The Preliminary Study Of Protection Against Hyperoxia-induced Lung Injury In Newborn Mice By Lentivirus-mediated TGF-β1 SiRNA In Vivo

Bronchopulmonary dysplasia(BPD) is an important cause of premature infants morbidity and mortality.Patients who survive with BPD often have obstructive airway disease,pulmonary hypertension,and delay of growth and development. Despite recent advances in the care of premature neonates,the morbidity of BPD didn't decline but even raised.There is no essential therapy for BPD at present which urge us to research pathogenesis of BPD and find effective treatments for it. Traditionally,Oxygen toxicity is one of important causes of BPD although the exact pathogenesis is not very clear.Previous studies have reported increased levels of transforming growth factor-β1(TGF-β1) in bronchoalveolar lavage fluid or lung tissues of BPD infants.However,there are fewer comprehensive studies about the role of TGF-β1 on BPD.Lentiviral vectors are capable of transducing a wide variety of dividing and nondividing cells,integrate stably into the host genome,and result in long-term expression of the transgene.And small interfering RNA(siRNA) technique is characterized by its specific silencing for taget gene.We constructed lentiviral vector expressing siRNA to explore whether inhibiting abnomal increased TGF-β1 in vivo does prevent lung from injury induced by hyperoxia in neonatal mice. Charpter one Construction and identification of lentiviral vector expressing siRNA of TGF-β1Objective:To construct the mouse TGF-β1-shRNA lentiviral vector and identify the knock-down efficiency of the TGF-β1 siRNA in alveolar epithelial cellsâ…¡(AECâ…¡).Methods:The shRNA sequence of TGF-β1 and negative control sequence were designed respectively.The gene homology of sequences were confirmed by BLAST sequence alignment in PubMed.Annealing of sequences was processed in vitro to form double-stranded oligonucleotides.Ligation reaction was set up between annealed fragments and pRNAT-u6.2 vectors which digested by the BamH1/Xhol.The products of ligation were transformed into DH5αcompetent cells.The recombinant clones were validated through sequence analysis when the plasmids were extracted by a small amount of extraction method.The positive plasmids were digested by Xhol and Nhel,and 7010bp gene band was recycled from gel.PKG-RFP vector was digested by Xhol and Nhel,and 1262bp RFP DNA fragments were recycled and purified.Recombinant vectors containing the 7010bp and 1262bp DNA fragments were ligated by T4 DNA ligation reaction and transformed into DH5αcells.Plasmid DNA from the positive colonies can be screened by digestion with BamH1 restriction enzyme and a 600bp band would be seen from the positive lentiviral vector.Three lentiviral plasmids from Lentiviral packaging system and the positive recombinant vector were mixed together and plasmid mix were transfected into 293T cells to package the virus.Virus preparation was titrated by gradient dilution method using G418 of drug screening from collected virus supernatant.The negative control vector was constructed by the same way.For identification of down-regulation efficiency of the TGF-β1 siRNA in vitro,the ACEâ…¡were randomly divided into 3 groups including interference group,negative control group and blank control group,for each set of five dishes.The packaged virus containing interference vector and negative vector were transfected into the plates of interference group and negative control group by polybrene method respectively,and the virus-free medium was added to the plate of blank control group.The down-regulation efficiency of TGF-β1 siRNA was verified to test the TGF-β1 protein expression by western blot after 96h transfection.Using the TGF-β1 polyclonal antibody as the first antibody,and FITC labeled goat anti-rabbit anti-IgG as the second antibody,the expression of TGF-β1 was observed by indirect immunofluorescence method on the AECâ…¡cells slices from 3 groups.Data were analyzed witt One-Way ANOVA and SNK method using SPSS13.0 software.Results:Sequencing analysis showed that,TGF-β1 shRNA silencing cassettes were inserted into pRNAT-u6.2 vector successfully.This recombinant plasmid was used to recombine with RFP fragments,and the positive Clones were identified by the procedures including transformation,screening of ampicillin-resistant and digestion of recombinant plasmid.The results of titration of virus preparation by gradient dilution method using G418 of drug screening showed that virus titer was 5.23×10⁸TU/ml.Western blot results showed that TGF-β1 protein expression of interference group was significantly lower than that of negative control group(P＜0.05) and blank control group(P＜0.05),and down-regulation efficiency of lentiviral vector was 73.6%.Indirect immunofluorecent indicated that negative control group and blank control group had a strong fluorescent expression, and the expression of green fluorescent of interfence group was obviously weakening.Conclusion:Lentivial Vector of pU6.2-TGF-RFP-Lenti expressing TGF-β1 shRNA for mouse has been constructed successfully and the results showed it