Research On The Blocking Effect Of Heat Shock Protein 70 To HIF-1α In Newborn Rats With Hypoxic Pulmonary Hypertension

Objective: 1.To explore the preparation of gene transfection liquid by adenovirus mediated HSP70. 2. To explore the best pathway,optimal transfection volume and transfection time of HSP70 gene in neonatal rat lung. 3. To observe the changes of the mPAP, pulmonary vascular structure and remodeling index and expression of HSP70、HIF-1α and its target genes ET-1、iNOS in newborn rats with HPH after transfection of with adenovirus mediated HSP70 gene by gene transfection technology,was to understand the effect of HSP70 on pulmonary arterial pressure and pulmonary vascular remodeling,to further infer whether HSP70 could downregulate the expression of HIF-1α and the expression of its target genes, could delay the development of neonatal rats with HPH for providing the preliminary theoretical basis for clinical treatment. Methods: 1. Recombinant adenovirus was produced, identified, purified and determined its titer by the construction of pHBAd-MCMV-GFP-HSP70 plasmid by using double plasmid co transfection of 293 cells. 2.(1) 48 neonatal rats were randomly divided into group A: tail intravenous injection, B: Intraperitoneal injection. Transfection fluid of 5ul×1010PFU/ml with Ad-HSP70 were injected into neonatal rats, the rats were sacrificed at the observation time and the specimens of lung tissue were collected, the pathway of HSP70 of transfected into lung tissue were taken to determine by immunofluorescence.(2) 54 neonatal rats were randomly divided into group: virus group,virus and HSP70 group,control group, 2.5ul×1010 PFU/ml、5ul×1010 PFU/ml、10ul×1010 PFU/ml were given respectively by tail vein. After transfection of 72 h on different dosage, the semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR) method and Western blot were used to detect the mRNA and protein levels of HSP70 to determine the optimal transfection of adenovirus mediated HSP70.(3) After determining the best way and the optimal transfection volume, 48 neonatal rats were randomly divided into groups 24 h group, 48 h group, 72 h group, 96 h group and the control group according to the different transfection time. At the time point of death of neonatal rats, the lung tissue were taken for RT-PCR and Western blot to detect the mRNA and protein levels of HSP70. 3.128 neonatal rats were randomly divided into 2 groups: HPH model group(H) and control group(C). Group H: divided into H1: saline group, H2: empty virus group(pHBAd-MCMV-GFP), H3: virus and HSP70 group(pHBAd-MCMV-GFP-HSP70) according to the transfection solution. The HPH model was established after injecting the virus or Sterile saline into the H group, and were observed after 3, 7, 10 and 14 days of hypoxia.(1) To observe of pulmonary arterial pressure(mPAP) at different time points, to compare the changes of mPAP in each groups, to understand the effect of HSP70 on the reduction of mPAP.(2) The structure and ultrastructure of pulmonary vessels were observed by optical microscope and electron microscope, and the indexes of pulmonary vascular remodeling(MA%, MT%) were measured.(3) the mRNA and protein expression of HSP70, HIF-1α, ET-1 and iNOS in the lung tissues of neonatal rats at different time points were detected by immunohistochemistry, RT-PCR and Western blot respectively, and the difference between the four factors was compared. To further infer the effect of HSP70 about blocking HIF-1α and iNOS, ET-1 in HPH of neonatal rats. Results: 1.The sequence of the recombinant plasmid pHBAd-MCMV-GFP-HSP70 were consistent with the sequence of Genebank, which was HSP70 gene. 2.(1)A small amount of green fluorescent signal labeled protein was observed in the 48 hours in the tail vein injection group, seen at 72 and 96 hours obviously.(2) Expression level of HSP70 mRNA and protein increased with Ad-HSP70 of 5ul×1010 PFU/ml.(3).Expression levels of HSP70 mRNA and protein in neonatal rats were significantly higher at 72 hours, was statistically significant than that of 24 h and 48h(P < 0.05), was not statistically significant than that of 96h(P>0.05).3.