| Background: Recently the prevention and treatment of idiopathic pulmonary fibrosis have been always the focus for most countries in the field of medical research. Currently glucocorticoids and cytotoxic drugs are still the main measures of idiopathic pulmonary fibrosis clinically. However, they have not been shown to halt progression of fibrosis, improve lung function or life expectancy and may be associated with harmful side effects. With the understanding of the pathogenesis of the disease over the years, many new drugs continue to be developed. Unfortunately the translation of these drugs into clinical practice has not been accomplished yet. Therefore, it is urgent to find an alternative method of prevention or treatment. Mesenchymal stem cells(MSCs) can always migrate to impaired tissue and play an important role in the tissue repair. And it’s easy for MSCs to be infected by exogenous gene. Thus they have shown great prospect in the treatment of idiopathic pulmonary fibrosis. Since idiopathic pulmonary fibrosis is found to be associated with oxidative stress, N-acetylcysteine, SOD and other antioxidants have gradually become a new treatment of the disease. In addition to scavenge free radicals, thioredoxin(TRX) has a very important biological role as an antioxidant in the inhibition of apoptosis and regulation of the cell cycle and so on. Thus,we constructed human umbilical cord mesenchymal stem cells(hucMSCs) expressing human thioredoxin(Trx-1) gene mediated by lentivirus, and also established the bleomycin mouse model of pulmonary fibrosis. This study will laid a experimental foundation in treatment of idiopathic pulmonary fibrosis with the gene-modified MSC.Methods: The CDS region sequences of Trx-1 were got through the NCBI website. Gene-specific primers was constructed for the CDS, and then around 15 bp homologous recombination sequences were added to both ends of the primers. After the vector was digested and linearized, and gene fragments and the vector were recombined. Finally the expression of plasmids was determined by direct observation of the fluorescence and the Western Blot results. Viral titer in 293 T cells was calculated through the fluorescent observation. The lentivirus carrying Trx-1 gene were prepared and the titer of recombinant lentivirus was determined. Firstly, set a certain multiplicity of infection(MOI) gradient such as 0,5,10,30 and 50 was set according to some literature. Then umbilical cord MSCs were infected with lentivirus at different MOI values. The optimal MOI value was determined by observing the fluorescence protein expression. With empty vector transfected human umbilical cord MSC and non-transfected human umbilical cord MSC as controls, hucMSCs were infected with the recombinant lentiviruses at the best MOI. Western Blot was applicated to detect the expression of cell protein Trx-1, cell cycle and immune phenotype were analysed by flow cytometry. Cell growth curve was drawn according to CCK-8 assay. With bleomycin as inducer and C57 BL / 6J mice as experimental animals, animal models of pulmonary fibrosis were respectively established by intratracheal instillation of bleomycin at the dosage of 5mg / kg or nasal instillation bleomycin at the dosage of 6mg / kg. Finally the two methods of modeling were compared through the observation of general conditions, body weight and HE staining or Masson staining of lung tissue.Results: The pGC-LV-Ubi-TRX-1-CMV-EGFP lentivirus vector plasmid was successfully constructed, and it was found that the plasmid expressed the target protein and post-translational modifications happened by Western Blot analysis. The virus titer was detected to be 1 × 108 TU/ml taking advantage of 293 T cells. As the MOI was 10 or above, the infection efficiency of MSC was more than 80%.Due to some possible toxicity of lentivirus to cells, the best MOI value was determined as 10. Western blot showed that Trx-1 was positively expressed in all hucMSCs,while it was highly expressed in hucMSCs infected with recombinant lentiviruses. The flow cytometry found that in line with the phenotypic characteristics of the MSC, each group of MSC highly expressed CD105, CD73, did not express CD34, CD80. Cell cycle analysis showed that before or after lentivirus infection, more than 80% of the human umbilical cord MSCs were in G0 / G1 phase, suggesting that people MSCs infected by lentivirus also displayed the typical characteristics of stem cells. However, cell growth was inhibited after transfection as the growth curve showed. Modeling results showed that the models of pulmonary fibrosis were both successfully established by intratracheal instillation and nasal instillation of bleomycin. However, compared with intratracheal administration, pathological pathological changes of the lung tissue was lighter after modeling through nasal administration, which meant that it was easier to establish a model of pulmonary fibrosis through the second method.Conclusion: The recombinant lentiviral vectors carrying human Trx-1 gene were successfully constructed. Mediated by the recombinant lentiviral, the gene-modified human umbilical cord MSCs were obtained, and they could highly express the target protein Trx-1 with no evident changes about the cytological characteristic. It demonstrated in the animal experiment that the mouse model developed at 28 days after intratracheal instillation of the BLM was relatively more successful, which indicated that it was a better model of pulmonary fibrosis. In this study, gene therapy combined with cell therapy may lay some experimental foundation and provide an alternative choice for the treatment of pulmonary fibrosis. |
PDF Full Text Request