| Objective: To observe the effects of different concentrations of adult CPB serum on AECⅡ in rats in vitro,and to establish a model of rat AECⅡ injury after CPB.At the same time,Annexin A1 pseudopeptide Ac2-26 was co-cultured with damaged rat AECⅡ to explore the protective effect on rat AECⅡ in vitro.Methods:(1)The lung of healthy 200-300 g SD male rats were isolated,purified and cultured by Ig G immune adhesion selection method,and the structure of cultured cells was observed by transmission electron microscope and the expression of SP-C protein was detected by immunofluorescence.(2)Sixteen patients with rheumatic heart disease undergoing valve replacement surgery were selected,aged(49.63±3.85)years old,and the operation duration was(4.20±0.38)hours,respectively,10 minutes after induction of anesthesia(before CPB),CPB was shut down and protamine neutralized heparin After(after CPB),10 ml of blood was collected aseptically,the serum was separated,the complement activity was inactivated,and it was stored at-80°C for later use.The two groups of serum before and after CPB were mixed separately for use,and the serum and DMEM medium were prepared at 2.5%,5.0%,7.5%,10%,and 20% respectively.(3)The rat AECⅡ cultured for 48 h was co-cultured with different concentrations of serum before and after CPB for 12 h,and the cells and supernatants of 12 h co-culture were selected.The proliferation rate of cells was detected by CCK-8,the apoptosis rate was detected by flow cytometry,and the concentrations of MCP-1 and TNF-α in the supernatant were detected by ELISA,so as to screen the modeling concentration of cell injury model in this experiment.(4)After screening the modeled concentration of 10% CPB post-serum concentration,model the model with this concentration(injury group),and the modeled concentration corresponding to the 10% pre-CPB serum concentration is used as the experimental concentration of the control group(control group).On the basis of the injury group,Ac2-26(group A)was added to the injury group at the same time.The cells and cell supernatant co-cultured for 12 h were selected,and the proliferation rate of cells was detected by CCK-8,and the rate of apoptosis was detected by flow cytometry.The concentrations of MCP-1 and TNF-α in the supernatant were detected by ELISA.Results:(1)The results of light microscope,electron microscope and SP-C protein expression of rat AECⅡ culture.Under the light microscope,the cells cultured for 0.5d-1d began to stretch and adhere to the wall,showing a round shape with obvious nuclei;the cultured cells for 2d-4d could grow in polygonal or spindle shape;after 5d of culture,the cells gradually lost their typical epithelial cell morphology and could not be identified.Under the transmission electron microscope,it can be seen that there are many microvilli of different thickness and length on the cell surface,and the cytoplasm contains concentric specific lamellar bodies.Immunofluorescence showed that the nucleus stained by DAPI was blue under ultraviolet excitation,SP-C positive expression was corresponding to fluorescein-labeled red light,and SP-C protein expression could be detected in AECⅡ.(2)CCK-8 to detect rat AECⅡ appreciation rate.There was no significant difference in the cell proliferation rate of different concentrations in each group before CPB(P>0.05);the cell proliferation rate of the20% serum concentration group after CPB was lower than that of the 2.5%,5.0%,7.5%,and 10% groups(P<0.05).There was no statistical significance among the other groups(P>0.05).Comparison of the same concentration of serum before and after CPB: The cell proliferation rate in the 7.5%,10%,and 20% groups was significantly lower than that in the pre-CPB group,and the difference was statistically significant(P<0.05);the cell proliferation rate in the 2.5% and 5.0% groups was not significant difference(P>0.05).After applying the annexin A1 peptidomimetic Ac2-26,the cell proliferation rate of A group was increased compared with the injured group,but the cell proliferation rate was lower than that of the control group(P<0.05).(3)Flow cytometry to detect rat AECⅡ apoptosis rate.There was no significant difference in the apoptotic rate of different concentrations in each group before CPB(P>0.05).With the increase of serum concentration after CPB,the apoptosis rate gradually increased,and the apoptosis rate in the 20% group was as high as 53.10%±1.14%,the difference was statistically significant(P<0.05).Comparing the apoptosis rate of the same serum concentration before and after CPB,the apoptosis rate of each serum concentration group after CPB was significantly higher than that before CPB,and the difference was statistically significant(P<0.05).After applying annexin A1 peptidomimetic Ac2-26,the apoptosis rate of A group was lower than that of the injured group,but it was higher than that of the control group(P<0.05).(4)ELISA to detect the content of MCP-1 and TNF-α.1)There was no significant difference in MCP-1 content in the cell supernatants of different concentrations in each group before CPB(P>0.05).With the increase of serum concentration after CPB,the MCP-1 content in the co-cultured cell supernatant gradually increased.The MCP-1 content in the cell supernatant of the 20% group was significantly higher than that of the other groups,the difference was statistically significant(P<0.05).Comparing the same concentration of serum before and after CPB,the MCP-1 content of cell supernatant in each group after CPB was significantly higher than that of each group before CPB(P<0.05).After applying the annexin A1 peptidomimetic Ac2-26,the MCP-1 content in the cell supernatant of the A group was lower than that of the injured group,but the MCP-1 content in the cell supernatant of the control group was increased(P<0.05).2)There was no significant difference in TNF-α content in the cell supernatant after co-cultivation at different concentrations in each group before CPB(P>0.05).After CPB,the TNF-α content in the 10% and 20% groups increased compared with the2.5%,5.0%,and 7.5% groups,and the difference was statistically significant(P<0.05).There was no significant difference in the content of TNF-α in the cell supernatant between the 10% group and the 20% group(P>0.05).Comparing the same concentration of serum before and after CPB,the content of TNF-α in cell supernatant of each group after CPB was significantly higher than that of each group before CPB(P<0.05).After applying the annexin A1 peptidomimetic Ac2-26,the TNF-α content in the cell supernatant of the A group was lower than that of the injury group,but the TNF-α content in the cell supernatant of the control group was increased(P<0.05).Conclusion:(1)Adult serum after CPB can cause AECⅡ damage in rats,and the cell injury model induced by serum after 10%CPB was successfully established.(2)Annexin A1 pseudopeptide Ac2-26 could reduce the concentration of MCP-1 and TNF-α in the supernatant of AECⅡ and attenuate the serum-induced AECⅡ damage in rats after adult CPB. |
PDF Full Text Request