Research On The Effects Of MiR-146a Causing Apoptosis Of Thymus Stromal Cells In The Thymus Aging Atrophy

Objective:The thymus is the primary lymphoid organ for generating self-restricted and self-tolerant functional T cells,where lymphatic hematopoietic progenitor cells from bone marrow differentiate and develop into mature T lymphocytes.In order to maintain a microenvironment that supports the normal differentiation of thymus cells,complex interactions between thymus cells and thymus microenvironment are required.The previous results showed that the failure of thymus matrix microenvironment played an important role in thymus aging atrophy and the aging atrophy of thymus was largely caused by the first change of thymus stromal cells.A large number of studies have shown that miRNA plays an important role in the development of thymus and T lymphocytes.For example,the abnormal expression of miRNA can destroy thymus microenvironment and cause T lymphocyte developmental disorder.There is a correlation between the decreasing trend of age-related thymus epithelial cells and the expression of miRNA,among which the tendency of miR-146a to decrease with thymus aging is the most obvious.It can be inferred that there is a link between the decrease of this expression and the failure of thymus microenvironment and thymus atrophy.In this experiment,we observed the effects of miR-146a on the expression of multiple genes and the apoptosis rate of thymus stromal cells of mice and tried to analyze the effect of miR-146a on thymus aging atrophy and its mechanism.Methods:1.Culture and identification of thymus stromal cells:The thymus stromal cells were isolated and purified after the thymus was taken.After a week of culturing the cells within DMEM/F12 culture fluid,the cells were collected and identified by flow cytometry.2.Mimics overexpression of miR-146a content in thymus stromal cells:Under LipoMax-mediated miR-146a mimics and negative control reagents were used to transfect the thymus stromal cells in the experimental group and control group.After 48hours,miRNA was extracted to detect miR-146a expression by RT-PCR.3.Detection of TRAF-6 protein,Smad4 protein and Bcl-2 protein concentration:After using LipoMax to transfect miR-146a mimics 72 hours,the protein in the thymus stromal cells of the experimental group and the control group was extracted respectively.Western blot was performed to detect TRAF-6 protein,Smad4 protein and Bcl-2 protein in cells.4.Apoptosis detection of thymus stromal cells:After transfection of miR-146a mimics72 hours,the cell culture solution of the experimental group and control group was extracted respectively.The IL-6 content was measured by using an Mouse IL-6Valukine^TM ELISA kit.The apoptosis rate of thymus stromal cells was detected by using flow cytometry in the experimental group and control group respectively.Results:1.Culture and identification of thymus stromal cells:The flow cytometry showed that more than 90%?90.78±0.428?of the cells were CD45-,indicating that most of the cells were thymus stromal cells and less than 10%?9.22±0.428?were CD45+lymphocytes.2.Mimics overexpression of miR-146a content in thymus stromal cells:After using LipoMax to transfect miR-146a mimics 48 hours,the expression of miR-146a in transfection group was significantly higher than that in the control group.The relative content of miR-146a in mimics group was 192.948±54.636,the control group was 1.The difference is statistically significant?P<0.05?.3.The content of TRAF-6 protein and Smad4 protein decreased with the increase of miR-146a:After using LipoMax to transfect miR-146a mimics 72 hours,the expression of TRAF-6 protein and Smad4 protein in transfection group was significantly lower than that in control group.The TRAF-6 protein of mimics group was 0.496±0.254 and the control group was1.The difference is statistically significant?P<0.05?.The Smad4 protein of mimics group was 0.377±0.103 and the control group was 1.The difference is statistically significant?P<0.01?.4.The content of IL-6 decreased with the increase of miR-146a:.After using LipoMax to transfect miR-146a mimics 72 hours,the IL-6 in cell culture medium of transfection group decreased obviously.The mimics group was 1536.843±528.624pg/ml and the control group was 7150.165±724.098pg/ml.The difference is statistically significant?P<0.01?.5.The apoptosis rate of thymus stromal cells decreased compared with that of control group:After using LipoMax to transfect miR-146a mimics 72hours,the expression of Bcl-2 protein in transfection group was significantly higher than that in control group.The mimics group was 2.430±0.292 and the control group was1.The difference is statistically significant?P<0.01?.And the apoptosis rate of transfection group cells decreased compared with the control group.The mimics group was?13.9±1.842?%and the control group was?22.833±3.435?%.The difference is statistically significant?P<0.05?.Conclusion:1.The expression of TRAF-6 protein and Smad4 protein in thymus stromal cells can be inhibited by miR-146a.2.miR-146a can inhibit the secretion of IL-6 in thymus stromal cells.3.miR-146a can increase the expression of Bcl-2 protein and inhibit apoptosis of thymus stromal cells to a certain extent.In addition,it can be considered that in the aging atrophy process,miR-146a can affect the expression of Bcl-2 protein and the apoptosis of thymus stromal cells by regulating its target gene TRAF-6 protein and Smad4 protein.