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OBJECTIVE To study the methodology of achieving stable co-expression of drug-metab?olizing enzymes in the HepG2 cells by the piggyBac (PB) transposon system. METHODS N-terminal attachment of enhanced green fluorscent protein plasmid (pEGFP- N2) and 2A peptide linked recombinant PB transposon plasmid containing dual-genes encoding drug metabolizing enzymes cyto?chrome P450 3A4 (CYP3A4) and CYP2C19 (pPB-CYP3A4-2A-2C19) were transfected into HepG2 cells respectively by Lipofectamine?LTX reagent, GenJetTM (Ver.Ⅱ) reagent and Neon?Transfection System reagent, which were widely used for large-sized DNA fragments transfection. 48 h later, the transfection efficiency and cell toxicity were detected and compared between the three methods so as to find a method with relatively high efficiency and low toxicity for later transfection.Then,three groups of recombinant PB transposons-single-gene transposon (PB-CYP3A4), 2A peptide linked dual-gene transposon (PB-CYP3A4-2A-2C19) and multiple single-gene transposon mixture〔PB-CYP3A4, PB-CYP2C8, PB-CYP2A6, organic anion transporting polypeptide 1B1 PB transposon (PB-OATP1B1)〕-were transfected into HepG2 cells respectively with the above established method.The puromycin (Puro)-resistant and GFP positive cell clones were picked up and further cultured. The mRNA, protein and metabolic levels of drug-metabolizing enzymes in monoclonal cell lines were detected by quantitative real-time PCR,Western blotting and high performance liquid chromatography-tandem mass spectrometry respectively after screening by Puro and green fluorescence. Comparisons of different groups were made using statistical analysis. RESULTS The comparison of three different transfection methods indi?cated that the transfection efficiency of GenJetTMwas up to(94.2±2.5)% and (89.3±3.3)%,significantly higher than those of the other two methods (P<0.01), along with lower cytotoxicity. Then GenJetTMwas chosen for later transfection. In the Puro-resistant monoclonal cell lines of single transposon PB-CYP3A4,PB-CYP3A4-2A-2C19 groups,the mRNA,protein and enzyme activity levels of drug-metabo?lizing enzymes were significantly increased respectively.The recombinant transposon (PB-CYP3A4-2A-2C19) containing 2A peptide could achieve stable and efficient co-expression of two metabolizing enzymes CYP3A4 and CYP2C19,while the expression of drug-metabolizing enzymes remained unbal?anced and random in those of multiple single-gene transposon mixture group (PB-CYP3A4, PB-CYP2C8,PB-CYP2A6,PB-OATP 1B1)(CYP3A4 was expressed in some cell clones only).CONCLUSION GenJetTM could be an effective method for the PB recombinant transposon transfection into HepG2 cells, by which the PB transposon could mediate stable expression of drug-metabolizing enzymes. In terms of multi-gene expression,a low and unbalanced expression is found by multiple transposons co-transfection method,which is different from that by virus mediated method.In contrast,mono-PB trans?poson linked by 2A peptide can achieve stable expression of multi-genes.
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Objective To investigate the impact of the regional cooperative chest pain center (CPC) on therapeutic time and short term outcome after primary percutaneous coronary intervention (PCI) of patients with ST segment elevated myocardial infarction.Methods 372 patients with ST segment elevated myocardial infarction were enrolled in the study who had received were operated primary PCI 18 months before and after the regional cooperative CPC was set up.There were 156 patients in the green channel group before the setup of CPC and 216 patients in the CPC group.Total ischemia time,first medical contact (FMC) time,FML-to-balloon (FMC2B) time,door-to-balloon (D2B) time,hospital mortality rates,cardiac failure rates on the next day after PCI,length of CCU stays and hospital stays were compared between the two groups.Results Compared to the green channel group,total ischemia time[(281.0±102.7)min vs.(365.2±115.6)min,P＜0.05],FMC time [(174.3±97.5) min vs.(225.4±104.6) min,P＜0.05],FMC2B time [(106.7±61.2) min vs.(139.8±75.7) min,P＜0.05] and D2B time [(75.2±45.4) min vs.(102.4±53.7) min,P＜0.05] of the CPC group were significant shorter.The rates of reaching the standard of FMC2B time (70.83％ vs.34.62％,P＜0.001) and D2B time (75.93％ vs.40.38％,P＜0.001)were significantly higher in the CPC group.Cardiac failure rates on the next day after PCI was lower in the CPC group (14.35％ vs.23.72％,P=0.021),and CCU stays was shorter [(64.3±13.72)h vs.(92.6±15.65)h,P=0.043].Conclusions Establishment of a standardized regional cooperative CPC requires combination and consideration of the characteristics of local resources FMC2B time and D2B time of STEMI patients can be shorten by a standardized CPC lending to further shortening of total ischemia time and improvement in cardiac function.
