| PartⅠ Study on the expression of mi R-223-3p in endothelial cells in vitroObjective:Lipopolysaccharides(LPS) stimulated human umbilical vein endothelial cells in vitro(HUVECs) with different concentration and different times. Observed the injury of endothelial cells that LPS induced, and Explored the correlation mi R-223-3p with the injury of endothelial cell.Methods:Using the concentration of LPS 0ug / ml, 1ug / ml, 5ug / ml, 10 ug / ml, 20 ug / ml stimulated endothelial cells 4 days, 5 days, 6 days, cell morphology was observed by inverted optical microscope, and recorded of the cytopathic effect(CPE);Using Reed-Muench’s two method to calculate the Tissue culture infective dose(TCID50); WST-1 method was used to determine the activity of endothelial cell, to evaluatethe endothelial cell growth; ELISA detected the concentrationsof tumor necrosis factor(TNF-α)and interleukin-6(IL-6) in cultured supernatant and the expression of mi R-223-3p in endothelial cells and culture supernatant by Real-time quantitative PCR(RT-q PCR).Result:1.LPS can induce endothelial cell morphology changes and the more higher the LPS concentration was, the more seriousthe cellsinjuries induced, we can see cell morphology stretched, cells gap increaseed, cells adhered loosely with different degrees, intracytoplasmic granules decreased, even some cells dissolved and disappeared; compared with the control group, the more higher the LPS concentration was, the lower optical densityvalue(OD) by WST-1, the differenceswere statistically significant(P < 0.05). With prolongation of stimulation time, the same concentration of LPS stimulation groups’ OD value was decreased(P < 0.0001).2.Results showed that with the increase of LPS concentration, the level of TNF-α expression gradually increased in the supernatant by ELISA assay compared with normal control group, the differences were statistically significant(P<0.05); with the increase of LPS concentration, the IL-6 concentrations in LPS stimulation groups in the culture supernatants were increased compared to the normal control group, the differences were statistically significant(P<0.01),LPS stimulation groups were statistically significant(F=63.6, P<0.01), Further analysis with the two-two comparison, no statistically significant difference between the two groups LPS 1ug/ml and LPS 20ug/ml, LPS 5ug/ml and LPS 10ug/ml, but LPS 5ug/ml and LPS 10ug/ml two groups’ were higher than that of LPS 1ug/ml group’s, the differences were statistically significant(P<0.0001).3.The LPS stimulation groups detected by RT q PCR, of the endothelial cellsof mi R-223-3p relative expression amount were increased obviously compared with the normal control group, the differences had statistical significance(P < 0.05), but among the stimulation groups, the differenceshad no statistical significance(F=3.84, P = 0.11). Mi R-223-3p did not expressed in the culture supernatant.Conclusion:Injuriesthat LPS on HUVECs were dependent on its concentration and time, with increasing LPS concentration, TNF-αconcentration gradually increased; all of LPS stimulation groups’ concentrations of IL-6 were increased compared to the normal control group, but had no regularitywith the LPS concentrations.It was a successful cell damage model by LPS stimulated HUVECs. The level of mi R-223-3p expression in the cells was up-regulated when LPS stimulated HUVECs, and the level of LPS did not correlated with the stimulated concentrations.PartⅡ Study on the mechanism of action of the mi R-223-3p in vitro endothelial cell injury model.Objective:To verify the regularity between the different levels of LPS stimulation and expression levels of the target gene IL6 ST. Using artificial synthesis pre-mi R-223-3p, mi R-223-3p-inhibition lentiviral transfected endothelial cells, detected the HUVECs’ growth,the concentrations of TNF-αand IL-6, the expression level of target gene IL6 STwhen mi R-223-3p was overexpressed and silenced.Methods:RT-q PCR detected the expression level of target gene IL6 ST in HUVECswith different concentrations of LPS; Virus transfection divided into several groups: mi R-223-3p over expression groups divided into mi R-223-3p overexpression group(hsa-mi R-223-3p), LPS stimulus(TCID50 value) + mi R-223-3p over expression group(abbreviated: LPS + mi R-223-3p); mi R-223-3p expression silencing group(mi R-223-3p inhibition), LPS stimulated cells group(LPS group,TCID50 value), normal cells, the empty lentiviral control group(LV-control). RT-q PCR detected the expression level of IL6 ST in endothelial cells; observed the changes of endothelial cells