| Idiopathic pulmonary fibrosis (Idiopathic pulmonary fibrosis, IPF) is a chronic,progressive lung interstitial disease, the incidence is rising. So far the cause of IPF isunknown, and lack of effective treatment, median survival of it is less than three years,the prognosis is poor, is hot and difficult clinical in studies. Corticosteroids andimmunosuppressive drugs is still the primary means of treatment of IPF, but the effectiverate is low. In recent years, thanks to the rapid development of molecular biologicalscience, treatment by cytokines of pulmonary fibrosis has been confirmed.Basic fibroblast growth factor (Basic fibroblast growth factor, bFGF) is amultifunctional damage repair factor, its biological significance in the developmentprocess of pulmonary fibrosis is still missing reported. In view of this, the experiment letbFGF connected with collagen-binding domain (Collagen-bind domain, CBD), after thereorganization of the CBD-bFGF to intervene at all stages of pulmonary fibrosis in mice,initially discovered given CBD-bFGF in the early stage of pulmonary fibrosis in mice hasno obvious biological effects, and given CBD-bFGF in later stage of pulmonary fibrosis inmicecan promote the expression of TGF-β1and collaboration with TGF-β1acclerlated thepulmonary fibrosis in mice.Interference the BFGF expression may become a new strategyof IPF in the future.Part1：Effetcs of CBD-bFGF on early stage of Bleomycin-inducedpulmonary fibrosis in miceObjective：To investigate the biological effects and mechanisms of CBD-bFGF on earlystage of bleomycin-induced pulmonary fibrosis in mice.Methods：Mice were randomly divided into3groups：（1）Sham group n=40;(2) BLMgroup n=60;(3) BLM+bFGF group n=60.The Sham group was received saline twice.TheBLM group was injected intratracheally with Bleomycin(3mg/kg) and immediately treated saline.BLM+bFGF group was injected intratracheally with Bleomycin (3mg/kg) andimmediately treated CBD-bFGF(2ug CBD-bFGF).Each group was sacrificed at7d、14d、21d、28d.Observe the general condition and weight changes of each group. The pathologicchanges of the lung tissue were obtained by HE and Masson staining,and the expression oftransforming growth factor β1（TGF-β1），α-smooth muscle actin (α-SMA)、collagenI(CO1-I)、collagenIII(CO1-III)were analyzed by immunohistochemistry.Results：（1）Sham mice breathing steady, move freely, eating, drinking normal; BLM mice3d effect from shortness of breath, slow, loss of appetite, peripheral cyanosis and otherperformance; BLM+bFGF group generally were similar with BLM;BLM+bFGF groupwere similar as BLM group;（2）Sham mice body weight continued to increase; BLM bodyweight continued to decline slightly after rising21d, there was significant differencecompared with the Sham group each observation point difference (p <0.01), BLM+bFGFdecreased body weight, compared with the BLM group the difference was not statisticallysignificant (p>0.05)（3）Sham group light microscope showed no abnormal pathology; InBLM group a lot of lung tissue fibroblast proliferation, excessive deposition ofextracellular matrix, and some structural damage to lung tissue, showing alveolar collapseor disappear, pulmonary fibrosis, Ashcroft score there were significant differencescompared with the Sham group(p<0.01);BLM+bFGF group pulmonary fibrosis in mice toform in light microscope, compared with the BLM group Ashcroft score was no significantdifference.(4)Lung tissue of mice in each group visible α-SMA、Collagen-I,、Collagen-IIIprotein expression, BLM+bFGF group and BLM group compared with sham group weresignificantly increased (p <0.01, p <0.05,p <0.05), but the comparison between the BLMgroup and the BLM+bFGF group, the difference was not statistically significant (p>0.05).In7d,14d observation point, BLM+bFGF group, BLM group TGF-β1expressioncompared with sham group was significantly increased (p <0.01, p <0.01),21d,28dobservation points BLM+bFGF group, BLM group TGF-β1expression decreased，compared with the sham group, the difference was statistically significant (p <0.05, p<0.05), BLM+bFGF group compared to the each time points the difference was not statistically significant with the BLM group(p>0.05).Conclusion:(1)BLM intratracheal administration successfully constructed pulmonaryfibrosis in mice.(2)Given CBD-bFGF on early stage of Bleomycin-induced pulmonaryfibrosis in mice without obvious biological effects.Part2：Effetcs of CBD-bFGF on later stage of Bleomycin-inducedpulmonary fibrosis in miceObjective：To investigate the biological effects and mechanisms of CBD-bFGF on laterphase of bleomycin-induced pulmonary fibrosis in miceMethods： Mice were randomly divided into2groups：(1) Sham group n=40;(2)Pulmonary fibrosis model group n=140.The Sham group was sham-operation byintratracheally with saline.Pulmonary fibrosis model was induced by single intratrachealinjection of bleomycin as part1.After6weeks administration,the pulmonary fibrosis modelgroup was randomly divided into2groups:(1)BLM group n=40;(2)BLM+bFGFgroup.n=40.Sham group and BLM group were sham-operation by intratracheally with0.1ml saline as part1.BLM+bFGF group was treated CBD-bFGF(2ug).The observed pointwas16w、22w、26w.The pathological mechanism of the lung tissue was similar as part1.The α-smooth muscle actin (α-SMA)、collagen I(CO1-I)、collagen III(CO1-III)wereanalyzed by immunohistochemistry. Lung function in mice was detection by animal lungfunction instrument and blood gas analyzer.Results:(1)Sham group, body weight decreased slightly after receive saline,then weightcontinued to increase; BLM group continued to drop weight during0-10w, at26w weightslightly recovery,compared with Sham group the difference was statistically significant (p<0.05); BLM+bFGF group: sustained decreased in body weight, each point were lowerthan BLM group after14w in corresponding time points, the difference was statisticallysignificant (p <0.05).(2)Sham group showed normal lung tissue in light microscope; BLMgroup18w,22w,26w lung tissue collagen fibers and fibroblast proliferation widespread, severe damage to the alveolar structure, part of alveolar collapse and disappear; BLM+bFGF group compared with BLM group pulmonary fibrosis score increased,18wobservation point difference was statistically significant (p<0.05),22w,26w observationpoint difference was significant.(p<0.01);.(3)The expression ofα-SMA、C01-I、C01-III inlung were more than BLM,the difference had statisticalsignificance(p<0.05,p<0.01,p<0.01). In26w observation point, BLM+bFGF group ofTGF-β1expression increased compared with BLM group, the difference was statisticallysignificant.(4)Compared with Sham group, BLM and BLM+bFGF group decreased lungcompliance group, the difference was statistically significant(p <0.05). BLM+bFGFgroup decreased more significantly than the BLM group, the difference was statisticallysignificant(p<0.05). In blood gas analysis, BLM group and the BLM+bFGF group haddecreased PO2, CO2retention performance, compared with the Sham group difference wasstatistically significant (p <0.05).BLM+bFGF group PO2decreased, CO2retentionremain higher than the level of BLM group, the difference was statistically significant(p<0.05).Conclusion: Given CBD-bFGF on later stage of bleomycin-induced pulmonary fibrosis inmice, does not have a role like wound repair, and can stimulate the expression of TGF-β1,increased pulmonary fibrosis in mice... |
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