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Objective:To investigate the impact of different hospitals on reperfusion time in acute ST-segment elevation myocardial infarction (STEMI) patients from regional cooperative chest pain center (CPC).Methods:A total of 364 STEMI patients received percutaneous coronary intervention (PCI) at 18 months before and after CPC establishment were enrolled.Based on hospital levels,the patients were divided into 2 groups:Initial PCI hospital group,n=197 and Initial non-PCI hospital group,n=167.According to hospital visiting time,Initial PCI hospital group was further divided into 2 subgroups as Green channel subgroup,n=91 and CPC subgroup,n=106;Initial non-PCI hospital group was further divided into 2 subgroups as Routine referral subgroup,n=71 and CPC referral subgroup,n=96.Total ischemia time,from onset to first medical contact (S-FMC) time,from S-FMC to balloon dilatation (FMC2B) time,from hospital visit to balloon dilatation (D2B) time were compared among relevant patients;the impact factors for total ischemia time were studied by multivariate regression analysis.Results:Compared with Routine referral subgroup,the following parameters were shortened in CPC referral subgroup:total ischemia time 325 (236,1185) min vs 367 (214,1340) min,P＜0.05;FMC2B time 115 (82,227) min vs 149 (94,483) min,P＜0.05;D2B time 69 (35,195) min vs 105 (55,260) min,P＜0.05.Compared with CPC referral subgroup,the following parameters were further shortened in Initial PCI hospital group:total ischemia time 283 (168,873) min vs 325 (236,1185) min,P＜0.05;FMC2B time 78 (45,265) min vs 115 (82,227) min,P＜0.05.Multivariate linear regression analysis presented that high school or above education (β=-0.117,P=0.047),arrived PCI hospital within 60 min of onset (β=-0.243,P=0.000)and using initial PCI hospital (β=-0.175,P=0.000) were the independent impact factors for total ischemia time in STEMI patients.Conclusion:Regional cooperative CPC may shorten FMC2B time by patients' referral;visiting PCI hospital within 60 min of onset was the best way to reduce total isehemia time in STEMI patients.
ABSTRACT
Objective:To investigate the impact of different hospitals on reperfusion time in acute ST-segment elevation myocardial infarction (STEMI) patients from regional cooperative chest pain center (CPC).Methods:A total of 364 STEMI patients received percutaneous coronary intervention (PCI) at 18 months before and after CPC establishment were enrolled.Based on hospital levels,the patients were divided into 2 groups:Initial PCI hospital group,n=197 and Initial non-PCI hospital group,n=167.According to hospital visiting time,Initial PCI hospital group was further divided into 2 subgroups as Green channel subgroup,n=91 and CPC subgroup,n=106;Initial non-PCI hospital group was further divided into 2 subgroups as Routine referral subgroup,n=71 and CPC referral subgroup,n=96.Total ischemia time,from onset to first medical contact (S-FMC) time,from S-FMC to balloon dilatation (FMC2B) time,from hospital visit to balloon dilatation (D2B) time were compared among relevant patients;the impact factors for total ischemia time were studied by multivariate regression analysis.Results:Compared with Routine referral subgroup,the following parameters were shortened in CPC referral subgroup:total ischemia time 325 (236,1185) min vs 367 (214,1340) min,P＜0.05;FMC2B time 115 (82,227) min vs 149 (94,483) min,P＜0.05;D2B time 69 (35,195) min vs 105 (55,260) min,P＜0.05.Compared with CPC referral subgroup,the following parameters were further shortened in Initial PCI hospital group:total ischemia time 283 (168,873) min vs 325 (236,1185) min,P＜0.05;FMC2B time 78 (45,265) min vs 115 (82,227) min,P＜0.05.Multivariate linear regression analysis presented that high school or above education (β=-0.117,P=0.047),arrived PCI hospital within 60 min of onset (β=-0.243,P=0.000)and using initial PCI hospital (β=-0.175,P=0.000) were the independent impact factors for total ischemia time in STEMI patients.Conclusion:Regional cooperative CPC may shorten FMC2B time by patients' referral;visiting PCI hospital within 60 min of onset was the best way to reduce total isehemia time in STEMI patients.