in a time and concentration dependent manner. Flow cytometry analysis indicated that 8 CA induced an appreciable increase of the cell population in G 1 phase with a marked reduction in S phase. Conclusion 8 CA can induce differentiation of HL 60 cells and block the cells at G 1 phase, thus inhibiting proliferation of HL 60 cells.展开更多
The in vitro anti-proliferative activity(pICi,i=hp,ca,hl)of fluoroquinolone(rhodanineα,β-unsaturated ketone)amide compounds,referred to as“fluoroquinolone amide derivatives(FQADs)”towards Hep-3B,Capan-1 and HL60 c...The in vitro anti-proliferative activity(pICi,i=hp,ca,hl)of fluoroquinolone(rhodanineα,β-unsaturated ketone)amide compounds,referred to as“fluoroquinolone amide derivatives(FQADs)”towards Hep-3B,Capan-1 and HL60 cells,was studied by the 3D-QSAR method of comparative molecular field analysis(CoMFA).Based on the training set of 14 compounds,the prediction model was established,which was further verified by the test set of 5 compounds with template molecule included.It is found that steric and electrostatic fields contribute 66.8%and 33.2%to pIChp,61.4%and 38.6%to pICca,and 61.5%and 38.5%to pIChl,respectively.The Rcv 2(i.e,cross-validation coefficient)is 0.324,0.381,and 0.421 for pIChp,pICca,and pIChl,respectively,while the corresponding R2(i.e,non-cross-validation coefficient)all reach 0.999.Then,the models were employed to estimate the activities of the training and test compounds,and the results show that the stability and predictability of developed models are very satisfactory.According to the contour maps of steric and electronic fields,bulky groups linked to 2-,3-,4-positions of phenyl ring,and electropositive groups near the 4-position and electronegative groups far away may increase the anti-proliferative activity.Using the information provided by the 3D contour maps,four new FQADs owing higher antiproliferative activity were designed,but their effectiveness should be further tested by experiments.展开更多
Aiming to better understand the physiochemical properties of lignite, we select Zhaotong lignite as object and adopt simulation and experiment data to construct its molecular structure. Firstly, the important paramete...Aiming to better understand the physiochemical properties of lignite, we select Zhaotong lignite as object and adopt simulation and experiment data to construct its molecular structure. Firstly, the important parameters including carbon skeleton, valence state and functional group of the sample are obtained by ultimate analysis, 13 C NMR, XPS and Py-GC/MS. Results indicate that the ratio of aromatic carbon and aromatic bridge carbon to surrounding carbon of the sample are 40.32% and 0.14, respectively. Such results imply that the aromatic structure of the sample is dominated by benzene and naphthalene. Moreover, the ratio of aliphatic carbon is 51.55%, and the aliphatic structure is mainly comprised by methyl, methylene, quaternary carbon and oxygen-aliphatic carbon. Oxygen atoms principally exist in ether, carbonyl and carboxyl groups, of which ether accounts for 70.2%. Additionally, the contents of pyridine, pyrrole and quaternary nitrogen are 25.2%, 46.3% and 13.0%, respectively. Based on the aforementioned results, the molecular structure model of Zhaotong lignite is constructed by the method of computer-aided molecular design. Subsequently, the molecular formula of Zhaotong lignite is calculated as C;H;O;N;. Finally, in order to verify the reasonability of the constructed model, the 13 C NMR of the molecular structure model is simulated by employing the basis set of GIAO/6-31G at the Gaussian 09 computing platform. These simulated results agree well with the experimental ones, which suggests that the molecular structure model of Zhaotong lignite is accurate and reasonable.展开更多
文摘Objective To study the differences and similarities of the antisense drugs with different structures on the biological functions of HL60 cells. Methods Cytotoxic effects were measured by cell viability assay. The expression levels of protein were assayed by immunofluorescence using fluoresce isothiocyanate label. The morphological changes in apoptotic cells were observed. Flow cytometric analysis of DNA fragmentation was also performed. Results Antisense peptide nucleic acid (PNA) targeting the coding region of the Bcl-2 mRNA could effectively inhibit the growth of HL60 cells, down-regulate the synthesis of Bcl-2 protein and induce apoptosis. After HL60 cells were treated with 10 μmol/L Bcl-2 antisense PNA or antisense oligonucleotide for 72 h respectively, apoptotic rates of HL60 cells were 17.80±1.53 and 13.17±1.12, respectively( P <0.05). Conclusion Antisense PNA targeting the coding region of Bcl-2 mRNA may have stronger antisense effects than the antisense oligonucleotides and could induce apoptosis of HL60 cells.
