The Construction And Primary Study For The Biological Character Of The Mouse Mesenchymal Stem Cell Line C3-H10T1/2with B7-H4Knocked Down By ShRNA Interference

Objective To construct B7-H4targeted shRNA stable transfected mousemesenchymal stem cell line C3H10T1/2(C3H10), and study the role and mechanism of theimmune inhibition of C3H10cells mediated by B7-H4.Methods The expression of B7-H4on C3H10was detected by flow cytometry,Western blot; the lentiviral vectors with mouse B7-H4target shRNA were transfected intothe C3-H10cells (C3H10-shRNA-B7-H4). The shRNA transfection efficiencies weremeasured by detecting the green fluorescence on C3-H10cells using the fluorescencemicroscope and flow cytometry (FCM). The transfected cells were sorted by puromycin.The knocked down effect of the different sequence of specific shRNAs were measured bydetecting the protein by flow cytometry and Western blot, or mRNA by RT-PCR. TheC3H10cells with highest B7-H4knocked down efficiency was defined asC3-H10-shRNA-B7-H4cell line, which could be passaged for many times. These cellswere co-cultured with the PHA activated mice spleen lymphocytes, and the activation,proliferation, cell cycle and apoptosis of the spleen lymphocytes were detected by flowcytometer analysis.Experimental allergic encephalitis (EAE) mice models were established, and the in vivoexperiment group were set as: sham operated group, EAE model group, C3H10cellsimplanted group, C3H10-shRNA-NC cells implanted group, and C3H10-shRNA-B7-H4cells implanted group. In the C3H10cells implanted group, C3H10-shRNA-NC cellsimplanted group, and C3H10-shRNA-B7-H4cells implanted group,8days after theimmunization, C3H10cells, C3H10shRNA-NC cells and C3H10-shRNA-B7-H4cellswere injection to EAE mice respectively, and the neurological impairment function scoreand weight were measured everyday. Result B7-H4were constitutively expressed on C3H10; the fluorescencemicroscope and FCM analysis showed that the transfection efficiency were above80%.Even more the transfection efficiency of puromycine sorted shRNA transfected C3H10were more than95%, and these cells could be passged stabily for many times. Among thefour pairs of B7-H4specific shRNA, shRNA-1was defined as the most effective one,which knock down B7-H4, both the expression of B7-H4mRNA and protein on C3H10were decreased to the lowest level.C3-H10can obviously inhibit the activation and proliferation of PHA stimulated micespleen lymphocyte. and most lymphocytes were arrested at G0/G1Phase, and these effectswere almost revised in the C3-H10cell tranfected by B7-H4target shRNA-1. C3-H10cellcan protect and trained mice spleen lymphocytes from AICD, while this effect was almostdisappeared when B7-H4was knocked down.In vivo experiments showed that the onset time of the attack was earlier and theneural function deficient score was higher in C3H10-shRNA-B7-H4cells implanted groupcomparing with C3H10cells implanted group and C3H10-shRNA-NC cells implantedgroup, but the onset time was later and the neural function deficient score was lower thanthat of the EAE group. The same trend could be seen in the weight detection.Conclusions1, The mouse mesenchymal stem cell line C3H10T1/2with stable transfected B7-H4targeting shRNA was constructed successfully;2, In vitro experiments showed B7-H4molecules involved in the immune inhibitoryeffect of C3H10cells on mice spleen cells, C3H10could inhibit the proliferation andactivation of PHA stimulated spleen cells, arrest the cells in the in G0/G1phase，couldprotect the mice lymphocytes from AICD;3,In vivo experiments showed that the co-inhibitor molecule B7-H4expressed onC3H10cell played a important role in the treatment of EAE by mesenchymal stem celltransplantation.