| Objective: Bronchopulmonary dysplasia(BPD)is a common and serious respiratory complication of premature infants,with the main feature of alveolar developmental arrest.The mortality rate is high,and even survival will be accompanied by long-term lung ventilation,ventilation and alveolar diffusion dysfunction,which seriously affects the quality of life of the child.But there is currently no effective treatment.Type Ⅱ alveolar epithelial cells(AEC Ⅱ)act as “progenitor cells” of alveolar epithelial cells.When the lung epithelium is damaged,it can not only renew itself,but also proliferate and differentiate into type I alveolar epithelial cells(AEC I).And functional restoration play an important role.A large number of studies have confirmed that oxidative stress is a key link in the occurrence and development of BPD disease,but the relationship with the outcome of BPD is still unclear.As the most serious manifestation of oxidative stress,DNA damage can cause permanent cell stagnation,and its repair is related to cell survival and outcome.Therefore,in order to explore the pathological mechanism of DNA damage of BPD lung epithelial cells,this study used animal ELISA,immunofluorescence,Western-blot,comet experiment and other experimental methods to detect from the animal level,primary cell level and in vitro cell hyperoxia exposure test.The expression changes of DNA damage markers 8-ohdG and γ-H2 AX in BPD rat lung tissue and AECⅡ,as well as DNA strand breakage,and on this basis,using gene chip technology to screen and verify the DNA damage repair signaling pathway Differentially expressed genes,and verified by cell transfection technology whether it can promote the repair of DNA damage of AECⅡ cells by changing the differential gene expression level,thereby reducing DNA damage,promoting cell proliferation and survival,intent to provide BPD biomarkers The theoretical basis provides a new intervention target for the clinical treatment of BPD.Methods:Part1: Detect the expression of DNA damage markers in lung tissue and AECⅡ of BPD rats.1.Establish a BPD animal model,and randomly divide newborn SD rats into a model group(FiO2=0.85)and a control group(FiO2=0.21),respectively,1d,3d,7d,and 14 d after birth.Specimens for subsequent experiments:(1)H & E staining was used to observe the morphological changes of lung tissue,and the RAC value was used to evaluate lung development;(2)The content of 8-ohdG in lung tissue was detected by ELISA;(3)The location and expression of γ-H2 AX in lung tissue were observed by immunofluorescence;(4)Application Western-blot was used to detect the expression of γ-H2 AX in lung tissue;(5)Double immunofluorescence was used to observe the co-expression of γ-H2 AX and AECⅡ marker p180 in lung tissue;2.Build a BPD animal model,at 3d,7d and 14 d after birth,take 8 random AECⅡs from each group and use Western-blot to detect the expression of γ-H2AX;3.Construct an in vitro hyperoxia-exposed cell model,and randomly divide the RLE-6TN cell line into a model group(FiO2=0.85)and a control group(FiO2=0.21),collect cells at 12 h,24h and 48 h,and perform subsequent experiments :(1)Observe the state of the cell under a microscope;(2)Use immunofluorescence to observe the location and expression of γ-H2 AX in the cell;(3)Use Western-blot to detect the expression level of γ-H2 AX in the cell.Part2: Screen and verify the differentially expressed genes in the DNA damage signaling pathway of BPD rats.1.Build a BPD animal model,randomly select 3 rats each in the model group and the control group at 14 days of age,and take lung tissue as a sample for subsequent chip experiments;2.Using gene chip technology to screen differentially expressed genes in DNA damage signaling pathways in the lung tissue of BPD rats;3.Apply Real-time PCR and Western-blot technology to verify the differential genes。 Part3: Observe the DNA damage and cell proliferation of AEC Ⅱ cells after overexpression /silence of rad1.1.Establish RLE-6TN cell line that overexpresses and silences Rad1,and divide the cells into 5 groups:(1)Control group: cells are placed in a common cell incubator(FiO2=0.85)for 48h;(2)Simple hyperoxia group: cells Placed in a hyperoxia cell incubator(FiO2=0.85)and cultivated for 48h;(3)empty group: empty vector transfected cells and placed in a hyperoxic cell incubator(FiO2=0.85)for 48h;(4)silent group: siRNA-Rad1 interference cells Then placed in a hypertoxic cell incubator(FiO2=0.85)for 48h;(5)Overexpression group: cells transfected with overexpressed Rad1 vector were placed in a hyperoxic cell incubator(FiO2=0.85)for 48 h for subsequent experiments;2.Using immunofluorescence to observe the location and expression of γ-H2 AX in each group of cells;3.Using Western-blot technology to detect the expression of γ-H2 AX in each group of cells;4.Using comet experiment to detect the DNA strand break of each group of cells;5.Using Western-blot technology to detect the expression of cell cycle regulatory protein p21 in each group of cells;6.Apply flow cytometry to detect the cell cycle of each group of cells;7.Observe the proliferation and survival of cells in each group using clone formation experiments.Results: Part1: 1.Morphological changes of lung tissue: 1)H & E results showed that the lung tissue morphology of the model group at 1d and 3d was not significantly different