The Construction Of SENP1 Gene Interfering Lentivirus Vectors And Its Affections On Alveolar Epithelial Cells Apoptosis Induced By Hyperoxia

Objectives: To construct a SENP1-RNAi lentivirus vector and establish human type Ⅱ alveolar epithelial cells(HEPApiC)that stably express SUMO specific protease 1(SENP 1).To research the relationship between SENP1 and apoptosis induced by oxidative stress after being exposed to hyperoxia.Methods : This experiment was divided into two parts.Part one:Preparing for virus particles with constructed plasmid LV3-SENP 1-RNAi to infect the HEPApiC cells,and detecting the expression of SENP 1 in different groups by qRT-PCR and Western blot.Part two: the experiment was based on SENP 1 stably silenced HEPApiC cells and the model of lung injury was induced by mixture gas formed with O2(900ml/L)and CO2(50ml/L)which were inleted at a speed of 3 litres a minute and continued for 10 minutes.Cells were randomly divided into 6 groups: control group,empty vector infected group,experimental group,hyperoxia group,hyperoxia and empty vector infected group,hyperoxia and experimental group.Control group: the HEPApiC cells were directly added DMEM sugar medium containing 10% fetal bovine serum and 1% mycillin,and then cultured in 37 degrees,5%CO2saturated humidity incubator.Empty vector infected group: the cells infected with lentiviral vector were added the same medium and cultured in the sameincubator with control group.Experimental group: SENP1-RNAi-HEPApiC cells were placed into the same condition as above.Hyperoxia group: the HEPApiC cells with same medium were dealt according to the hyperoxia model mentioned above and then put into the same environment as previous groups.Hyperoxia and empty vector infected group: the cells infected with lentiviral vector were dealt with mixtured gas as hyperoxia group and then grew in the same condition.Hyperoxia and experimental group: SENP1-RNAi-HEPApiC cells were treated as the hyperoxia group did.After culturing for 12 h,24h and48 h,cells would be collected and the indicators would be detected.One,observing morphological changes and taking pictures with inverted phase contrast microscope.Two,obtaining cells apoptosis situation through flow cytometry instrument after 24 h.Three,testing the transposition of SIRT1 by immunofluorescence technique after 24 h.Four,measuring the expression of SENP1,SIRT1 separately in cytoplasm and nucleus,P53 and AC-P53 by Western Blot after 24 h.Results: Part one: LV3-SENP 1-RNAi was successfully constructed and virus was correctly packaged.Furthermore,high titer virus particles were obtained and then infected HEPApiC cells successfully.As PCR showed,the 2-ΔΔCt of SENP1 in experimental group was 0.026 while in control group was 0.050,and in Empty vector infected group was 0.057.As western blot showed,the expression of SENP1 at protein level in experimental group,control group and empty vector infected group were 0.161±0.015 、0.781±0.046、0.811±0.008.Compared with the control group and the emptyvector infected group,the expression of SENP 1 in experimental group was significantly decreased at the protein and mRNA levels(p<0.05).Part two:Firstly,under inverted phase contrast microscope,the cells in control group,empty vector infected group and experimental group grew well,sticked the wall well,uniform distribution and less suspension cells were observed,in addition,the majority of cells were fusiform.In hyperoxia group,the cells grew poorly and sticked the wall badly.Morphology variation happened and most of cells were circular and Ellipse.More suspension cells appeared in medium.In hyperoxia and empty vector infected group,the situation of cells was similar to that in the hyperoxia group.The cell gap increased,and the suspended cells were more.In hyperoxia and experimental group,the growth of cells was poor,but the morphological changes and the number of suspension cells were fewer compared to those in hyperoxia group and hyperoxia and empty vector infected group.Secondly,the apoptosis rate increased when cells were dealt with by hyperoxia 24 h later according to the results of flow cytometry instrument,with significant difference(P < 0.05).The apoptosis rate in control group was lower than that in the hyperoxia group,and the apoptosis rate in empty vector infected group was lower than that in hyperoxia and empty vector infected group,the apoptosis rate of experimental group was also lower than that in hyperoxia and experimental group.Compared with each other,the differences were significant(P < 0.05).Furthermore,the apoptosis rate in hyperoxia and experimental group was lower than another two groups disposed by hyperoxia,while it was stillhigher than control group(P < 0.05).Compared hyperoxia group with hyperoxia and empty vector infected group,there was no diference(P > 0.05).Thirdly,Immunofluorescence results showed that blue fluorescence represented nuclei,while red fluorescence was related to SIRT1 protein and the purple was fusion fluorescence.It was obviously that SIRT1 protein translocated more in those groups dealt with hyperoxia and the difference between six groups was statistically significant(χ2=99.34,P=0.000<0.05).Compared with control group,the translocation increased significantly in hyperoxia group,and the difference was statistically significant(χ2=45.36,P=0.000<0.05);compared with hyperoxia group,the translocation in hyperoxia and experimental group decreased significantly,but did not reach the level of control group,with significant difference in statistics(χ2=30.75,P=0.000<0.05).Fourthly,compared with control group,at the level of protein,the expression of SENP1,SIRT1 in cytoplasmic,AC-P53 increased,while the expression of SIRT1 came from nucleus and P53 decreased obviously in hyperoxia group.In hyperoxia and experimental group,SENP1,SIRT1 in cytoplasmic,AC-P53 expressed less as SIRT1 in nucleus expressed more than that in hyperoxia group,but both were failed to meet the level of control group(P < 0.05).Conclusion: SENP1 silenced HEPApiC cells were successfully constructed.SENP1 and SIRT1 were involved in the oxidative stress induced by hyperoxia.Hyperoxia led to higher expression of SENP1.Then the translocation of SIRT1 increased.SIRT1 decreased in nucleus and increased in cytoplasm,which induced acetylation ofP53 and apoptosis.While silencing SENP1,in the same conditions,the expression of SENP1 decreased as well as the translocation of SIRT1.Then,the acetylation of P53 declined and apoptosis decreased.In the opposite direction,it was proved that SENP1 could promote the damage of HEPApiC cells induced by hyperoxia.