[Establishment of mouse stably knockout of MYSM1 in MSC cell line C3H10T1/2 and its effect on the immune regulation in vitro].

OBJECTIVE: To aimed at the establishment of mouse stably knockout of MYSM1 mesenchymal stem cell(MSC) line C3H10T1/2, and to investigate its immunological capacity of MSC in vitro.

METHODS: To establish the stably transfected MSC cell line by using CRISPR-Cas9 technology. Then the Flow cytometry, quantitative PCR and Western blot were employed to detect whether the MYSM1 have been knockout yet. Furthermore, the immune modulatory effect of MYSM1-/- MSC was tested by addition of MYSM1-/- MSC supernatant into spleen lymphocyte and Foxp3 culture. The mRNA expression of inflammatory cytokines such as interleukin-4, interferon-γ and interleukin-17 were detected by quatitatine PCR.

RESULTS: The expression of MYSM1 was steadily knock out in MSC. In addition, MYSM1-/- MSC showed a stronger inhibitory effect on the expression of inflammatory cytokines. Therefore, the MYSM1 has been stably knocked out in C3H10T1/2.

CONCLUSION: The mouse stably knockout of MYSM1 mesenchymal stem cells has been successfully established, the knock-out of MYSM1 in MSC can induce more potent immunosuppressive effects on cellular immune reaction in vitro. Our data laid a foundation for the further MSC-based applications in immune related diseases.