| Objective:Based on the multiple functions of alveolar type Ⅱ epithelial(AT2)cells,including proliferation,differentiation into type Ⅰ epithelium,and immune regulation,we investigated the effect of autologous fibroblasts on induced AT2 cells(ciAT2)transformed by direct cell reprogramming on acute lung injury mice.Protective effects.By studying the biological behavior of ciAT2 and its protective effect on the lung tissue of acute lung injury after transplantation,it provides an experimental basis for the treatment of acute lung injury by cell transplantation.Part Ⅰ Acquisition and Characterization of ciAT2 CellsMethods:Mouse embryonic fibroblasts(MEFs)were treated with transdifferentiation chemical small molecule combination(SMC)for 14 days to obtain MEF transdifferentiated induced AT2 cells(ciAT2).Using MEF as a negative control and eAT2 as a positive control,the cellular characteristics of ciAT2 were identified.Experimental methods:The cytological characteristics of ciAT2 cells were identified by cell morphology,immunofluorescence staining of special markers(SPC)of AT2 cells,detection of SPC protein content in cells,and electron microscopy detection of special organelles(lamellar bodies)of AT2 cells.Results:The ciAT2 group had similar morphology and characteristics of eAT2:the cell shape was round-like,the immunofluorescence staining found that SPC(unique to AT2 cells)had strong positive expression,and electron microscopy revealed that the cells had a lamellar structure(unique to AT2 cells),with a diameter of about 0.6 μm;by Western blot experiment,it was found that SPC protein was highly expressed in ciAT2.These results suggest that ciAT2 cells have similar morphological characteristics to eAT2 cells.Part Ⅱ Preparation of mouse model of acute lung injury with LipopolysaccharideMethods:Forty male C57 mice were randomly divided into 4 groups,the normal control group and the observation group at different time after acute lung injury(24h,48h,72h).Experimental methods:The acute lung injury model was established by intratracheal instillation of endotoxin LPS(2 mg/kg)combined with vertical and uniform shaking(30S)after instillation,and the relevant indicators of acute lung injury were observed at 24h,48h,and 72h after modeling.Changes,including inflammatory-related indicators such as the level of inflammatory factors in bronchoalveolar lavage fluid,mRNA expression of inflammatory factors in lung tissue,etc.,and the damage to the alveolar and lung tissue structure of the model mice at three time points was detected by pathological sections.Results:Compared with the normal control group mice,the acute lung injury mice showed obvious mental fatigue and laziness 24h,48h and 72h after modeling.The levels of inflammatory factors in bronchoalveolar lavage fluid were significantly increased,and the levels of anti-inflammatory factors were significantly decreased;among them,the levels of inflammatory factors IL-6 and TNF-a reached a peak at 48h.The mRNA expression of inflammatory genes in lung tissue was significantly increased,and the mRNA expression of anti-inflammatory factors was significantly decreased,and the mRNA expression levels of inflammatory factors IL-1β and TNF-α reached a peak at 48h.In addition,the pathological section of the lung tissue showed that the alveolar and lung tissue structures were significantly damaged.The damage occurred around the trachea at 24h,spread to the trachea and most of the lungs at 48h,and diffused to the entire lung at 72h.Part Ⅲ Protective effect of ciAT2 cells on acute lung injury in miceMethods:60 healthy C57 mice were randomly divided into 5 groups.