| Objective: To investigate the effects transplantation of bone marrow mesenchymal stem cells genetically modified hepatocyte growth factor in Bleomycin-induced lung fibrosis in rats on efficacy.Methods: Study was carried out on 140 male Fisher 344 rats weighing 200 to 250 g,randomly classified into 4 groups(n=35): Control group, BLM group(bleomycin treated group), EGFP-MSCs group(MSCs were transduced with empty vector), h HGF-MSCs group(MSCs were transduced with lentiviral vector modified with human HGF). Bleomycin was inhaled by endotracheal as a single dose(5 mg/kg) dissolved sterile saline in BLM group, EGFP-MSCs group and h HGF-MSCs group; Control group received sterile saline only. EGFP-MSCs group and h HGF-MSCs group received EGFP-MSCs or h HGF-MSCs 5×105 in DMEM 1 ml by injection into left jugular vein 2 days after administration of bleomycin,while Control group and BLM group received equal DMEM. The survival rate within 28 days of 15 rats was monitored selected from each group. MSCs were observed in lung tissue frozen section by Laser scanning confocal microscope selected from each group at day 28 of 5 rats.Rat(5 rats/ group) were killed on days 7, 14 and 28 after BLM treatment. The general conditions of the rats were observed and recorded the weight loss. The lungs were harvested for histopathological studies by HE or Masson staining. The Ashcroft score was used to quantify the histologic analysis. Lung Tissue hydroxyproline(HYP) content was measured by the way of hydroxyproline alkaline. The content of rat HGF(r HGF), h HGF and SP-A protein in serum and TGF-β1 in lung tissue were measured by ELASA. The expression of TGF-β1、SP-A in the lung tissue were determined by immunohistochemistry.Result:(1) A large number of green fluorescent cells were observed in the lung of EGFP-MSCs group and h HGF-MSCs group.No green fluorescent cells were observed in BLM group.(2) h HGF and r HGF concentration of serum was assessed by enzyme immunoassay. There was no h HGF detected in other groups except in h HGF-MSCs group. The r HGF concentration of serum showed non significantly difference at day 7( p > 0.05), increased at day 14,28 in the EGFP-MSCs group and h HGF-MSCs group compared to BLM group(p < 0.05). Furthermore, there was a further increase in h HGF-MSCs group compared to EGFP-MSCs group(p < 0.01, p < 0.05).(3) There was a decrease in weight loss in EGFP-MSCs group and h HGF-MSCs group that was significantly decrease when compared to BLM group(p < 0.01).In h HGF-MSCs group,it showed non significantly difference at day 7,14 and greater significantly difference at day 28 when compared to EGFP-MSCs group(p < 0.01).(4) In the BLM group,the mortality rate of BLM group was significantly increased, which was mainly concentrated on 14 day ago. Survival rates for EGFP-MSCs group and h HGF-MSCs group were significantly higher than that for the BLM group(p < 0.05), the survival time of h HGF-MSCs group was longer than that for the EGFP-MSCs group.(5) The degree of alveolitis in the EGFP-MSCs group and h HGF-MSCs group showed a significant decreased compared to BLM group at different time points. Moreover, there was a further reduced in h HGF-MSCs group compared to EGFP-MSCs group. The collagen deposition and Ashcroft score were non significantly difference at day 7( p > 0.05), reduced at day 14,28 in the EGFP-MSCs group and h HGF-MSCs group compared to BLM group(p < 0.05).Furthermore, there was a further reduce in h HGF-MSCs group compared to EGFP-MSCs group(p < 0.05).(6) The hydroxyproline content of the lung treated with EGFP-MSCs was reduce compared to BLM group at different time points(p < 0.01, p < 0.05).In rats treated with h HGF-MSCsthe hydroxyproline content was even further reduce compared to EGFP-MSCs group(p < 0.01).(7) Immunostaining using TGF-β1 antibody showed significantly reduce in EGFP-MSCs group compared to BLM group at different time points(p < 0.05). The tissue concentration of TGF-β1 in h HGF-MSCs group was significantly lower than in EGFP-MSCs group(p < 0.05).A significant and marked increase in the tissue concentration of lung SP-A was seen in EGFP-MSCs group. The expression levels of lung SP-A revealed a significant increase in h HGF-MSCs group compared to EGFP-MSCs group(p < 0.05).(8) The expression levels of lung TGF-β1 and serum SP-A significantly reduce in EGFP-MSCs group compared to BLM group at different time points(p < 0.01, p < 0.05). Furthermore, there was a further reduce in h HGF-MSCs group compared to EGFP-MSCs group(p < 0.05).Conclusion:(1) In our study, a lung fibrosis model was successfully induced by endotracheal inhalation of bleomycin in rats.(2) MSCs were delivered to rats by injection into left jugular vein may survived in lung detected in lung fibrosis in rats.(3) MSCs implantation ameliorated the general condition of rats and pathological changes and collagen deposition of lung tissue prolong the survival time of rats; h HGF-MSCs contribute a more effective treatment of lung fibrosis. |
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