| Objective To study the therapeutic effect of hemofiltration on sepsis and the effect of GEF-H1 / Rho A signaling pathway on sepsis endothelial cells during hemofiltration.Methods 1.According to the diagnostic criteria for clinical sepsis patients(2.1.1),serum from patients with sepsis was collected and subjected to continuous hemofiltration.10%serum before and after hemofiltration was used to interfere with pulmonary vascular endothelial cells(PVECs)at 0h,2h,4h,6h,and 8h,respectively.Then use cell monolayer cell impedance experiments and permeability experiments to compare and analyze the effects of serum on cell impedance and permeability before and after hemofiltration.2.Intervening PVECs with serum before and after hemofiltration at a concentration of 10%.Real-time PCR and Western Blot methods were used to detect changes in m RNA and protein expression levels of ICAM-1 and VCAM-1,respectively,and statistical analysis was performed.3.Real-time PCR and Western Blot methods were used to detect the changes of GEF-H1 m RNA and protein expression levels in serum before and after hemofiltration,and statistical analysis was performed.4.Construction of a GEF-H1 interference vector and cell transfection using Lipofectamine 3000 for 24 h.Real-time PCR and Western Blot were used to detect the expression levels of GEF-H1 m RNA and protein,respectively,and the vector with the highest interference efficiency was selected for stable cell line selection.Purinemycin was selected at a concentration of 1.0 ? g / m L for 5 weeks to construct a stable GEH-H1 cell line(sh RNA-GEF-H1).At the same time,a negative control group(sh RNA)was set up in the experiment,and cell impedance and permeability detection and statistical analysis were performed.5.Construction of GEF-H1 overexpression vector and cell transfection,Then use G418 at a concentration of 200 ? g / m L to screen for 6 weeks to construct a stable GEH-H1 over-expressing cell line(pc DNA-GEF-H1).At the same time,a negative control group(pc DNA)was set in the experiment,and the cell impedance and permeability of the two groups were detected for statistical analysis.Results(1)Cell impedance test to measure the transcellular resistance of the two groups of cells at 0h,2h,4h,6h,and 8h,respectively.Compared with the serum intervention group before hemofiltration,the impedance of pulmonary vascular endothelial cells in the serum intervention group Increasing and showing time dependence,there is a significant difference between 6h and 8h(F = 3.498,P <0.01;F = 2.671,P <0.001).At the same time,the cell permeability test was used to detect the cell permeability of the two groups of cells at 0h,2h,4h,6h,and 8h.Compared with the pre-hemofiltration serum intervention group,the post-filtration hemofiltration serum intervention group pulmonary vascular endothelial cells The transmittance is decreasing and showing time dependence.There is a significant difference between 6h and 8h(F = 10.150,P <0.01;F = 7.465,P <0.001).(2)Compared with serum before hemofiltration,the m RNA expression of ICAM-1 and VCAM-1 in serum after hemofiltration was significantly reduced(F = 1.755,P <0.05;F = 1.538,P <0.01),and The protein expression level was also significantly reduced(F = 1.156,P <0.001;F = 3.288,P <0.001);the differences were statistically significant.(3)Compared with before hemofiltration,serum after hemofiltration can significantly inhibit the expression of GEF-H1 m RNA and protein in PVECs(F = 8.533,P <0.001;F = 16.260,P <0.05),and the differences were statistically significant.(4)Compared with the control group,the expression of both GEF-H1 m RNA and protein in the GEF-H1 interference group was significantly reduced(F = 4.213,P <0.01;F = 1.541,P <0.01);at the same time,Rho A protein expression It decreased(F = 1.025,P <0.01);the resistance of PVECs increased(F =34.820,P <0.01),and the permeability decreased(F = 3.484,P <0.01).The differences were statistically significant.(5)Compared with the control group,the expression levels of GEF-H1 in m RNA and protein were significantly enhanced after GEF-H1overexpression(F = 12.850,P <0.01;F = 6.183,P <0.01),and the expression level of Rho A protein was also Significantly increased(F = 2.650,P <0.01).At the same time,the resistance of pulmonary vascular endothelial cells decreased(F = 2.591,P <0.01)and permeability increased(F = 5.707,P <0.01).The differences were statistically significant.Conclusions(1)The sepsis serum after hemofiltration can increase the resistance of endothelial cells and reduce the permeability of endothelial cells compared with the sepsis serum before hemofiltration.(2)ICAM-1 and VCAM-1 may be involved in the effect of hemofiltration on the permeability of endothelial cells in sepsis.(3)GEF-H1 /Rho A signaling pathway is involved in the regulation of endothelial cells by continuous hemofiltration sepsis serum. |
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