①Control group(same volume of NaCl,n=12),②LPS group(LPS 2mg/kg,n=12),③LPS+MEF transplantation group(LPS 2mg/kg,n=12),④LPS+ciAT2 transplantation group(LPS 2mg/kg,n=12),⑤LPS+eAT2 transplantation group(n=12).The mice in the cell transplantation experimental group were all transplanted 6-8h after tracheal instillation of LPS or the same volume of NaCl and at the same time point(30-32h)the next day,the dose was 0.3X106/mice,and the samples were collected after 72h of cell transplantation treatment,to detect changes in relevant indicators.Elisa was used to detect the changes of inflammatory factor-related indexes in bronchoalveolar lavage fluid;fluorescence quantitative PCR instrument was used to detect the mRNA expression of inflammatory factors in lung tissue;the ratio of lung body ratio and wet to dry weight(wet/dry lung weight)was calculated using To evaluate the abnormal permeability of alveolar wall blood vessels;HE staining was used to evaluate the pathological changes of lung tissue.Results:The results of inflammatory indicators in the bronchoalveolar lavage fluid showed that compared with the normal control mice,the contents of TNFα and IL-6 in the LPS group were significantly increased(P<0.001),and the contents of IL-10 and TGF-βwere significantly decreased(P<0.01,P<0.05).Compared with the LPS group,the contents of TNFα and IL-6 in the LPS+eAT2 group and the LPS+ciAT2 group were significantly decreased(P<0.001),while the contents of TNF-α and IL-6 in the LPS+MEF group decreased,but not statistically significant(P>0.05).The contents of IL-10 and TGF-β in the three groups were increased,but there was no statistical significance(P>0.05).The mRNA expression levels of inflammatory factors in the lung tissue showed that compared with the normal control group,the mRNA expression levels of TNF-αand IL-1β in the LPS group were significantly increased(P<0.001,P<0.01).The mRNA expression levels of-10 and TGF-β2 were significantly decreased(P<0.001),and the mRNA expression levels of TGF-β1 were decreased,but there was no statistical difference(P>0.05).Compared with mice in LPS group,the mRNA expression levels of TNF-α and IL-1β in LPS+eAT2 group and LPS+ciAT2 group were significantly decreased(P<0.001,P<0.01),the mRNA levels of TGF-β1 and TGF-β2 were significantly decreased(P<0.001,P<0.01).The expression levels were significantly increased(P<0.05,P<0.01,P<0.001),and the mRNA expression levels of TNF-α and IL-1β in the LPS+MEF group were decreased,but not statistically significant(P>0.05).The mRNA expression level of IL-10 was significantly decreased(P<0.001),the mRNA expression level of TGF-β2 was significantly increased(P<0.05),and the mRNA expression level of TGF-β1 was increased,but there was no statistical significance(P>0.05).The results of lung-to-body ratio and wet-dry weight showed that compared with the normal group,the lung-to-body ratio and wet-to-dry weight ratio of the LPS group were significantly increased(P<0.001).The ratio and wet-dry weight of LPS+ciAT2 group were significantly decreased(P<0.001),and the lung body and wet-dry weight of LPS+ciAT2 group were significantly decreased(P<0.001),while the lung-body ratio and wet-dry weight of LPS+MEF decreased,but there was no statistical Academic significance(P>0.05).The results of HE staining showed that the lung tissue damage in the ciAT2 group and the eAT2 cell transplantation group was significantly improved,some alveolar structures were restored,and the number of alveoli with normal structures increased.Conclusion:In this study,by comparing the cell morphology,immunofluorescence staining and protein content detection of SPC,a characteristic marker of AT2 cells,and the presence of unique organelles(lamellar bodies)found by electron microscopy,we determined the MEF transdifferentiation induced by the combination of small molecule drugs.ciAT2 cells had similar morphology and structure of AT2 cells and expressed AT2 cellspecific marker SPC.Based on the ability of AT2 cells to proliferate,differentiate into type I epithelium and regulate immune function,we established a mouse cell transplantation model for acute lung injury,and compared the protective efficacy of eAT2 cells and ciAT2 cells on acute lung injury.The results showed that both eAT2 cells and ciAT2 cells can improve pulmonary edema caused by acute lung injury,reduce inflammatory factors and increase the secretion of anti-inflammatory factors,improve acute inflammation to a certain extent,repair lung injury and protect lung function.Research provides experimental basis for cell transplantation therapy of acute lung injury. |
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