from that of the control group.The difference in lung tissue began to be obvious at 7d,with the number of alveoli decreasing significantly and irregular morphology,the alveolar crest and stimulation interval were not obvious.The structure of the alveoli is simplified;2)The RAC value showed that compared with the control group,the RAC value in the model group decreased significantly from 7d(P<0.05),and was more obvious at 14d(P<0.01).2.Expression of DNA damage markers 8-OHdG and γ-H2 AX in lung tissue: 1)ELISA results showed that compared with the control group,the 8-OHdG content of the model group increased from 3 days,the difference was statistically significant(P> 0.05),the difference was more significant at 7d and 14d(P <0.01);2)Immunofluorescence results show that γ-H2 AX is mainly expressed in the nucleus,and the enlarged γ-H2 AX focal point is clearly gathered in the nucleus after magnification.Compared with the control group,the number of γ-H2 AX positive cells in the model group increased from 3d,and the difference was more obvious at 7d and 14d;3)Western-blot results showed that compared with the control group,the expression level of γ-H2 AX in the model group increased from 3d,and was significantly higher than the control group at 7d,the difference was statistically significant(P<0.05),the difference was even more at 14 d Significantly(P<0.01).3.Expression of DNA damage marker γ-H2 AX in lung AECⅡ: 1)Immunofluorescence double staining results show that γ-H2 AX is expressed in the nucleus and p180 is expressed in the cytoplasm.In the model group,the percentage of γ-H2AX/P180 double-stained cells in P180 positive cells was significantly higher in the model group than that in the control group,and the difference was statistically significant(P<0.01);2)Western-blot results showed that the expression of γ-H2 AX protein in BPD lung primary AEC Ⅱ cells was higher than that in the control group,and the difference was more significant with age.4.Expression of DNA damage marker γ-H2 AX and DNA strand breakage in a cell model of hyperoxia exposure in vitro: 1)The results of cell culture showed that the cells in the control group grew well,and the number of cells increased continuously as the culture time increased.Compared with the control group,the model group showed a decrease in the number of cells at 12 h,and the cell state became worse.At 24 h and 48 h,the number of cells decreased more significantly and the cell state became worse;2)Immunofluorescence results show that γ-H2 AX is expressed in the nucleus.The control group showed less expression of γ-H2 AX.The expression of γ-H2 AX in the model group increased from 12 h,and it increased more obviously at 24 h and 48h;3)Western-blot results showed that compared with the control group,the expression level of γ-H2 AX in the model group increased from 12 h,the difference was statistically significant(P<0.05),the difference was more significant at 24 h and 48h(P<0.01);4)The results of the comet experiment showed that: only a small number of cells in the control group migrated without significant changes in DNA;the model group showed significant DNA migration from the nucleus at 12 hours,forming a "comet tail",and with time,"more obvious.Compared with the control group,the tail length and Olive tail moment of the model group began to increase from 12 h,the difference was statistically significant(P <0.01),and with the extension of time,the difference was more significant,the largest difference at 48h(P <0.01).According to the DNA content of the comet tail,the DNA damage degree of the cells was graded.The results showed that: the percentage of DNA content in the tail of the control group had no significant change,and the degree of damage was all 0 grade;Significance(P <0.01),the degree of damage increased to level 2,at 24 h and 48 h,the percentage of DNA content in the tail of the comet increased more obviously,and the degree of damage reached level 3.Part2: 1.RT-qPCR chip screening results showed that according to the standard of P<0.05 and the fold difference >1.5,there were 19 differentially expressed genes in the two groups of DNA damage signaling pathways,of which 8 genes were highly expressed in the model group,respectively.Brca1,Cdc25 c,Cdkn1a,Dclre1 a,Fanca,Fancd2,Gadd45 g,and Mgmt.Eleven low expression genes are Ddit3,Fancc,Mbd4,Msh3,Nthl1,Pms1Ppm1 d,Rad1,Rad52,Smc3,and Wrn;2.Real-time PCR and Western-blot results confirmed that the mRNA and protein levels of Cdc25 c and Cdkn1 a in the model group were significantly up-regulated t(P<0.01),consistent with the chip results;3.The primary AEC Ⅱ cells were extracted and the cell cycle was detected by flow cytometry.The experimental results showed that the ratio of G0 / G1 phase of the primary AEC Ⅱ cells in the model group increased significantly t(P<0.01),the proportion of S phase decreased significantly(P<0.01),and the cell proliferation was slow;4.Real-time PCR and Western-blot results confirmed that the mRNA expression of Rad1,Rad52,and Smc3 in the lung tissue of the model group were significantly lower than those of the control group t(P<0.01),which was consistent with the chip results.The protein expression of Rad1 and Smc3 were significantly lower than those of the control group t(P <0.01),which was consistent with the chip results.However,the expression