can knock down the AECâ…¡TGF-β1 protein expression effectively.Charpter two Influence of knock-down TGF-β1 gene on development of alveolar in neonatal miceObjective:To explore the method importing the lentiviral vector that expresses TGF-β1 siRNA into the lungs of neonatal mice and its impacts on alveolar development.Methods:75 1-day-old Kunming newborn mice were randomly divided into interference group,negative control group and blank control group,and 25 for each group.TGF-β1 shRNA lentivirus vector and control vector was administered intranasally to lungs of newborn mice in interference group and negative control group at postnatal days 3(Postnatal days 3,to express P3,the same below) respectively.No interference treatment was processed in blank control group. Lung samples of 5 mice from each group were excised at P 4,7,14,21,28 respectively for detecting the expression of red fluorescent protein on frozen sections using Laser scanning confocal microscope.Histomorphology analysis of lung tissues on paraffin sections was processed to check the mean linear intercept(MLI)of alveolar and the radical alveolar counts(RAC).The expression of TGF-β1 mRNA and TGF-β1 protein in lung tissues from each group mice were determinated by RT-PCR and Western blot. Data were analyzed with univariate of general linear model and SNK of SPSS13.0 Software.Results:The expression of red fluorescent protein in lung tissue could be observed at P4～P28.The MLI and RAC in interference group were no significant difference compared with other two groups at P4(P＞0.05).Compared with the other two groups,MLI of mice in interference group was increased while the RAC was reduced significantly at PT～P28(P＞0.05).The expression of TGF-β1 mRNA in interference groups at all time point were lower significantly than those of blank control group and negative control group(P＜0.05).The expression of TGF-β1 protein in interference groups were lower significantly than those of ohther two groups at all time point(P＜0.05) except P4.All results showed no significant difference between the blank control group andnegative control group at all time-point(P＞0.05).Conclusion:The Intranasal administration is an efficacious method to import the lentiviral vector into the lungs of neonatal mice.Knock-down the expression of TGF-β1 gene by lentivirus vector-mediated small interfering RNA may lead to the developmental arrest of alveoli in neonatal mice.Charpter three The expression changes of TGF-β1 and Smad 4 in lung tissues of bronchopulmonary dysplasia miceObjective:Construction of mouse model of bronchopulmonary dysplasia induced by Oxygen exposoure and investigation changes of TGF-β1 and Smad 4 in lung tissues.Methods:50 newborn KM mice were randomly divided into hyperoxia group and control group.Each group contained 25 mice.Mice in hyperoxia group were exposed to 600mL/L oxygen from postnatal days 4 to days 14,and then restored to room air condition.The volume fraction of CO₂ was limited within 5ml/L.Mice in control group were exposed to room air condition.5 mice per group were randomly sacrificed and lung tissues were excised at postnatal days 4,7,14,21,28 respectively.Left lobes were processed for paraffin embedding and sectioning.Mean linear intercept(MLI) and radical alveolar counts(RAC) were counted.Ultrathin sections were made for observing ultrastructure of lung tissues by transmission electron microscope.SABC immunohistochemistry method was used to detect TGF-β1 in lung tissues.Right lobes were excised and frozen for detecting expression of TGF-β1 and Smad4 mRNA by Reverse transcription-Polymerase chain reaction.Data were analyzed with Independent-Sample T Test method using SPSS13.0 software.Results:Alveolar structure was irregular with thick alveolar wall and less alveoli number in every group at postnatal days 4.Mice in control group presented lung development from postnatal days 7 to days 28 characterized by increasing alveolar number along with more secondary septal crests formation.The thickness of alveolar wall and alveolar size decreased persistently with age.However,mice in hyeroxia group appeared impaired alveolar development characterized by a simplification of acinar structure, with a decreased number of alveoli and greatly enlarged terminal airways after postnatal days 7.Lung fibrosis was evidenced by increased thickness of interstitial after postnatal days 14.Some alveolar structure were ruined and fused after postnatal days 14.MLI of alveolar increased and RAC decreased in hyperoxia group compared with air group at postnatal days 7,14,21,28(P＜0.05).In hyperoxia group, ultrastructure of lungs tissues showed that alveolar typeâ…¡epithelial cell were swollen along with the electron density decreased.Lamellar bodies,with a loose structure,reduced even dispeared.Swollen Mitochondria were also viewed. Microvilli exfoliated and arranged in disorder.Obvious swollen of capillary endothelial cells was observed.TGF-β1-staining mainly located at bronchial epithelial,vascular endothelial and interstitial of lungs at P4～P28 in control group.However,alveolar epithelial cells also were stained in