The Therapeutic Study Of Human Hepatocyte Growth Factor-enhanced Umbilical Cord Mesenchymal Stem Cells On Bleomycin-induced Pulmonary Fibrosis In Rats

Part Ⅰ The improvement of a pulmonary fibrosis rat model induced by bleomycinBackground:Idiopathic pulmonary fibrosis（IPF）is a chronic progressive interstitial pneumonia primarily characterized by dyspnea.The presence of fibrotic lesions in the lungs leads to restrictive ventilatory dysfunction in patients.Due to the limited therapeutic methods,the prognosis of the patients is poor.Therefore,it is necessary to prompt the basic research of pulmonary fibrosis to find a novel way of therapy for IPF patients.The model induced by bleomycin is the most frequently used animal model of pulmonary fibrosis,which has similar pulmonary fibrotic lesions with IPF.However,few studies have comprehensively elaborated the pulmonary function of this model currently in terms of pulmonary volume,pulmonary compliance and pulmonary ventilatory function.In the minority of these studies,the impairment of pulmonary function may be mild.Thus,there are some pulmonary function features that are not similar to IPF in the existing models,and it is difficult to study the mechanism and therapeutic effect through pulmonary function.In addition,the model established in the way used in the previous studies showed no significant impairment of pulmonary function in our preliminary experiments.Therefore,it is needful to improve the establishment method of the pulmonary fibrosis model and carry out a more comprehensive evaluation of this improved model,especially the pulmonary function test,in order to make sure that its characteristics are more similar to that of IPF.Objective:The aim of this part is to establish an improved animal model of pulmonary fibrosis,in which the pulmonary function characteristics of the model are more similar to that of IPF,to provide a better animal model for the future study.Methods:1.The grouping and modeling of animals:Rats were randomly divided into control group 1（CTRL1）,bleomycin group 1（BLM1）,control group 2（CTRL2）,and bleomycin group 2（BLM2）（12 rats in each group）.On the day of the establishment of pulmonary fibrosis,after anesthesia,dissecting the neck of rats until the cervical part of the trachea was exposed and making a small incision between two trachea rings,CTRL1 and BLM1 used the method reported in the previous papers（i.e.,the conventional way）,that is,CTRL1 received normal saline（1ml/kg body weight）,while BLM1 received bleomycin dissolved in normal saline（1ml/kg body weight,5mg/ml）,both endotracheally through the incision by a 1ml syringe;CTRL2 and BLM2 adopted an improved method（i.e.,the improved way）,that is,CTRL2received normal saline（2ml/kg body weight）,while BLM2 received bleomycin dissolved in normal saline（2ml/kg body weight,5mg/ml）,both endotracheally through the incision by an intrapulmonary spraying device which can convert the liquid into the aerosol.After completing each operation of every rat,the rat was shaking to promote the even distribution of the drug in the lungs.2.Pulmonary function test:On day 21 after bleomycin challenge,pulmonary function was tested in all groups under anesthesia.The indexes included:（1）Pulmonary volume:total lung capacity（TLC）,vital capacity（VC）,inspiratory capacity（IC）,inspiratory reserve volume（IRV）,residual volume（RV）,functional residual capacity（FRC）,RV/TLC%,FRC/TLC%.（2）Pulmonary compliance:dynamic pulmonary compliance（Cdyn）,static pulmonary compliance（Cchord）,pulmonary compliance at 50%of FVC（Cfvc50）.（3）Pulmonary ventilatory function:maximum mid-expiratory flow（MMEF）,forced expiratory flow after 10%of FVC has been exhaled(FEF_10%),FEF_25%,FEF_50%,FEF_75%.3.The analysis of pulmonary histopathology and the detection of hydroxyproline:After the pulmonary function test,rats were euthanatized.The left lungs were collected for H&E staining,Masson staining and Ashcroft scoring,while the right lungs were collected for the detection of hydroxyproline（HYP）.Results:1.The pulmonary fibrosis model established by the conventional way:（1）Pulmonary function:（1）Pulmonary volume:comparing to CTRL1,RV/TLC%and FRC/TLC%were significantly increased in BLM1（P<0.05）,but there was no significant difference in the change of the remaining indexes of pulmonary volume in BLM1（P>0.05）.