In the present study, packaging system composed of pAAV-CMV-GFP, pAAV-RC and pHelper were transfected into human embryonic kidney 293 cells (HEK293 cells) mediated by polyethyleneimine (PEI) to explore an optimal transfection condition. Different total plasmid DNA dosages (1, 2, 3, 4, 5, 6 μg) and different PEI/Plasmid ratios (1:1, 3:1, 5:1, 7:1) were tested with detection of green fluorescence protein (GFP) with ImagePro Plus6. 0 Software. Then transfection efficiency of the optimized transfection system was further observed for different time periods(12, 24, 36, 48, 60, 72 h). The results showed that total plasmid dosage of 4 μg/well with PEI/plasmid ratio of 3 : 1-5 : 1 was an efficient transfection condition. Transfection efficiency-time curve was an S-shaped curve. Transfection efficiency reached a plateau at 60 h after transfection. The optimized conditions for PEI-mediated transfection at the optimal time result in enhanced transfection efficiency of triple plasmid into HEK293 cells.
Green Fluorescent Proteins ; HEK293 Cells ; Humans ; Plasmids ; Polyethyleneimine ; Transfection ; methods

Objective To observe the effects of vaporized perfluorocarbon (PFC) combined with exogenous pulmonary surfactant (PS) inhalation on rabbit models of acute lung injury (ALI).Methods Thirty-two New Zealand rabbits were randomly divided into four groups: ALI group, combination treatment group, PFC group, and PS group (each groupn = 8 rabbits). The rabbit model of ALI was induced by the whole lung normal saline lavage. After modeling, in the combined group, 3 mL/kg vaporized perfluorooctyl bromide/dipalmitoylphosphatidylcholine (PFOB/DPPC) emulsion was inhaled, the rabbits in PFC and PS groups were treated with vaporized PFOB emulsion and vaporized DPPC emulsion 3 mL/kg inhalation respectively, and in the ALI group was given the same amount of vaporized normal saline inhalation. In each group, before modeling for 30 minutes (basic value), after modeling for 1 hour and after treatment at 0 minute, 30 minutes, 2 hours, 4 hours, the respiratory rate (RR), oxygenation index (OI), dynamic lung compliance (Cdyn) were observed, and the lung coefficient (LI) and lung permeability index (LPI) were calculated; the levels of serum tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were measured by double antibody sandwich enzyme-linked immunosorbent assay (ELISA); the lung tissue was collected and the lung pathological changes were observed under macroscopic and microscopic observation.Results Aftermodeling, the levels of OI, Cdyn were quickly lowered, RR became significantly elevated, and there were obvious edema, hemorrhage and exudation in lung tissue of ALI group. The levels of OI were significantly increased in combined group and PFC group compared with the level in ALI group after treatment at 0 minute initially [mmHg (1 mmHg = 0.133 kPa): 231.0±16.7, 221.4±19.0 vs. 189.5±21.0, both P < 0.05], while the level of OI in PS group was increased significantly until 4 hours after treatment, being higher than that in ALI group (mmHg: 297.0±20.7 vs. 243.3±36.7,P < 0.05); RR was decreased significantly in combined treatment group at 30 minutes after treatment compared with that in ALI group (bpm: 151.1±13.3 vs. 178.5±32.0,P < 0.05), while the RR in PFC group and PS group were not increased significantly until 4 hours after treatment being higher than that in ALI group (bpm: 129.3±14.3, 133.1±13.9 vs. 157.5±32.5, bothP < 0.05). Compared to ALI group, the three treatment groups resulted in significant improvement in Cdyn right at 0 minute (mL/cmH2O: 1.64±0.10, 1.45±0.10, 1.43±0.09 vs. 0.57±0.05, allP < 0.05), their LPI, LI and inflammatory cytokines were significantly decreased [LPI (×10-5): 4.21±0.42, 4.76±0.55, 4.87±0.49 vs. 5.56±0.52, LI: 8.04±0.58, 8.90±0.88, 9.22±0.71 vs. 10.85±0.73, TNF-α (ng/L): 50.05±4.91, 56.18±5.54, 63.60±5.96 vs. 73.60±5.27, IL-1β (ng/L): 34.27±4.55, 40.29±5.03, 48.13±6.38 vs. 54.71±4.26, allP<0.05], and pulmonary edema, congestion and inflammatory cell infiltration were obviously ameliorated (pathological scores: 3.74±0.58, 4.50±0.75, 5.29±0.72 vs. 6.13±0.72, P < 0.05). Cdyn levels were increased significantly in combined treatment group at 0 minute, 30 minutes, 4 hours after treatment compared with thosein PFC and PS group, but there were no significant differences between PFC and PS group. Levels of LI, LPI, inflammatory factors and pathological scores were decreased significantly in combined treatment group compared with those in PFC and PS group, the degrees of improvement of inflammatory factors and pathological scores in PFC group were more obvious than those in PS group (allP < 0.05).Conclusions PFOB combined with DPPC inhalation can provide greater oxygen delivery, reduce the pro-inflammatory cytokines, supplement PS and influence its distribution on the surface of lung, which might lead to a marked and sustained improvement in oxygenation, pulmonary function and amelioration of lung edema and inflammatory reaction in saline lavage induced lung injury of rabbits.