文摘Objective To optimize experimental parameters for the photosensitization of 5-aminolevulinic acid (ALA) in promyelocytic leukemia cell HL60 and compare them with normal human peripheral blood mononuclear cell (PBMC). Methods ALA incubation time, wavelength applied to irradiate, concentration of ALA incubated, irradiation fluence may modulate the effect of 5-aminolevulinic acid based Photodynamic Therapy (ALA-PDT).The high-pressure mercury lamps of 400W served as light source, the interference filter of 410nm, 432nm, 545nm, 577nm were used to select the specific wavelength. Fluorescence microscope was used to detect the fluorescence intensity and location of protoporphyrin IX (PpIX) endogenously produced by ALA. MTT assay was used to measure the survival of cell. Flow cytometry with ANNEXIN V FITC kit (contains annexin V FITC, binding buffer and PI) was used to detect the mode of cell death. Results ① 1mmol/L ALA incubated 1×105/mL HL60 cell line for 4 hours, the maximum fluorescence of ALA induced PpIX was detected in cytomembrane. ② Irradiated with 410nm for 14.4J/cm2 can result in the minimum survivability of HL60 cell. ③ The main mode of HL60 cell death caused by ALA-PDT is necrosis. Conclusion ALA for 1mmol/L, 4 hours for dark incubation time, 410nm for irradiation wavelength, 14.4J/cm2 for irradiation fluence were the optimal parameters to selectively eliminate promyelocytic leukemia cell HL60 by ALA based PDT. The photosensitization of ALA based PDT caused the necrosis of HL60 cell, so it could be used for inactivation of certain leukemia cells.
文摘Objective: To clone the gene associated with apoptosis induced by an inhibitor of polyamine biosynthesis, a-difluoromethylornithime(DFMO). Methods: The differential sufbtraction sereening was used for gene cloning from cDNA library of HL60 cells treated by DFMO. Northern blot,morphological observation, FCM assays and ladder map of DNA electrophoresis were performed. Results: The transfectiong gene expression and activity of inducing apoptosis in the cell transfected from recombinant plasmid containing the cloned fragment df4 wasproved. Conclusion: It is suggest that df4 gene cloned in the study coul be a gene regulating apoptosis of HL60 cells.
文摘To explore the effect of NF κB on bcl x gene transcription in extended drug resistance leukemia cell line HL 60/E6, drug resistant subline HL 60/E6 was derived by intermittently exposing HL 60 cells to 6 ng/ml epirubicin. Indirect immunofluorescence was used to demonstrate the location of NF κB RelA in HL 60/E6 cells. FCM analysis and RT PCR were used to detect the efficiency of liposome mediated ODN transfection and the change of bcl x L mRNA levels after 5 μmol/L phosphorothioate (PS) derivatized antisense (AS) oligodeoxynucleotide (ODN) directed to RelA was transferred into HL 60/E6 cells. The results showed that RelA remained persistently active and located at the nuclei of HL 60/E6 cells,but in the cytoplasm of HL 60 cells, the efficiency of liposome mediated ODN transfection was significantly higher than that of null ODN ( P <0.01 in 4 h, 6 h, 12 h, 24 h). Exposure of HL 60/E6 cells to 5 μmol/L AS PS ODN directed to RelA led to a maximal 40 % decline of bcl x L mRNA levels within 8 h. The inhibition rate of bcl x L mRNA was (15±1.79) %, (28±2.34) %, (40±3.47) %, (20±1.54) % in 4 h, 6 h, 8 h, 15 h, respectively, but it was less than 15 % in control group. It was concluded that NF κB was involved in regulating bcl x transcription. It was suggested that NF κB was an important factor for drug resistance in leukemia cells.
