| 【Objective】To investigate the role of NANCI-NKX2.1 signaling pathway in the pathogenes of bronchopulmonary dysplasia(BPD)in neonatal mice,by using adenovirusmediated lnc RNA NANCI interference and overexpression techniques.【Methods】This study explored the role of NANCI-NKX2.1 signaling pathway in the pathogenes of BPD by using NANCI interference and overexpression techniques.ⅠAdenovirus-mediated NANCI interference technique was used to treat normal newborn mice.A total of 32 neonatal C57BL/6J(B6)mice were randomly divided into 4 groups:interference group,negative control group,normal saline control group and blank control group,with 8 pups in each group.1ul normal saline with lnc NANCI sh RNA adenovirus vector and control vector was administered intranasally to lungs of newborn mice in interference group and negative control group at postnatal days 2 respectively.1ul normal saline was administered intranasally to lungs of newborn mice in normal saline control group.No interference treatment was processed in blank control group.The mice were sacrificed at 7 days after birth and lung tissue samples were collected.All the operations were in accordance with the regulations of laboratory animal management,and the pain was minimized when executed.The fluorescence expression of lung tissue was observed in frozen blank section.Hematoxylin-eosin(HE)staining was used to observe pathological changes in lung tissues.RT-q PCR was used to measure the expression of NANCI.RT-q PCR and Western Blot were used to measure the mRNA and protein expression of NKX2.1,SPC and AQP5.ⅡAdenovirus-mediated NANCI overexpression technique was used to interfere with hyperoxy-induced BPD newborn mice.A total of 32 neonatal B6 mice were randomly divided into 4 groups:interference group,negative control group,normal saline control group and blank control group,with 8 pups in each group.All four groups of mice were kept in a high oxygen environment(Fi O2≥95%).1ul normal saline with lnc NANCI adenovirus vector and control vector were administered intranasally to lungs of newborn mice in interference group and negative control group at postnatal days 2 respectively.1ul normal saline was administered intranasally to lungs of newborn mice in normal saline control group.No interference treatment was processed in blank control group.The mice were sacrificed at 7 days after birth and lung tissue samples were collected.All the operations were in accordance with the regulations of laboratory animal management,and the pain was minimized when executed.The fluorescence expression of lung tissues were observed in frozen blank section.HE staining was used to observe pathological changes in lung tissues.RT-q PCR was used to measure the expression of NANCI.RT-q PCR and Western Blot were used to measure the mRNA and protein expression of NKX2.1,SPC and AQP5.【Results】ⅠAdenovirus-mediated NANCI interference technique was used to treat normal newborn mice1.Lung tissue immunofluorescence: neonatal mice inhaled adenovirus vectors,and a week later the lung tissues of the experimental group and the negative control group showed green fluorescent expression,and no fluorescent expression was observed in the normal saline and the blank control group.2.Lung histopathology:(1)The alveolar size of the control group,the normal saline control group and the blank control group were basically uniform,the shape of the alveoli was more regular,and the alveolar septum was thinner;but in the experimental group: the new septum of the lung tissue was reduced,the alveolus enlarged and partially fused,the alveoli were divided in two times,the number of alveoli was less and the alveoli were different,the alveolar structure was disturbed and the wall of the bronchi was thickened.(2)RAC values: There was a significant difference among the four groups(F=7.26,P=0.001);and the decrease of RAC value in the experimental group was statistically significant compared with the other three groups(all P < 0.05);compared with the negative control group,there was no significant difference in the RAC value between the saline and the blank control group(P >0.05,P>0.05).3.The expression of NANCI : there was significantly different among the four groups(F=37.96,P=0.000);The relative expression of NANCI in the experimental group was lower than that of the control group,which was 0.26 times to the negative control group(P < 0.05);compared with the negative control group,there was no significant difference in the relative expression of NANCI between the normal saline and the blank control group(P > 0.05,P>0.05).4.The mRNA and protein expression of NKX2.1: there was significantly different among the four groups(F=20.38,P=0.000);the relative expression of the mRNA of NKX2.1 in the experimental group was lower than that of the control group,which was 0.50 times to the negative control group(P < 0.05);compared with the negative control group,there was no significant difference in the relative expression of the mRNA of NKX2.1 between the normal saline and the blank control group(P >0.05,P > 0.05).The expression of NKX2.1 protein in the lung tissues of the experimental group was lower than that in the control groups(all P < 0.05).5.The mRNA and protein expression of SPC: there was significantly different among the four