To set up a three-parameter method for cell cycle analysis by two-laser flowcy-tometer, which can detect two types of cyclin plus DNA content in one measurement, and thatanalyze unscheduled expression of cyclins. Meth...To set up a three-parameter method for cell cycle analysis by two-laser flowcy-tometer, which can detect two types of cyclin plus DNA content in one measurement, and thatanalyze unscheduled expression of cyclins. Methods: Three-color fluorescence was used for analysisof two types of cyclins and DNA content simultaneously in individual cells by two-laser flowcytometry. MOLT-4 cells were used to study the expression of major cyclins in mammalian cells. ATriton-X100 permeabilization procedure was optimized for detection of two types of cyclins. Onecyclin was stained directly with a FITC-conjugated monoclonal antibody (mAb), and the other,indirectly with RPE-Cy5-conjugated secondary antibody, while DNA was stained with the fluorochromeDAPI. mAMSA and mimosine treated MOLT-4 cells were used to test this three-parameter method.Results: Permeabilization with 0.5% Triton-XlOO in PBS containing 1% BSA for 5 min on ice providedoptimal conditions for the simultaneous labelling of two cyclins plus DNA in single cells. It wasfound that the emission spectrum of the three dyes (DAPI, FITC and RPE-Cy5) could be measured withno compensation. Based on cyclinA/cyclinE/DNA flow cytometric analysis, asynchronously growingMOLT-4 cells could be divided into 6 compartments (G1o, G1e, G1l, S, G2, and M) simultaneously,allowing for analysis of cell cycle phase specific perturbations without the necessity of cellsynchronization. Unscheduled cyclin B1 expression was observed in G1 cells treated with mimosine andcyclin E in G2 cells treated with mAMSA. We found that unscheduled cyclin expression paralleledexpected cyclin expression. Conclusion: Thus, three-color FCM analysis of cells may not only beapplied to measure unscheduled vs. expected cyclin expression but may also be used to estimate thefraction of cycling cells in up to 6 cell populations.展开更多
Circulating free nucleic acids; cell free DNA and circulating micro-RNA, are found in the plasma of patients with hematologic and solid malignancies at levels higher than that of healthy individuals. In patients with ...Circulating free nucleic acids; cell free DNA and circulating micro-RNA, are found in the plasma of patients with hematologic and solid malignancies at levels higher than that of healthy individuals. In patients with hematologic malignancy cell free DNA reflects the underlying tumor mutational profile, whilst micro-RNAs reflect genetic interference mechanisms within a tumor and potentially the surrounding microenvironment and immune effector cells. These circulating nucleic acids offer a potentially simple, non-invasive, repeatable analysis that can aid in diagnosis, prognosis and therapeutic decisions in cancer treatment.展开更多
To investigate the dynamics of plasma cAMP/cGMP in patients during cardiac surgery, and its relationship to traumatic stress. Methods: Sixteen patients, aged 19.31 years± 10.4 years , who underwent an open heart ...To investigate the dynamics of plasma cAMP/cGMP in patients during cardiac surgery, and its relationship to traumatic stress. Methods: Sixteen patients, aged 19.31 years± 10.4 years , who underwent an open heart operation with cardiopulmonary bypass (CPB) and hy pother mia were served as subjects. The arterial plasma concentrations of cyclic adenos ine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were measured by radioimmunoassay 2 hours before operation, after heparinization, 20 minutes following CPB, at the end of the operation, and 24 and 72 hours postoperatively, respectiv ely. The patients preoperative blood samples were heparinized and the venous b lood samples of 30 healthy blood donors were taken to measure the levels of cAMP and cGMP as heparin and normal controls separately. Results: There were no statistical difference among the heparin control, preoperative level and normal control. The peak values of cAMP and cGM P occurred during CPB and plasma cAMP levels changed synchronously with intensit ies of operative stimulus to human body. However cGMP level was mainly related t o the operative stimulus to the heart and CPB. The cAMP value was positivel y correlated with the cGMP value (r= 0.6313 , P< 0.001 ).Conclusions: Dynamic variation of plasma cyclic ribonucleotide can be considered as a reference parameter for intensity of traumatic stress.展开更多
基金This project was supported by grants from China Key Basic Research Program Grant (No. G1998051212) the National Natural Sciences Foundation of China (No. 39670265, 39730270 and 39725027) grants from the Science Foundation of Ministry of Public Health, China (No. 202-01-06).
