| Objective:Ourstudy aimed to explore the effection of lung inflammation and fibrosis of BPD kunming white mice by over-expressing mir-206 with hydrodynamic injection.Methods:BPD of neonatal mice models were established by exposing in constant hyperxia condition. 4 litters of 1 day of birth were booked,they were randomly divided into High oxygen group, high oxygen mi R-206 group, hyperoxia in blank plasmid group and air control group. Each group contained 6-8 mouse, The three groups of high oxygen environment were exposed in 80% oxygen concentration.To Observe and record the weight and the general state of mice on3 thd, 8thd, 22 thd, 29 thd, 35 thd after birth; Beginning in 21 thd, High oxygen mi R-206 group and hyperoxia in blank plasmid group was intervened treatment respectively. The details are as follows: The mmu-mi R-206 recombinant plasmid and blank plasmid sh NC were dissolved in physiological saline.We injected the Physiological saline dissolving recombinant plasmid mmu-mi R-206 via hydrodynamic injection(30 mg/kg/ time, 2 times / week, for 2 weeks).3days after the injection, mice were killed andorbital blood was got for the assessment of blood inflammatory factor IL-6,TGF-βby ELISA,Lungs were dissected and divided into 4parts,one part wasfixed inthe 4% poly Formaldehyde Solution for making paraffin section;one part was storedin liquid nitrogen preservation for total RNA extraction in lung tissue, onepart was put in-20 ℃ for frozen section,onepart was for weighingbefore and afteroven treatment.The Pathological changes of lung tissue,inflammatory reaction and fibrosis degree were observed via HE staining and Masson trichrome staining,.The mice weight, lung wet weight, lung wet / dry weight ratio, radial alveolar count(RAC) and serum interleukin(IL-6), transforming growth factor beta(TGF- β) concentration changeswere determined.At the same time, mi R-206 m RNA and the main extracellular matrix associated protein fibronectin 1(FN1) m RNA were surveillancedby Real-time PCR.Lung tissue frozen section immunity fluorescence was conducted to observe whether the expression of FN1 protein changes to explore the effection of lung inflammation and fibrosisof BPD kunming white mice by over-expressing mir-206.Results:1.After the recombinant plasmid mmu-mi R-206 was injected via the tail into mice,The expression of mi R-206 m RNA in lung tissues was detected by RT-PCR.The difference of the expression of mi R-206 in high oxygen mi R-206 treatment group and high oxygen plasmid control group mi R-206 was statistically significant(P<0.01).The results suggestedthat exogenous gene transfer is successful.2.Theweight of mice inhigh oxygen mi R-206 treatment group and high oxygen plasmid control group mi R-206 showed no difference in statistics.However,the mice from the high oxygen environment treatment group showed better activity endurance than hyperoxia control groups.Lung wet weight lower, wet / dry weight ratio relatively higher,obvious Subcapsular inflammatory reaction had not been observed in lung tissue paraffin section HE staining;but Mason’s trichrome stain showed collagen sediment was no significant differencebetween each group.3. The difference of IL-6, TGF-β in mice surum between high oxygen mir206 and high oxygen mir206 treated group showed statistical significance(P < 0.05)..Real-time PCR test showed that the FN1 gene m RNA expression was decreased in high oxygen mir206 treated, but there was no significant decrease in FN1 protein expression..Conclusion:ourstudy implemented the over-expression of mir-206 gene in mice lung throug h the tail vein of high pressure injection. After intravenous recombinant plasmidmm u-mi R-206 was injected,the expression of mi R-206 in lung tissue of mice increased,Through the down-regulation of the pro-inflammatory cytokine levels of IL-6 inhib it the reaction of the inflammatory response from expanding and decreas the expres sion of m RNA FN1 gene. The above shows that pulmonary fibrosis have improving trend at the molecular level. Because the starting time and duration of delayed tr eatment of short treatment time, pathological structure is improved obviously, It ma y indicate that the anti fibrosis effect of mi R-206 in a time-dependent manner. The se changes resulted in the inhibition fo inflammation and the decrease of airway inf lammation and remodeling index TGF-β. |
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