(1)The mPAP level were significantly higher between H1, H2 groups and control group at 3, 7, 10, 14 days, the difference was statistically significant(P<0.05), The mPAP in H3 group at 3, 7, 10 days level and the control group were not statistically significant(P>0.05), The mPAP level between H3 group and H1, H2 groups at 14 days were not statistically significant(P > 0.05).(2).Pulmonary vascular remodeling were not observed under the light microscope in H3 group at 3, 7, 10 days by HE, VG staining, the pulmonary vascular remodeling were reduced at 14 days.The ultrastructure of the lung under electron microscope showed that the pulmonary vascular remodeling were reduced at 7, 10, 14 days of hypoxia.MT%and MA% in pulmonary vessels in each group of hypoxia were significantly different(P<0.05), were significantly different(P<0.05) between H3 group and H2 group at7, 10days; at 14 days of hypoxia in each group(P<0.01), were significantly different(P<0.01) between C group and HPH groups.(3).Reaction intensity of HSP70,HIF-1α,ET-1 and iNOS in the lung tissue was negative by immunohistochemistry in control group, reaction intensity of HSP70,HIF-1α, ET-1 and iNOS in HHP groups was positive reaction in different degrees, and its expression was more common in pulmonary vascular endothelial cytoplasm and cell. The immunohistochemistry expression intensity of HSP70 in each group was statistically significant(P<0.05) at 3、7、10days of hypoxia,the comparation between HPH groups and C group was statistically significant(P<0.01). The comparation between H3 group and H1, H2 groups were statistically significant(P<0.01). The immunohistochemistry expression intensity of HIF-1α, ET-1and iNOS in each group at 3,7,10 days of hypoxia were statistically significant(P<0.01), HPH groups were all enhanced, the comparation between H3 group and H1, H2 groups were statistically significant(P<0.01), H3 group were decreased.(4).The expressions of HSP70 mRNA were statistically significant(P<0.01) in each group at 3, 7, 10 days of hypoxia, the comparation between HHP groups and C groups were enhanced, statistically significant(P<0.05), The expression of H3 group and H1 group was enhanced, statistically significant(<0.05). the comparation between HPH groups and C group at 14 days was statistically significant(P<0.05). The expression of HSP70 protein in hypoxia 3, 7, 10 days was statistical significance(P<0.05), and the comparation between H1, H2 groups and C group was enhanced, statistically significant(P < 0.01), and the expression between H3 group and H1, H2 groups was enhanced, statistically significant(P<0.05).(5). The expressions of HIF-1αmRNA, ET-1 mRNA, iNOS mRNA at 3, 7, 10 days of hypoxia were statistically significant(P<0.01), the comparation between H1, H2 groups and C group was statistically significant(P<0.05), the comparation between H3 group and H1, H2 group were statistically significant(P<0.05),the expression of H3 group was decreased. The expression of HIF-1α, ET-1,iNOS protein were statistically significant(P<0.05) in each group, the comparation between H3 group and H1, H2 groups was reduced, statistically significant(P<0.05). The expression of iNOS mRNA at 14 days of hypoxia was statistically significant(P<0.05) in each group,the comparation between HPH groups and C group was enhanced, statistically significant(P<0.01).Conclusion: 1.The recombinant adenovirus vector of HSP70 gene and the recombinant adenovirus were successfully constructed. 2. Determine the optimal way: tail vein,volume: 5ul×1010PFU/ml and the earliest observation time :72 hour of recombinant adenovirus mediated by HSP70 in the body of neonatal rats. 3. Hypoxia stress can induce endogenous HSP70 expression, adenovirus mediated HSP70 can improve the expression of exogenous HSP70 in HPH, downregulate the expression of HIF-1α, ET-1, iNOS, reduce pulmonary arterial pressure, reduce pulmonary vascular remodeling, can become a new strategy for treatment of neonatal HPH.