（2）Pulmonary compliance:comparing to CTRL1,Cchord was significantly decreased（P<0.05）,but Cdyn and Cfvc50 showed no significant change（P>0.05）in BLM1.（3）Pulmonary ventilatory function:there was no significant difference in the change of the indexes of pulmonary ventilatory function in BLM1 compared with CTRL1（P>0.05）.（2）Pulmonary histopathology:according to H&E staining and Masson staining,lung tissue showed a normal structure and clear pulmonary alveoli in CTRL1,while the lung tissue displayed damaged alveolar structure,the mild to moderate deposition of interstitial cells and collagen fibers in the mesenchyme and thickened alveolar septa in BLM1.The Ashcroft scoring showed that BLM1 had significantly higher scores than CTRL1 did（P<0.05）.（3）Lung HYP:comparing to CTRL1,lung HYP of BLM1 was significantly increased（P<0.05）.2.The pulmonary fibrosis model established by the improved way:（1）Pulmonary function:（1）Pulmonary volume:comparing to CTRL2,TLC,VC,IC and IRV were significantly decreased,while RV,FRC,RV/TLC%and FRC/TLC%were significantly increased in BLM2 compared to CTRL2（all P<0.05）.（2）Pulmonary compliance:comparing to CTRL2,Cdyn,Cchord and Cfvc50 were significantly decreased in BLM2（all P<0.05）.（3）Pulmonary ventilatory function:there was significant difference in the change of FEF_10%,FEF_25%,FEF_50%and MMEF in BLM2 compared with CTRL2（P<0.05）.But FEF_75%showed no significant change in BLM2 compared with CTRL2（P>0.05）.（2）Pulmonary histopathology:H&E staining and Masson staining showed that there was a normal pulmonary structure in CTRL2,but lungs in BLM2 displayed severely damaged alveoli,the moderate to severe deposition of lots of interstitial cells and collagen fiber bundles in the mesenchyme,significantly thickened alveolar septa and enlarged alveoli in BLM2.Comparing to CTRL2,the Ashcroft scores of BLM2 were significantly increased（P<0.05）.（3）Lung HYP:lung HYP in BLM2 significantly increased compared to that in CTRL2（P<0.05）.Conclusion:1.The pulmonary fibrosis rat model established by the conventional way showed no obvious pulmonary function damage which means no restrictive ventilatory dysfunction,though some fibrotic lesions existed in the lungs.Therefore,the pulmonary fibrosis was mild,and the similarity of pulmonary function between this model and IPF is low.2.The pulmonary fibrosis rat model established by the improved way not only carried the histopathological features of pulmonary fibrosis,but also showed restrictive ventilatory dysfunction.Hence,the pulmonary fibrosis was severe,and this model has a high similarity with IPF.Part Ⅱ The therapeutic effect of human hepatocyte growth factor-enhanced umbilical cord mesenchymal stem cells on an improved pulmonary fibrosis rat model induced by bleomycin Background: IPF is a progressively exacerbating interstitial lung disease.For IPF,pirfenidone is the recommended drug which has positive therapeutic effect,but the efficacy is limited.Thus,it is necessary to find a more effective way to treat them.Mesenchymal stem cells（MSCs）have been proved that they can provide a therapeutic effect to pulmonary fibrosis.A number of studies have confirmed that most of the MSCs can home to pulmonary fibrosis areas after entering the body through veins,and play an important role in suppressing pulmonary fibrosis,by secreting a group of factors（e.g.,hepatocyte growth factor）.In the clinical trials,the treatment of MSCs had been proved to be safe,but the efficacy still needs to be improved.Hepatocyte growth factor（HGF）is the most studied anti-fibrotic factor in the study of pulmonary fibrosis.Recent studies have confirmed that HGF contributes to the repair of damaged alveolar epithelium and the inhibition of the pro-fibrotic effect of fibroblasts in lungs,and mediates the anti-fibrotic effect of MSCs.HGF-modified bone marrow MSCs had been proved to have better anti-fibrotic efficacy than normal ones,but the duration of the efficacy of this modified MSCs was limited.Among the MSCs from multiple tissue,umbilical cord MSCs（UCMSCs）have a higher level of HGF secretion,faster self-expansion in vitro and weaker immunogenicity,which has attracted much attention in recent years.Our previous study（unpublished data）found that UCMSCs had a stronger inhibitory effect on the deposition of collagen than bone marrow MSCs