TRIM28 Regulates Immortalized Bone Marrow Mesenchymal Stem Cells To Reverse Liver Fibrosis In Mice

Liver fibrosis exists in most chronic liver diseases.Long-term liver fibrosis can lead to cirrhosis,and even will be liver cancer.Mouse Bone Marrow Mesenchymal Stem Cells（BMSCs）are adult stem cells with multi-directional differentiation potential,which can exert immune regulation,secrete nutritional factors and other functions,and can be used to reverse liver fibrosis and promote the repair and regeneration of damaged tissues.The cost of BMSCs isolation and culture is high,the number of passages is limited,and with the increase of passages,the cells willgradually be old,the ability of cell viability and differentiation decreases significantly,and it is not easy to make the genetic modification.These characteristics limit the basic research and clinical application based on BMSCs.Therefore,it is significant to construct an immortalized mouse Bone Marrow Mesenchymal Stem cell line（im BMSC）.SV40 large T antigen（SV40LT）is a protein encoded by the SV40（simian virus 40）virus genome,and continuous expression of SV40LT leads to immortalized proliferation of cells.Small hairpin RNA（sh RNA）can generate the corresponding si RNA to silence the expression of the target gene by RNA interference（RNAi）.In this study,CRISPR Cas9 gene editing technology was used to knock in the SV40LT gene at the Rosa26 site of mouse BMSC to construct an im BMSC.The lentiviral system was used to construct im BMSC cell line with TRIM28 exhausted（im BMSC-sh TRIM28）.The carbon tetrachloride（CCl₄）-induced liver fibrosis model was used to study the effect of TRIM28 on the reversal of liver fibrosis by im BMSCs.The results were as follows:（1）Primary mouse BMSCs were isolated by the bone.The isolated BMSCs were in a monolayer adherent state.The P0～P5generations were long fusiform and the boundary was clear.As the algebra increased,it gradually became polygonal and the boundary gradually blurred.Flow cytometry showed that both positive and negative surface markers of BMSC stem cells accounted for more than 99%of the total cells.（2）Construction of im BMSC cell lineThe target plasmid lenti CRISPR v2-Rosa26 and the donor plasmid p UC19-Donor were co-transfected into the first generation of mouse BMSCs,and the G418-resistant cells had been obtained by continuous screening with 150μg/m L G418 for 7 days.The im BMSCs were enriched by immunomagnetic beads,and monoclonal cells were selected by a limited dilution method.The results showed that SV40 LT was inserted into the Rosa26 site by PCR.Flow cytometry showed that the surface markers and negative surface markers of im BMSCs accounted for more than 99%of the total cells.The im BMSC cell line showed monolayer adherent growth and long spindle shape,and the proliferation rate was faster than that of wild-type BMSC.The non-tumorigenicity of im BMSCs was detected by subcutaneous tumor-bearing mice.Im BMSC cell line can be passaged for more than 40 generations and maintain the original cell morphology and stem cell function,which can be used for cell therapy.（3）Knockdown of TRIM28 inhibited im BMSCs apoptosis.The TRIM28 of im BMSCs was knocked down by the lentivirus system,and the im BMSC-sh TRIM28 cells were obtained by continuous screening with 1μg/m L puromycin for 3 days.The effect of TRIM28 on the phenotype of im BMSC was detected by cell proliferation and apoptosis experiments.The results showed that the expression of TRIM28 in im BMSC-sh TRIM28 was down-regulated compared with im BMSC-sh NC.CCK-8 test showed that there was no significant difference in the effect of TRIM28 knockdown on the proliferation of im BMSCs.Annexin V-FITC/PI staining showed that knockdown of TRIM28inhibited im BMSCs apoptosis.（4）Knockdown of TRIM28 promotes im BMSC to reverse liver fibrosis.The mouse liver fibrosis model was constructed by carbon tetrachloride induction（CCl₄）.The ability of im BMSC to reverse liver fibrosis and the effect of TRIM28 on im BMSCs reverse liver fibrosis were detected by tail vein infusion of 1×10^6 cells.The results showed that the im BMSCs could reverse liver fibrosis,and the knockdown of TRIM28 could promote the im BMSCs to reverse liver fibrosis.In conclusion,TRIM28 knockdown could significantly inhibit the apoptosis of im BMSCs and promote the reversal of liver fibrosis induced by CCl₄ in mice.The im BMSC cell line constructed in this study can be used as seed stem cells for cell therapy and tissue engineering,which provides convenience for basic and clinical research.