| Objective:Mesenchymal stem cell is adult stem cell that can be extracted from varied kinds of tissues.Because adipose tissue is abundant in subcutaneous tissue and easy to obtain,the study of adipose-derived mesenchymal stem cell has become a research hotspot in recent years.Currently,relevant teams have developed the anti-rejection treatment of patients after clinical transplantation by MSC due to its characteristics such as low immunogenicity,strong ability of immune regulation and exosome secretion et.al.And some studies have suggested that the mechanism may be related to the inhibitory effect of MSC on CD4~+T cell proliferation.In this study,we investigate the effect of ADSC combined with preconditioning on the proliferation of CD4+T cells by changing the oxygen volume fraction and glucose concentration during cell culture which can provides experimental basis for further improving the biological efficiency of ADSC.Materials:W In this study,we extract ADSC of C57BL/6 mice with collagenase,detect the expressions of surface antigens CD29?SCA-1?CD34 and CD45 in ADSC by flow cytometry,and then detect the adipogenic,chondrogenic and osteogenic differentiation by culture induction method.We purify the CD4+T cells of C57BL/6 mice by magnetic activated cell sorting(MACS)and labeled with CFSE at the same time the spleen cells of C57BL/6 mice were extracted,then CD4+T cells were used to construct a mixed lymphocyte reaction with spleen cells.ADSC were cultured in medium with glucose concentration of 17.5mmol/L at oxygen volume fraction of 21%to the third generation for subsequent experiments.We divide the ADSC into 6 groups,including 3 groups in hypoxic environment(5%oxygen volume fration),respectively,which the glucose concentration of medium are 5.56mmol/L,17.5mmol/L and 30mmolL,and the other 3groups in normoxic environment also use glucose concentration 5.56mmol/L,17.5mmol/L and 30mmol/L medium.After the six groups of ADSC pretreatment in their respective culture environment for 5 days,inoculate them in 96-well plates,and the original culture environments of each group were used to continue the culture for adherence.The previously constructed mixed lymphocyte responses were divided into three groups,among which the ADSC group was composed of six experimental groups with ADSC under six different pretreatment conditions and CD4~+T cells in a 1:4 ratio;the MLR group was a mixed lymphocyte response without ADSC;the control group was CD4~+T cells cultured alone.Each group was co-cultured for 5 days,and then detect the changes of CFSE peak value by flow cytometry to determine the changes of CD4~+T cell spPtrraootlliiisffteiercraaattlii ooannn ailiny neshiaiscb,i ht pigoarinro uwrpias.tee sc oarmep aerixsporne ssoef dm uwlittihpl ex s±asm,p lae ndm euasnes cSaPnS Sus es oLftSwDa-rte t efsotr,which P<0.05 for the difference is statistically significant.Results:1.The ADSC used were identified as ADSC2.The inhibition rate of CD4~+T cell proliferation in hypoxic group was higher than that in normal oxygen group(P<0.05).3.In the low-oxygen environment,the inhibition rate of CD4+T cell proliferation in the group with glucose concentration of 30mmol/L was higher than that in the group with glucose concentration of 17.5mmol/L and 5.56mmol/L(P<0.05).There was no significant difference in the proliferation inhibition rate of CD4+T cells between the groups with glucose concentration of 17.5mmol/L and 5.56mmol/L(P>0.05).4.There was no statistically significant difference in the proliferation inhibition rate of CD4~+T cells between the three groups with different glucose concentrations in a normoxic environment(P>0.05).Conclusion:1.Hypoxic pretreatment of ADSC can enhance its ability to inhibit the proliferation of CD4~+T cells.2.In the normoxic environment,the effect of short-term changes in medium glucose concentration on the immunomodulation ability of ADSC was not obvious.3.30mmol/L glucose medium can enhance the effect of hypoxic pretreatment on the immunomodulatory ability of ADSC. |
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