level of Rad52 protein was higher than that of the control group t(P<0.05),which was inconsistent with the mRNA level,suggesting that there may be post-transcriptional regulation.Part3: 1.Expression of γ-H2 AX in 5 groups of cells: 1)Immunofluorescence results show that: the fluorescence intensity of γ-H2 AX in the control group is very weak,and the expression level is very low;the fluorescence intensity and number of γ-H2 AX focus in the hyperoxia group and the empty group are significantly increased;compared with the empty group,the The expression group γ-H2 AX fluorescence intensity and expression level were significantly reduced,while the silence group γ-H2 AX expression level was significantly increased;2)Western-blot results showed that compared with the control group,the expression levels of γ-H2 AX in the cells of the pure hyperoxia group and the empty group were significantly increased,and the difference was statistically significant(P<0.01).Compared with the empty group,the expression level of γ-H2 AX in the cells of the overexpression group was significantly reduced,the difference was statistically significant(P <0.01);while the expression level of γ-H2 AX in the silent group was significantly increased,the difference was statistically significant(P<0.01).2.DNA strand breakage in 5 groups of cells:The results of the comet experiment showed that the cells in the control group had no comet tail,and the degree of damage was grade 0.The cells in the hyperoxia group and the no-load group had obvious comet tails.The length of the comet tail,the Olive tail moment,and the DNA content of the comet tail were significantly higher than the control In the group(P<0.01,P<0.05 and P<0.01,respectively),the degree of cell damage was grade 3;compared with the empty group,the over-expression group cells had significantly shorter comet tails,comet tail length,Olive tail moment and comet tail DNA The content was significantly reduced,and the difference was statistically significant(P <0.01,P <0.05 and P <0.05,respectively).The degree of cell damage was significantly alleviated and became grade 2;the tail of the cells in the silent group was significantly longer,the length of the tail of the comet,Olive The tail moment and comet tail DNA content increased significantly,the difference was statistically significant(P <0.05,P <0.01 and P <0.01,respectively),and the degree of cell damage was aggravated,which was grade 3.3.The expression of cyclin and cell cycle changes in 5 groups: 1)Western-blot results showed that compared with the control group,the expression level of p21 in the cells of the pure hyperoxia group and the empty group was significantly increased,the difference was statistically significant(P<0.01);compared with the empty group,the overexpression group The expression level of p21 in the cells was significantly reduced,the difference was statistically significant(P <0.01);while the expression level of p21 in the silent group was significantly increased(P <0.01);2)The cell cycle results show that the ratio of cells in the G0 / G1 phase of the hyperoxia group and the no-load group is significantly increased(P<0.01),the ratio of the S phase is significantly reduced(P<0.01),and the cell cycle is blocked.Cell proliferation was inhibited;compared with the empty group,the G0/G1 phase ratio of the overexpression group was significantly reduced(P<0.01),the S phase ratio was significantly increased(P<0.01),cell cycle arrest was reduced,and cell proliferation was improved;and In the silent group,the G0/G1 phase ratio increased significantly(P <0.01),and the S phase ratio decreased significantly(P <0.05).Cell cycle arrest increased and cell proliferation was inhibited.4.Cell proliferation and survival in 5 groups: The results of clone formation experiments showed that the cells in the control group had a strong ability to form clones.Compared with the control group,the cell formation ability of the pure hyperoxia group and the no-load group was significantly weakened,and the colony formation rate was significantly reduced(P<0.01).Compared with the unloaded group,the cloning ability of cells in the overexpression group was significantly enhanced and the cloning formation rate was significantly increased(P<0.01);the cloning ability of cells in the silent group was significantly weakened and the cloning formation rate was significantly reduced(P<0.01)Conclusions: 1.The expression of DNA damage markers in the lung tissue and AEC Ⅱ of BPD rats increases,and it becomes more and more significant with the increase of age,suggesting that the cumulative DNA damage may be an important cause of the developmental arrest of BPD alveoli The serum biomarkers provide a theoretical basis;2.Gene chip screening and verification of the down-regulation of important DNA repair factor Rad1,suggesting that down-regulation of Rad1 expression inhibits the repair of DSB of AECⅡ leading to the accumulation of damage may be the key link of AECⅡ cell cycle arrest and cell proliferation inhibition in BPD;3.The up-regulated expression of Rad1 can promote the repair of DSB of AEC Ⅱ,reduce cell DSB damage,improve cell cycle arrest,enhance cell proliferation and survival,and provide a new intervention target for future clinical treatment of BPD. |
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