文摘To set up a three-parameter method for cell cycle analysis by two-laser flowcy-tometer, which can detect two types of cyclin plus DNA content in one measurement, and thatanalyze unscheduled expression of cyclins. Methods: Three-color fluorescence was used for analysisof two types of cyclins and DNA content simultaneously in individual cells by two-laser flowcytometry. MOLT-4 cells were used to study the expression of major cyclins in mammalian cells. ATriton-X100 permeabilization procedure was optimized for detection of two types of cyclins. Onecyclin was stained directly with a FITC-conjugated monoclonal antibody (mAb), and the other,indirectly with RPE-Cy5-conjugated secondary antibody, while DNA was stained with the fluorochromeDAPI. mAMSA and mimosine treated MOLT-4 cells were used to test this three-parameter method.Results: Permeabilization with 0.5% Triton-XlOO in PBS containing 1% BSA for 5 min on ice providedoptimal conditions for the simultaneous labelling of two cyclins plus DNA in single cells. It wasfound that the emission spectrum of the three dyes (DAPI, FITC and RPE-Cy5) could be measured withno compensation. Based on cyclinA/cyclinE/DNA flow cytometric analysis, asynchronously growingMOLT-4 cells could be divided into 6 compartments (G1o, G1e, G1l, S, G2, and M) simultaneously,allowing for analysis of cell cycle phase specific perturbations without the necessity of cellsynchronization. Unscheduled cyclin B1 expression was observed in G1 cells treated with mimosine andcyclin E in G2 cells treated with mAMSA. We found that unscheduled cyclin expression paralleledexpected cyclin expression. Conclusion: Thus, three-color FCM analysis of cells may not only beapplied to measure unscheduled vs. expected cyclin expression but may also be used to estimate thefraction of cycling cells in up to 6 cell populations.
文摘Circulating free nucleic acids; cell free DNA and circulating micro-RNA, are found in the plasma of patients with hematologic and solid malignancies at levels higher than that of healthy individuals. In patients with hematologic malignancy cell free DNA reflects the underlying tumor mutational profile, whilst micro-RNAs reflect genetic interference mechanisms within a tumor and potentially the surrounding microenvironment and immune effector cells. These circulating nucleic acids offer a potentially simple, non-invasive, repeatable analysis that can aid in diagnosis, prognosis and therapeutic decisions in cancer treatment.
文摘To investigate the dynamics of plasma cAMP/cGMP in patients during cardiac surgery, and its relationship to traumatic stress. Methods: Sixteen patients, aged 19.31 years± 10.4 years , who underwent an open heart operation with cardiopulmonary bypass (CPB) and hy pother mia were served as subjects. The arterial plasma concentrations of cyclic adenos ine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were measured by radioimmunoassay 2 hours before operation, after heparinization, 20 minutes following CPB, at the end of the operation, and 24 and 72 hours postoperatively, respectiv ely. The patients preoperative blood samples were heparinized and the venous b lood samples of 30 healthy blood donors were taken to measure the levels of cAMP and cGMP as heparin and normal controls separately. Results: There were no statistical difference among the heparin control, preoperative level and normal control. The peak values of cAMP and cGM P occurred during CPB and plasma cAMP levels changed synchronously with intensit ies of operative stimulus to human body. However cGMP level was mainly related t o the operative stimulus to the heart and CPB. The cAMP value was positivel y correlated with the cGMP value (r= 0.6313 , P< 0.001 ).Conclusions: Dynamic variation of plasma cyclic ribonucleotide can be considered as a reference parameter for intensity of traumatic stress.