hyperoxia group after P7. Compared with control group,the expression of TGF-β1-staining of lung tissues increased in hyperoxia group at postnatal days 7,14,21,28.There were no difference between two groups on expression of TGF-β1 and Smad 4 mRNA at P4.The expression of TGF-β1 and Smad 4 mRNA were higher significantly in hyperoxia mice than those of control mice at postnatal days 7,14,21,28.Conclusion:The mouse animal model for bronchopulmonary dysplasia was constructed successfully.High expressions of TGF-β1 and Smad 4 in lung tissues of newborn mice induced by hyperoxia suggest a key role of enhanced TGF-β1/Smad signaling in pathogenesis of bronchopulmonary dysplasia.Charpter four The protection against hyperoxia-induced lung injury in newborn mice by lentiviral vector-mediated TGF-β1 siRNA in vivoObjective:To explore whether down-regulating high levels of TGF-β1 by lentivirus vector-mediated small interfering RNA(siRNA) should protect against hyperoxia-induced lung injury in neonatal mice.Methods:100 newborn KM mice were randomly divided into hyperoxia group,interference group,negative control group and air group.Each group contained 25 mice.Lentiviral vectors containing TGF-β1 shRNA or nagetive control sequences were administered intranasally to lungs of mice in interference group and negative control group respectively at postnatal days 3.Mice in hyperoxia group,interference group,and negative control group were exposed to 600mL/L oxygen from postnatal days 4 to days 14 before restoring to room air condition.The volume fraction of CO₂ was limited within 5ml/L.Mice in air group were exposed to room air.5 mice per group were randomly sacrificed and lung tissues were excised at postnatal days 4,7,14,21,28 respectively. Left lobes were processed for paraffin embedding and sectioning.Mean linear intercept(MLI) and radical alveolar counts(RAC) were analyzed.SABC immunohistochemistry method was used to detect SP-C and AQP5 in lung tissues. Frozen sections were processed and incubated with FITC-UEA-1 for detecting microvascular proliferation and assembly by laser confocal microscope.Right lobes were excised and frozen for detecting expression of TGF-β1 and collagenâ… mRNA and TGF-β1 protein by Reverse transcription-Polymerase chain reaction and Western blot methods respectively.Data were analyzed with univariate of general linear model and SNK of SPSS13.0 software.Results:The MLI of alveolar in interference group was smaller than that of hyperoxia group but larger than that of air group at postnatal days 7,14,21,28(P＜0.05).While the RAC was higher than that of hyperoxia group but lower than that of in air group at postnatal days 7,14,21,28(P＜0.05);The expression of AQP-5 increased gradually from postnatal days 4 to days 28 in air group(F=614.440,P=0.000).In hyperoxia group the AQP-5 expression were higher than that of air group at postnatal days 7 but lower than that of air group after that (P＜0.05) Interference group showed higher level of AQP-5 from P14～P28 compared with hyperoxia group but lower level compared with air group(P＜0.05); The expression of SP-C increased gradually from postnatal days 4 in air group untill reaching peak value at postnatal days 14,then decreased and sustained a stable level after that(F=290.029,P=0.000).SP-C staining in hyperoxia group was lower significantly than that of air group at postnatal days 7(P＜0.05) but higher than that of air group at postnatal days 14～21.The expression of SP-C in interference group was lower compared with hyperoxia group but higher compared with air group at postnatal days 14～21(P＜0.05);Lectin-binding test showed the pattern of fluorescence was anfractuous and fluorescence intensity was enhanced in hyperoxia group and negative control group.However,fluorescence of FITC in interference group was consistent with alveolar structure and relatively weak;There were no difference among four groups on collagenâ… mRNA expression at postnatal days 4～7(P＞0.05). At postnatal days 14～28,the expression of collagenâ… mRNA in lungs of interference group were lower significantly than those of hyperoxia group and negative control group(P＜0.05) but higher than those of air group (P＜0.05);Compared with other three groups,the expression of TGF-β1 mRNA in lungs decreased significantly in interference group at postnatal days 4(P＜0.05).At postnatal days 7,14,21,28,the expression of TGF-β1 mRNA and TGF-β1 protein in lungs of interference group were lower significantly than those of hyperoxia group and negative control group(P＜0.05) but higher than those of air group(P＜0.05).The above indexes showed no significant difference between hyperoxia group and negative control group at every time-point.Conclusion:knock-down TGF-β1 gene by lentivirus vector-mediated small interfering RNA may protect against hyperoxia-induced lung injury in neonatal mice.Recovery of normal differentiation from AECâ…¡to AECâ… ,promotion of normal degradation of extracellular matrix and improvement of proliferation and assembly of pulmonary microvascular via down-regulating excessive levels of TGF-β1 may be important mechanisms of this protection.