| Many acute and chronic lung disorders with variable degrees of pulmonary inflammation and fibrosis are collectively referred to as interstitial lung diseases(ILDs) or diffuse parenchymal lung diseases.Idiopathic pulmonary fibrosis(or cryptogenic fibrosing alveolitis)(IPF or CFA) is one of several idiopathic interstitial pneumonias.Treatment options recommended by the American Thoracic Society(ATS) and the European Respiratory Society(ERS) include corticosteroids and immunosuppressive agents (azathioprine and cyclophosphamide) alone or in combination.But physicians lack enough evidence of the efficiency of the present treatment.We study the therapeutic effect of over one times transplantation with S8 embryonic stem cell's(SS-ESC) on the pulmonary injury of mouse induced by bleomycin and their mechanisms.Part 1Objectives 1.To look for a reasonable and reliable dosage for mice pulmonary fibrosis model induced by bleomycin.2.To look for a reasonable quantity of S8-ESC after comparing with different transplanting quantity of S8-ESC for mice pulmonary fibrosis model induced by bleomycin in small scale.3.To look for a reasonable times and interval of transplanting S8-ESC after comparing with different times and interval of it for mice pulmonary fibrosis model induced by bleomycin in small scale.Methods 1.60 mice were divided into four groups randomly.2 mg/kg,3.5 mg/kg and 5.0 mg/kg of bleomycin were given to the mice intratracheally in group BLM-1, BLM-2 and BLM-3 respectively and the mice in control group were administrated the same volume of saline.All of the mice were sacrificed 4 weeks later and their lung tissues were harvested for histological study and the hydroxyproline contents were measured.2. Twenty-one C57BL/6J female mice were divided randomly into five groups,they were control group,bleomycin model group,treatment group-1,treatment group-2 and treatment group-3.Those mice except mice in control group were administrated bleomycin intratracheally to induce model of lung injury,then 1.0×105,1.0×106 and 1.0×107epithelial progenitor cells from S8-ESC were transplanted into mice respectively in three treatment groups and the mice of the control group and the bleomycin model group were administrated 0.9%sodium chloride respectively.After twenty-eight days,the life-span, hydrocyproline concentration in lung tissue,transforming growth factorβ1(TGF-β1) and interferon-γ(IFN-γ) concentration in lung tissue bomogenate were examined and the pathologic changes in the injured lung tissue were observed.3.Sixty-six C57BL/6J female mice were divided randomly into three big groups:sequential transplantation group, every-other-day transplantation group and every-two-day transplantation group.Each big group mice were divided randomly into six groups,they were control group,bleomycin model group,treatment group-1,treatment group-2,treatment group-3 and treatment group-4.Those mice except mice in control group were administrated bleomycin intratracheally to induce model of lung injury,then 1.0×107epithelial progenitor cells from S8-ESC were transplanted into mice respectively in four treatment groups from once to four times and the mice of the control group and the bleomycin model group were administrated 0.9%sodium chloride respectively.After twenty-eight days,the life-span, hydrocyproline concentration in lung tissue,transforming growth factorβ1(TGF-β1) and interferon-γ(IFN-γ) concentration in lung tissue bomogenate were examined and the pathologic changes of the lung and the engraftment of the epithelial progenitor cells in the injured lung were observed.Result 1.With the increasing of the bleomycin does administrated to the mice,in three groups the extent of the pulmonary fibrosis became severe one by one,while the mice in control group was normal,and the mortalities were 0%,60.0%,73.3%and 93.3% respectively.2.With the increasing of epithelial progenitor cells from S8-ESC administrated to the mice,in four groups the extent of the pulmonary fibrosis became light one by one,while the mice in control group was normal and the mortalities, hydrocyproline concentrations and TGF-β1 concentrations in lung tissue bomogenate from high to low were bleomycin model group,treatment group-1,treatment group-2,treatment group-3 and control group respectively.However,IFN-γconcentrations in lung tissue bomogenate were inverse.3.The extent of the pulmonary fibrosis in five groups from severe to light were bleomycin model group,treatment group-1,treatment group-2, treatment group-3 and treatment group-4 while the mice in control group were normal.The expression was more apparent in every-other-day transplantation group than other big groups. And the mortalities,hydrocyproline concentrations and TGF-β1 concentrations in lung tissue bomogenate from high to low were bleomycin model group,treatment group-1, treatment group-2,treatment group-3,treatment group-4 and control group