after inverted fluorescence microscope morphology; cell activity was detected by WST-1 assay. The concentration of TNF-αand IL-6 was determined by ELISA.Results:1. Compared with the normal control group, the IL6 ST expression detected by RT q PCR significantly increased with affection on LPS concentration of 1ug / ml 5ug / ml, 10 ug / ml and 20 ug / ml after 4 days of the endothelial cells,the differenceswere statistically significant(P < 0.05), but among the stimulated groups, the differenceshad no statistical significance(F=1.5, P = 0.27).2.Lentiviral transfection efficiency can reach more than 90% in 24 h.3.The result of the Lentiviral transfection part,:(1) hsa-mi R-223-3p group: The levels of IL6 ST expression of hsa-mi R-223-3p group wasdown-regulated than the normal cells group and LV-control group, the differenceswere statistically significant(P < 0.05).The difference of IL6 ST expression between the normal group and LV-control had no statistical significance(P > 0.05). Under fluorescent microscope, compared with the normal group,the hsa-mi R-223-3p group and LV-control group were performed for 15%- 20% of the outline of the cellswere not full, the morphology was normal, the intercellular space without broadening and cytoplasmic granules was notreductive. hsa-mi R-223-3p group and LV-control group had low OD value compared with normal cells group by WST-1 assay, the difference was statistically significant(P < 0.05). The differencesbetween hsa-mi R-223-3p group and LV-control group had no statistical significance(P > 0.05). In hsa-mi R-223-3p group and LV-control group TNF-αand IL-6 levels in the supernatant were increased than the normal group by ELISA, the difference is statistically significant(P < 0.05); the differencesbetween hsa-mi R-223-3p group and LV-control group had no statistical significance(P > 0.05).(2) LPS+ mi R-223-3p: The levels of IL6 ST expression of LPS+ mi R-223-3p group was down-regulatedthan the LPSgroup, the difference was statistically significant(P<0.05). Under fluorescent microscope, cells in LPS+mi R-223-3p group occurred lesser morphological changes,crinkled cells reduced, loosely adhered relieved, no large areas of cell fusion, dissolved and disappearedcompared with LPS group. LPS+ mi R-223-3p group detected higher OD value than the LPS group by WST-1, the difference was statistically significant(P<0.05). LPS+ mi R-223-3p group detected lower TNF-αand IL-6 levels than the LPS group by ELISA, the difference was statistically significant(P<0.05), speculated that overexpression of mi R-223-3p negatively regulated the expression of TNF- alpha, IL-6.(3) hsa-mi R-223-3p inhibition group: The levels of IL6 ST expression of hsa-mi R-223-3p inhibitiongroup was up-regulated than the normal cells group and LV-control group, the differenceswere statistically significant(P < 0.05). Under fluorescent microscope, the hsa-mi R-223-3p inhibition group was the san with LV-control group. hsa-mi R-223-3p inhibition group had low OD value compared with normal cells group by WST-1 assay, the difference was statistically significant(P < 0.05). The differencesbetween hsa-mi R-223-3p inhibition group and LV-control group had no statistical significance(P > 0.05). In hsa-mi R-223-3p inhibitiongroup,TNF-αand IL-6 levels in the supernatant were increased than the normal group by ELISA, the difference is statistically significant(P < 0.05); but the differencesbetween hsa-mi R-223-3p inhibition group and LV-control group had no statistical significance(P > 0.05).(4)Compared withhsa-mi R-223-3p inhibition group, IL6 ST gene expression level in hsa-mi R-223-3p group was down regulated, the difference is statistically significant(P < 0.05), but under fluorescent microscope, cell morphology difference was not obvious. Cell activity, TNF-α, IL-6 levels in the supernatant as well(P > 0.05).Conclusion:1. IL6 ST expression level was up-regulatedwhen LPS stimulated HUVECs, but it did not depend on the concentration of LPS.2.Lentiviral transfection efficiency can reach more than 90% in 24 h, but the virus transfection on HUVECs had a certain degree of damage.3.Mi R-223-3p overexpression can induce IL6 ST gene expression down regulation; mi R-223-3p expression silencing can lead to IL6 ST gene expression up regulation. In the LPS damage model, mi R-223-3p may play a protective role by inhibiting the overexpression of IL6 ST gene.4. Overexpression of mi R-223-3p could negative regulate the expression of TNF-α, IL-6,that can reduce the endothelial damage by LPS stimulation. |
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