Objective: To explore the risk factors for Type 1 cardio-renal syndrome (CRS1) atfer ST-segment elevation myocardial infarction (STEMI). Methods: A total of 378 patients with STEMI were divided into two groups: a CRS1 group (n=98) and a non-CRS1 group (n=280). Clinical characteristics in the 2 groups were compared, and independent risk factors for CRS1 after STEMI were analyzed, and the effect of emergency Results: In the 378 STEMI patients, CRS1 was found in 98 patients (25.9%). Between the 2 groups, there was significant difference in 12 parameters, including age, history of diabetes, admission mean arterial pressure, admission systolic blood pressure, admission heart rate, Killip classification, left ventricular ejection fraction, baseline serum creatinine, baseline evaluated glomerular ifltration rate (eGFR), emergency PCI, β-blockers and angiotensin converting enzyme inhibitor/angiotensin, receptor antagonist (ACEI/ARB) application (allP<0.05). Multivariate logistic regression showed that age, history of diabetes, admission systolic blood pressure, Killip classification, reduced left ventricular ejection fraction, reduced eGFR, emergency PCI non-undergo and ACEI/ARB non-use were independent risk factors for CRS1 atfer STEMI. In the 256 patients undergoing emergency PCI, 50 patients (19.5%) had CRS1. hTe door-ball time and the amount of contrast agent in the CRS1 group were signiifcantly higher than those in the non- CRS1 group (bothP<0.05), but there was no signiifcant difference in the blood lfow in the “culprit vessel”atfer the PCI (P>0.05). Conclusion: CRS1 is a common complication of STEMI, which is associated with many factors. Immediate revascularization can reduce the incidence of CRS1 in patients with ST-segment elevation myocardial infarction.

Pulmonary embolism (PE) refers to the endogenous or exogenous emboli blocking pulmonary trunk or branches, causing clinical and pathophysiological syndrome of pulmonary circulation disorder, the incidence rate is high. Sometimes PE patients were lack of specific symptoms and signs, or without any symptoms, which often result in misdiagnosis, un-timely diagnosis, and the delay of treatment. A PE case with syncope, vomiting and shock, which was proved to be pulmonary artery trunk and branch wide embolism later, was presented so as to improve the understanding of the disease.

Aim To investigate the role of cholesterol in the regulation of lipid raft and the function of platelets.Methods Using in vitro incubation of methyl β-cyclodextrin(MβCD)and in vivo elevation of peripheral blood total cho-lesterol levels to remove and load platelet cholesterol,respectively.Cholera toxin B staining combined with flow cytometry was used to detect platelet lipid raft content,fluorescence antibody staining combined with flow cytometry was used to detect the expression levels of P-selectin and activated integrin α Ⅱ bβ3,annexin Ⅴ labeling combined with flow cytometry was used to detect the level of phospholipid efflux,in vitro experimental system and rat tail bleeding experiment were used to detect platelet aggregation ability.Results The content of lipid raft on B lymphocytes decreased with the removal of cholesterol,while in vitro incubation of MβCD to remove platelet cholesterol significantly increased its lipid raft level(P＜0.05).Consistent with this,in vivo cholesterol loading increased the lipid raft content of B lymphocytes but decreased the lipid raft content of platelets(P＜0.05).The increase in lipid raft after removing cholesterol was not conducive to platelet activation and aggregation function.In vivo cholesterol loading downregulated platelet lipid raft content(P＜0.05),enhanced its ability to respond to low concentration stimulant for activation aggregation and coagulation,and this enhancing effect disappeared after cholesterol removal.Conclusion Platelet cholesterol is a key regulator of platelet lipid raft content and platelet function,which can negatively regulate lipid raft,promote platelet activation,and enhance their coagulation function.