Construction And Experiment In Vitro Of Mouse HSCs With Chimeric MHC-I Gene

Objective:To construct molecular chimeric hematopoietic stem cell (HSC) and explore its effect on stimulating the proliferation of T cell in vitro and to lay the foundation for inducing transplantation immune tolerance.Methods:In this study,H-2Db gene was amplified by RT-PCR, and it was subcloned into plasmid pMSCV-neo by double enzyme digestion. After the recombinant plasmid carrying H-2Db gene was confirmed by DNA sequencing, it was co-transfected with two packaging plasmids into 293T cells and obtain the recombinant retrovirus.②The HSCs of BALB/c mouse were separated and collected by density gradient separation. HSCs were transfected by recombinant retrovirus. a non-transfected and an empty retrovirus transfected HSCs were control groups.Real-time fluorescence quantitative PCR and flow cytometry were used to observe the expression of H-2Db gene respectively. The reaction cells derived from BALB/c mouse spleen cells and the stimulation cells derived from C57BL/6 mouse spleen cells were collected on the 7th day respectively after HSCs were transfused into Balb/c mouse. One-way mixed lymphocyte culture were operated and the proliferation of T cells was detected by MTT assay.Results:①H-2Db gene was successfully gained by RT-PCR from C57BL/6 mouse liver.And the construction of plasmid pMSCVneo-H-2Db were confirmed by restriction enzyme digestion and sequence analysis.②The recombinate retrovirus carrying H-2Db gene was successfully harvested.③BALB/c mouse HSCs were successfully separated and cultured in vitro.The rate of CD34+ cells was up to(35.60±2.19)%.④Compared with the non-transfected group and the empty plasmid transfected group, the expression of H-2Db gene at mRNA level and protein level showed an significant increase in the recombinant retrovirus transfected group (P<0.01), but there was no obvious difference in the non-transfected group and the empty retrovirus transfected group (P>0.05).⑤The stimulation index in the recombinant retrovirus transfected group was lower than in the non-transfected group and the empty retrovirus transfected group(P<0.01). However, There was no statistical difference between the non-transfected group and the empty retrovirous transfected group (P>0.05).Conclusions:①ouse HSCs can be successfully separated from the bone marrow cells by density gradient separation.②BALB/c mouse HSCs can stably express H-2Db antigen after transfection with recombinant retrovirus.③Molecular chimeric HSCs can obviously inhibit the proliferation of T cells in vitro. It may lay the basis for further study of transplantation immune tolerance induced by molecular chimerism.