did,suggesting that UCMSCs may be more suitable for the treatment of pulmonary fibrosis.For the reasons mentioned above,it may be more helpful for the treatment of pulmonary fibrosis to further enhance the capacity of HGF secretion of UCMSCs by genetic modification.Objective: The aim of the present study is to evaluate the therapeutic effect of human HGF-UCMSCs on bleomycin-induced pulmonary fibrosis rat model established in an improved way and to preliminary explore the therapeutic mechanism of HGF-UCMSCs.Methods: 1.Production of the UCMSCs and HGF-UCMSCs: The primary UCMSCs were isolated from a healthy puerpera under 35 years old by the technicians of BEIJING SH BIOTECHNOLOGY CO.,LTD.After the identification of the morphology,phenotype,osteogenic and adipogenic differentiation,the UCMSCs were used for routine culture.The HGF-UCMSCs provided by the company were produced by infecting UCMSCs with recombinant adenovirus Ad-HGF which is a patented technology belonging to this company.2.Identification of the sixth generation of UCMSCs and HGF-UCMSCs:The UCMSCs and HGF-UCMSCs were resuscitated,seeded and taken photos.After culturing for three days,the supernatant of the culture medium was collected for the detection of HGF by an ELISA kit,and the cells were collected for the phenotype identification.The identification markers included CD73,CD90,CD105,CD45,CD19,CD34.3.The grouping and modeling of animals: SD rats were randomly divided into control group（CTRL）,bleomycin group（BLM）,pirfenidone group（PFD）,UCMSCs treatment on the third day group（MSC-D3）,HGF-MSCs treatment on the third day group（EMSC-D3）,UCMSCs treatment on the seventh day group（MSC-D7）and HGF-MSCs treatment on the seventh day group（EMSC-D7）（12 rats in each group）.The way to establish pulmonary fibrosis model was consistent with the improved way used in part I.On the day of the establishment of pulmonary fibrosis（day 0 after bleomycin challenge）,after anesthesia,dissecting the neck of rats until the cervical part of the trachea was exposed and making a small incision between two trachea rings,CTRL received normal saline（2ml/kg body weight）,while other groups received 5mg/ml bleomycin（2ml/kg body weight）,both via an intrapulmonary spraying device.After completing each operation of every rat,the rat was shaking to promote the even distribution of the drug in the lungs.4.Therapeutic schedule: On day 3 and day 7 after bleomycin challenge,both CTRL and BLM were injected with normal saline（400μl/rat/day * 2 days）through tail veins,while MSC-D3 and EMSC-D3 were respectively injected with UCMSCs and HGF-UCMSCs at a dose of 2*10^6/400μl/rat/day through tail veins for a two-day therapy.For MSC-D7 and EMSC-D7,respectively,UCMSCs and HGF-UCMSCs at the same dose mentioned above were injected into tail veins for a two-day therapy（on day 7 and day 11）after bleomycin challenge.For PFD,pirfenidone（100 mg/kg body weight/day）was given orally in drinking water from day 3 to 20 post bleomycin challenge（100 mg/kg body weight/day * eighteen days）.5.Pulmonary function test: On day 21 after bleomycin challenge,pulmonary function was tested in all groups under anesthesia.The indexes included:（1）Pulmonary volume: total lung capacity（TLC）,vital capacity（VC）,inspiratory capacity（IC）,inspiratory reserve volume（IRV）,residual volume（RV）,functional residual capacity（FRC）,RV/TLC%,FRC/TLC%.（2）Pulmonary compliance: dynamic pulmonary compliance（Cdyn）,static pulmonary compliance（Cchord）,pulmonary compliance at 50% of FVC（Cfvc50）.（3）Pulmonary ventilatory function: maximum mid-expiratory flow（MMEF）,forced expiratory flow after 10% of FVC has been exhaled(FEF_10%),FEF_10%,FEF_10%,FEF_10%.6.The analysis of pulmonary histopathology and the detection of hydroxyproline: After the pulmonary function test,rats were euthanatized.The left lungs were collected for H&E staining,Masson staining and Ashcroft scoring,while the right lungs were collected for the detection of hydroxyproline（HYP）.7.The detection of the lung inflammatory factors: Some of the right lung tissue was used to detect the inflammatory factors including granulocyte-macrophage colony-stimulating factor（GM-CSF）,interleukin-4（IL-4）,IL-1β,IL-10,IL-17 and vascular endothelial growth factor（VEGF）.Results: 1.The identification of primary UCMSCs: The primary UCMSCs were plastic-adherent and