| Objective: Investigate the role of Wnt/beta-catenin signaling pathway in differentiation of primary bone marrow mesenchymal stem cells(BMSCs)into type II alveolar epithelial cells(AEC2s)by establishing pulmonary fibrosis(PF)model in rats and extracting different drug serum.METHODS: Animal experiment: SD rats were divided into normal group,model group,high concentration group,medium concentration group and low concentration group,every group had 15 rats.In addition to the normal group,SD rats were exposed to trachea after anesthesia and injected with Bleomycin(BLM)(5mg/Kg)to establish pulmonary fibrosis model.Two normal and model rats were randomly selected for HE staining on 1,7,14 and 28 days.The rats in the model group were fed with normal saline(1ml/100 g volume)and the other three groups were respectively fed with high,medium and low concentrations of Maimendong Decoction(1ml/100 g volume).The serum containing drugs was taken through abdominal aorta after 2 weeks of continuous gastric administration.Cell experiment part: The tibia and femur of 2-week-old suckling mice were taken out and immersed in PBS,which without calcium and magnesium ions.The epiphysis was cut,washed and whitened with complete medium containing 2% bis-antibody.The cells washed out were cultured.The cells were divided into normal group(abbreviated as normal alpha group),SAE group,SAE + model serum group(abbreviated as SMX group),SAE + high concentration serum group(abbreviated as SGM group),SAE + medium concentration serum group(abbreviated as SZM group)and SAE + low concentration serum group(abbreviated as SDM group).The cells with stable growth state were plated,and 24 cells were added to serum-free SAE medium for culture.After 24 hours,the corresponding serum was added until the 5th day and the 10 th day,and then performed to Western blot to detect SP-C,β-catenin,Wnt3 a and WISP1 protein expression level.Result: 1.According to HE staining results,compared with the normal group,there were some changes of alveolar wall thickening and a few neutrophils and eosinophils proliferation on the first day after modeling;inflammatory cells increased significantly on the seventh day after modeling,and the area of thickened alveolar wall increased significantly;the lung tissue was significantly changed in the 14 th day after modeling,and the thickening of the alveolar wall in most areas was more significant and a large number of inflammatory cells and slightly pulmonary fibrosis were observed;On the 28 th day,the alveoli were significantly enlarged,and the range of substantial changes was larger than that on the 14 th day,basically all of them were thickened alveolar walls.The inflammatory cells were slightly lighter than those on the 14 th day,but the fibrosis was more obvious.2.Growth morphology of BMSCs: The primary bone marrow mesenchymal stem cells of the 2nd and 4th generations were obviously spindle-shaped,and arranged in scaly or radial or spiral shape.Some of them were round or polygonal,with abundant cytoplasm,large and clear nucleolus and obvious nucleolus.Most of the cells had more than 2-3 nucleolus.However,about 90% of the 3rd and 4th generations of cells were spindle-shaped cells,and the volume of cells became larger.A few round or polygonal cells could be seen and arranged more closely.3.MTT results showed that the cell survival rate on the 5th day was significantly different from that in the normal group(10% FBS MEM alpha complete medium culture).When the concentration of drug-containing serum was 5%,there were significant differences except for normal group and SMX group(P<0.05);when the concentration of drug-containing serum was 10%,there were significant differences except SDM(P<0.05);when the concentration of drug-containing serum was 15%,there were significant differences except for normal group(P<0.05).The difference between SAE group and normal group was significant(P<0.05).Compared with SAE group,the concentration of each group had significant difference(P<0.05).On the 10 th day of intervention,when the concentration of serum containing drugs was 5%,the survival rate of each group had significant difference(P<0.05);when the concentration of serum containing drugs was 10%,the difference between normal group A and SZM group was significant(P<0.05),but there was no significant difference between SMX,SGM and SDM groups;when the concentration of serum containing drugs was 15%,the difference was significant except SGM and SDM groups(P<0.05).The difference between the two groups was significant(P<0.05).Compared with SAE group,there were also significant differences(P<0.05).4.Under the microscope,the SAE group was mostly short spindle-shaped;and the normal group,SAE model group,SAE high-barley group,SAE mid-wheat group and SAE lowbarley group were mostly long spindle-shaped.In SAE group,the spiral arrangement structure was sparse and the cells were scattered,while in the other groups,the fish scale or spiral arrangement was obvious and the structure was compact.5.Western blot analysis showed that: the expression of SP-C protein: The SP-C protein content in the SMX group and the SGM group was higher than that on the 10 th day(P<0.05),but the difference between the SMX group and the SZM group was not obvious.Wnt3 a protein expression: When the cell intervention on the 5th day,the expression levels of SMX group,SGM group and SDM group were significantly higher than those of SAE group(P<0.05);the expression of SZM group was lower than that of the normal α group(P<0.05)and there was no statistically significant difference between the other groups and the normal α group,when cell intervention was on the 10 th day.The expression level on the 10 th day in the SAE group and the normal α group was significantly higher than that on the 15 th day(P<0.05).The expression of β-catenin: When the cell intervention was on the 5th day,there was no significant difference between the normal α group and the SAE group,and then the protein content of the other 4 groups was significantly higher than those(P<0.05);When the intervention reached the 10 th day,only the content of the SZM group was higher than that of the normal α group(P<0.05).The expression levels in the groups of each group were comparable on the 5th and 10th(P>0.05).The expression of WISP1: there was a significant difference between the SGM group and the SAE group at the 5th day of intervention(P<0.05).There was no significant difference between the other groups and the SAE group or the normal α group.On the 10 th day,the protein content of the SAE group was significantly higher than that of the normal α group,and the expression levels of the normal α group,SGM group and SZM group were lower than those of the SAE group(P<0.05).The protein content of the SDM group was higher than that of the normal α group.(P<0.05).The expression level of the SAE group and the SDM group on the 5th day was higher than that on the 10 th day(P<0.05).Conclusion(s): 1.The whole bone marrow mesenchymal stem cells(BMSCs)were extracted from the femur of the suckling rats by whole bone marrow adherence method,and the 3th generation cells had the best growth state.2.Through statistical analysis of MTT results,the concentration of drug-containing serum was 10%,which is conducive to cell growth.3.The results of this experiment indicated that serum-free small airway epithelial cell culture medium combined with model serum or Maimendong decoctioncontaining serum medium could induce BMSCs to differentiate into type II alveolar epithelial cells;Maimendong Decoction could activate classical Wnt/β-catenin signaling pathway to regulate the mechanism by which BMSCs differentiate into alveolar epithelial cells. |
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