文摘Aim To study the effect of 8 chloroadenosine (8 CA)on undifferentiatied HL 60 cell line. Methods The IC 50 of cancer cell proliferation was determined using a microculture plate reader at 570 nm (MTT) and 540 nm (SRB). Morphology of HL 60 cells was observed under a scanning electron microscope and a transmission electron microscope. The differentiation of HL 60 cells was examined by nitro blue tetrazolium reduction (NBT) and acid phosphatase assay. The cycle of HL 60 cells was analyzed by flow cytometry. Results 8 CA inhibited proliferation of eight human cancer cell lines. The IC 50 ranked in the following order: KB (0 05 μmol·L -1 ) < HL 60 (0 25 μmol·L -1 ) < Bel 7402 (0 56 μmol·L -1 ) < MCF 7 (0 65 μmol·L -1 ) < HCT (0 79 μmol·L -1 ) < HeLa (0 89 μmol·L -1 ) < BGC 823 (1 149 μmol·L -1 ) < PG (2 50 μmol·L -1 ). The scanning and transmission electron microscopy showed that the microvilli of HL 60 cell surface shortened, and the shape of HL 60 cells nuclei changed to kidney shaped, horse shoe shaped and bilob ated after treatment with 8 CA. Meanwhile, 8 CA promoted NBT reduction and increased activity of acid phosphatase in HL 60 cells in a time and concentration dependent manner. Flow cytometry analysis indicated that 8 CA induced an appreciable increase of the cell population in G 1 phase with a marked reduction in S phase. Conclusion 8 CA can induce differentiation of HL 60 cells and block the cells at G 1 phase, thus inhibiting proliferation of HL 60 cells.
基金supported by the National Natural Science Foundation of China (21676292, 21075138)special fund of State Key Laboratory of Structure Chemistry (2016028)
文摘The in vitro anti-proliferative activity(pICi,i=hp,ca,hl)of fluoroquinolone(rhodanineα,β-unsaturated ketone)amide compounds,referred to as“fluoroquinolone amide derivatives(FQADs)”towards Hep-3B,Capan-1 and HL60 cells,was studied by the 3D-QSAR method of comparative molecular field analysis(CoMFA).Based on the training set of 14 compounds,the prediction model was established,which was further verified by the test set of 5 compounds with template molecule included.It is found that steric and electrostatic fields contribute 66.8%and 33.2%to pIChp,61.4%and 38.6%to pICca,and 61.5%and 38.5%to pIChl,respectively.The Rcv 2(i.e,cross-validation coefficient)is 0.324,0.381,and 0.421 for pIChp,pICca,and pIChl,respectively,while the corresponding R2(i.e,non-cross-validation coefficient)all reach 0.999.Then,the models were employed to estimate the activities of the training and test compounds,and the results show that the stability and predictability of developed models are very satisfactory.According to the contour maps of steric and electronic fields,bulky groups linked to 2-,3-,4-positions of phenyl ring,and electropositive groups near the 4-position and electronegative groups far away may increase the anti-proliferative activity.Using the information provided by the 3D contour maps,four new FQADs owing higher antiproliferative activity were designed,but their effectiveness should be further tested by experiments.
基金supported by the National Natural Science Foundation of China (21766013)Analysis and Testing Foundation of Kunming University of Science and Technology (2020M20192208021)
文摘Aiming to better understand the physiochemical properties of lignite, we select Zhaotong lignite as object and adopt simulation and experiment data to construct its molecular structure. Firstly, the important parameters including carbon skeleton, valence state and functional group of the sample are obtained by ultimate analysis, 13 C NMR, XPS and Py-GC/MS. Results indicate that the ratio of aromatic carbon and aromatic bridge carbon to surrounding carbon of the sample are 40.32% and 0.14, respectively. Such results imply that the aromatic structure of the sample is dominated by benzene and naphthalene. Moreover, the ratio of aliphatic carbon is 51.55%, and the aliphatic structure is mainly comprised by methyl, methylene, quaternary carbon and oxygen-aliphatic carbon. Oxygen atoms principally exist in ether, carbonyl and carboxyl groups, of which ether accounts for 70.2%. Additionally, the contents of pyridine, pyrrole and quaternary nitrogen are 25.2%, 46.3% and 13.0%, respectively. Based on the aforementioned results, the molecular structure model of Zhaotong lignite is constructed by the method of computer-aided molecular design. Subsequently, the molecular formula of Zhaotong lignite is calculated as C;H;O;N;. Finally, in order to verify the reasonability of the constructed model, the 13 C NMR of the molecular structure model is simulated by employing the basis set of GIAO/6-31G at the Gaussian 09 computing platform. These simulated results agree well with the experimental ones, which suggests that the molecular structure model of Zhaotong lignite is accurate and reasonable.