mainly characterized by spindle shape.They could differentiate into osteoblasts and adipoblasts after induction in vitro,and expressed CD105,CD73,CD90 and CD44,HLA-ABC but not CD34,CD45,CD11 b,CD19 and CD14.2.The identification of the sixth generation of UCMSCs and HGF-UCMSCs: The sixth generation of UCMSCs and HGF-UCMSCs seeded were all plastic-adherent and mainly characterized by spindle shape,and expressed CD73,CD90 and CD105 but not CD45,CD19 and CD34.HGF was not detected in the culture supernatant from CTRL Well.Comparing to UCMSCs,HGF-UCMSCs secreted a significantly higher level of HGF（P < 0.05）.The concentration of HGF in the culture supernatant from HGF-UCMSCs Well was 30 times higher than that from UCMSCs Well.3.Pulmonary histopathology: In the pictures of H&E and Masson staining of lung tissue,it could be seen that the structure of CTRL lungs was normal,while the alveolar structure of BLM was severely damaged accompanied by moderate to severe collagen fiber bundles deposited in lung mesenchyme,significantly thickened alveolar septa and enlarged alveoli.H&E and Masson staining of lung tissue appeared to show decreased injury when the treatments were given.In terms of the Ashcroft scoring,BLM had significantly higher scores compared to CTRL（P < 0.05）.Comparing to BLM,the scores in all treatment groups were reduced significantly（P < 0.05）.Both PFD and EMSC-D7 showed significant difference in the Ashcroft scores compared with other three treatment groups（all P <0.05）,but there was no significant difference between PFD and EMSC-D7（P > 0.05）.4.Lung HYP: Comparing to BLM,lung HYP of CTRL was significantly decreased,also,that of PFD and EMSC-D7 were significantly decreased（all P < 0.05）.There was no difference in lung HYP between PFD and EMSC-D7（P > 0.05）.MSC-D3,EMSC-D3 and MSC-D7 showed no significant difference compared with BLM（P all > 0.05）.5.Pulmonary function:（1）Pulmonary volume: comparing to CTRL2,TLC,VC,IC and IRV were significantly decreased,while RV,FRC,RV/TLC% and FRC/TLC% were significantly increased in BLM2 compared to CTRL2（all P < 0.05）.Comparing to BLM2,there was no difference in the above pulmonary volume indexes in the groups treated with pirfenidone,UCMSCs or HGF-UCMSCs（all P > 0.05）.（2）Pulmonary compliance: comparing to CTRL2,Cdyn,Cchord and Cfvc50 were significantly decreased in BLM2（all P < 0.05）.Pirfenidone,UCMSCs or HGF-UCMSCs did not significantly change the pulmonary compliance indexes compared with BLM2（all P > 0.05）.（3）Pulmonary ventilatory function: there was significant difference in the change of FEF_10%,FEF_10%,FEF_10% and MMEF in BLM2 compared with CTRL2（P > 0.05）.But FEF_10% showed no significant change in BLM1 compared with CTRL2（P > 0.05）.All treatment groups did not show a significant therapeutic effect towards the indexes of pulmonary ventilatory function.6.Lung inflammatory factors: The results of the expression of inflammatory factors in lung tissue showed that the levels of GM-CSF,IL-4,IL-1β,IL-10 and VEGF in BLM were significantly lower than those in CTRL,while the level of IL-17 was significantly higher than that in CTRL（all P < 0.05）;GM-CSF was significantly increased in MSC-D7 compared with BLM（P < 0.05）.Compared with BLM,the level of IL-4 in MSC-D3 was significantly increased（P < 0.05）.Comparing to MSC-D7,both of GM-CSF and IL-4 decreased significantly in EMSC-D7（all P < 0.05）.Conclusion: 1.HGF-UCMSCs,which had a higher level of HGF secretion than UCMSCs did and carried similar basic characteristics of morphology and phenotype compared with UCMSCs,were successfully prepared by the way of infecting UCMSCs with recombinant adenovirus Ad-HGF.2.After bleomycin challenge,the treatment of HGF-UCMSCs on day 7 showed stronger inhibition towards pulmonary fibrosis than that on day 3.3.Starting the treatment on the seventh day after the establishment of the pulmonary fibrosis model,HGF-UCMSCs exerted more significant influence in inhibiting pulmonary fibrosis than UCMSCs did,and the efficacy of UCMSCs was similar to pirfenidone treatment,but it is still difficult for HGF-UCMSCs and pirfenidone to reverse severely impaired pulmonary function.4.The treatment of HGF-UCMSCs on day 7 after bleomycin challenge showing a more significant therapeutic effect was probably due to the suppression of lung tissue GM-CSF and IL-4.