groups(F=18.28,P=0.000);the relative expression of the mRNA of SPC in the experimental group was lower than that of the control group,which was 0.46 times in the negative control group(P < 0.05);compared with the negative control group,there was no significant difference in the relative expression of the mRNA of SPC between the normal saline and the blank control group(P > 0.05,P>0.05).The expression of SPC protein in the lung tissues of the experimental group was lower than that in the control groups(all P < 0.05).6.The mRNA and protein expression of AQP5: there was significantly different among the four groups(F=21.84,P=0.000);the relative expression of the mRNA of AQP5 in the experimental group was lower than that of the control group,which was 0.48 times in the negative control group(P < 0.05);compared with the negative control group,there was no significant difference in the relative expression of the mRNA of AQP5 between the normal saline and the blank control group(P > 0.05,P>0.05).The expression of AQP5 protein in the lung tissues of the experimental group was lower than that in the control groups(all P < 0.05).ⅡAdenovirus-mediated NANCI overexpression technique was used to interfere with hyperoxy-induced BPD newborn mice1.Lung tissue immunofluorescence: neonatal mice inhaled adenovirus vectors,and a week later the lung tissues of the experimental group and the negative control group showed green fluorescent expression,and no fluorescent expression was observed in the normal saline and the blank control group.2.Lung histopathology:(1)In the experimental group,the alveolar size in the lung tissue was basically uniform,the morphology was more regular,and the alveolar space was thinner.There were more coronal protrusions in the alveolar cavity,extending for the newborn alveolar septum,decreasing the average intercept of the alveoli,increasing alveolar count,reducing the degree of fibrosis,and alveolar size.The size of the alveoli was more homogeneous than the control group,the negative control group and the saline control group.New septum formation was reduced,the alveolar space was enlarged and partially fused,and the number of alveoli was less in the control group,the negative control group and the saline control group.(2)RAC values: There was a significant difference among the four groups(F=8.40,P=0.000);and the increase of RAC value in the experimental group was statistically significant compared with the other three groups(all P <0.05);compared with the negative control group,there was no significant difference in the RAC value between the saline and the blank control group(P >0.05,P>0.05).3.The expression of NANCI : there was significantly different among the four groups(F=57.18,P=0.000);the relative expression of NANCI in the experimental group was higher than that of the control group,which was 2.70 times to the negative control group(P < 0.05);compared with the negative control group,there was no significant difference in the relative expression of NANCI between the normal saline and the blank control group(P > 0.05,P>0.05).4.The mRNA and protein expression of NKX2.1: there was significantly different among the four groups(F=28.04,P=0.000);the relative expression of the mRNA of NKX2.1 in the experimental group was higher than that of the control group,which was 2.36 times to the negative control group(P < 0.05);compared with the negative control group,there was no significant difference in the relative expression of the mRNA of NKX2.1 between the normal saline and the blank control group(P > 0.05,P>0.05).The expression of NKX2.1 protein in the lung tissues of the experimental group was higher than that in the control groups(all P <0.05).5.The mRNA and protein expression of SPC: there was significantly different among the four groups(F=9.22,P=0.000);the relative expression of the mRNA of SPC in the experimental group was higher than that of the control group,which was 1.78 times to the negative control group(P < 0.05);compared with the negative control group,there was no significant difference in the relative expression of the mRNA of SPC between the normal saline and the blank control group(P > 0.05,P >0.05).The expression of SPC protein in the lung tissues of the experimental group was higher than that in the control groups(all P < 0.05).6.The mRNA and protein expression of AQP5: there was significantly different among the four groups(F=12.94,P=0.000);the relative expression of the mRNA of AQP5 in the experimental group was higher than that of the control group,which was 2 times to the negative control group(P < 0.05);compared with the negative control group,there was no significant difference in the relative expression of the mRNA of AQP5 between the normal saline and the blank control group(P > 0.05,P>0.05).The expression of AQP5 protein in the lung tissues of the experimental group was higher than that in the control groups(all P < 0.05).【Conclusion】Adenovirus-mediated NANCI interference technique was successfully used to construct neonatal mice model of BPD,and adenovirus-mediated overexpression of NANCI significantly protected lung tissues of hyperoxia-induced neonatal mice with BPD,suggesting that the NANCI-NKX2.1 signaling pathway may play an important role in neonatal mice with BPD. |
PDF Full Text Request