respectively. However,IFN-γconcentrations in lung tissue bomogenate are inverse in each group.The expression was also more apparent in every-other-day transplantation group than other big groups.With the increasing of the time of transplanting epithelial progenitor cells administrated to the mice,field planting time in the lung became longer and longer.Preliminary conclusions 1.Considering both effectiveness and death rate,the better dosage of bleomycin that induced pulmonary fibrosis model by endotracheal injection in mice is 3.5mg·kg-1.2.The transplanting epithelial progenitor cells from ESCs may relieve injured lung induced by bleomycin and mechanism of action is associated with cell factors such as TGF-β1 and IFN-γand so on while with the increasing of the transplanting epithelial progenitor cells administrated to the mice(105-107),The expression was also more apparent.3.Over one time transplanting epithelial progenitor cells from S8-ESC may relieve injured lung induced by bleomycin and mechanism of action is associated with cell factors such as TGF-β1 and IFN-γand so on.Part 2Objective The aims of this study were to be further to observe the therapeutic effect of over one times transplantation with S8-ESC on the pulmonary injury of mouse induced by bleomycin and their mechanisms,in larger scale.Methods Sixty C57BL/6J female mice were divided randomly into six groups,they were control group,bleomycin model group,treatment group-1,treatment group-2, treatment group-3 and treatment group-4.Those mice except mice in control group were administrated bleomycinintratracheally to induce model of lung injury,then epithelial progenitor cells from S8-ESC were transplanted into mice from one time to four times respectively in four treatment groups and the mice of the control group and the bleomycin model group were administrated 0.9%sodium chloride respectively.After twenty-eight days,the life-span,hydrocyproline concentration in lung tissue,transforming growth factorβ1(TGF-β1) and interferon-γ(IFN-γ) concentration in lung tissue bomogenate were examined and the pathologic changes of the lung and the engraftment of the epithelial progenitor cells in the injured lung were observed.Result After twenty-eight days,the results in six groups were as follows respectively.1.The death rates were 0%(in control group),60%(in bleomycin model group),50%(in treatment group-1),40%(in treatment group-2),40%(in treatment group-3) and 20%(in treatment group-4) respectively.The survival curves are separated between bleomycin model group and treatment groups and are separated among treatment groups except that those of close groups are overlapped partly.2.Hydrocyproline concentrations in lung tissue were 0.390±0.027μg/mg(in control group), 0.748±0.043μg/mg(in bleomycin model group),0.674±0.071μg/mg(in treatment group-1), 0.604±0.027μg/mg(in treatment group-2),0.554±0.050μg/mg(in treatment group-3) and 0.509±0.034μg/mg(in treatment group-4) respectively.Comparisons between each group have significant difference(P＜0.05).3.TGF-β1 and IFN-γ,concentrations in lung tissue bomogenate were 53.889±16.052 pg/mg and 15.770±3.112 ng/mg(in control group), 142.247±99.564pg/mg and 53.135±9.513 ng/mg(in bleomycin model group), 188.645±43.491pg/mg and 40.946±5.881ng/mg(in treatment group-1),289.102±55.243 pg/mg and 36.016±6.290 ng/mg(in treatment group-2),339.512±82.648 pg/mg and 34.871±4.929 ng/mg(in treatment group-3) and 517.198±138.608 pg/mg and 28.041±7.248 ng/mg(in treatment group-4) respectively.Comparisons of TGF-β1 and IFN-γ,concentrations in lung tissue bomogenate between each group have significant difference(P＜0.05) except those between treatment group-1 and treatment group-2. Comparisons of TGF-β1 and IFN-γconcentrations in lung tissue bomogenate between each group have no significant difference(P＞0.05 ) except for control group.4.With the increasing of the time of transplanting epithelial progenitor cells administrated to the mice, the extent of the injured lung in pathologic slice by HE stain in five groups became slighter and slighter,while the mice in control group was normal.Average rank's comparisons of the extent of the injured lung in pathologic slice by HE stain between each group have significant difference(P＜0.05) except that between bleomycin model group and treatment group-1.5.With the increasing of the time of transplanting epithelial progenitor cells administrated to the mice,field planting time in the lung became longer and longer.The field planting average time was 2 days(in treatment group-1),3 days(in treatment group-2),7 days(in treatment group-3),10 days(in treatment group-4).Comparisons between each group have significant difference(P＜0.05).ConcLusion Over one time transplantation with embryonic stem cell S8-ESC may relieve injured lung induced by bleomycin and mechanism of action is associated with cell factors such as TGF-β1 and IFN-γand is not associated with TIMP-1 and MMP-9. |
