The Preliminary Study For The Role And Mechanism Of B7-H4for The Treatment Of EAE By Mesenchymal Stem Cell C3H10T1/2Transplantation

Objective To investigate the role of B7-H4molecules for the proliferation, existence,migration and the other biologic characteristic of mouse mesenchymal stem cell C3H10T1/2(C3H10) by in vitro experiments, and to explore the effects of B7-H4in the treatmentof EAE by C3H10cell transplantation.Methods1、Construct B7-H4targeted shRNA stable transfected mouse mesenchymalstem cell C3H10(C3H10-shRNA). The effect on the proliferation ability, and apoptosis ofthe C3H10cell by B7-H4knocked down were detected by immunofluorescence and flowcytometry (FCM). To study the ability of migration of C3H10indirectly, the expression ofchemokine receptor CXCR4was detected. C3H10cell, C3H10-shRNA cell wereco-cultured with the PHA activated mice spleen lymphocytes, and the proliferation wasdetected by flow cytometer analysis.2、 Experimental allergic encephalitis (EAE) mice models was constructed byMOG35-55with CFA subcutaneous injection on a single point, pertuss toxin intravenousinjection of vaccine, and the in vivo experiment group were set as:(1) sham operatedgroup,(2) EAE model group,(3) C3H10cells implanted group,(4) C3H10-NC cellsimplanted group,(5) C3H10-shRNA cells implanted group. In the C3H10cells implantedgroup, C3H10-NC cells implanted group, and C3H10-shRNA cells implanted group,6days after the immunization, C3H10cells, C3H10-NC cells and C3H10-shRNA cells wereinjected about1×106to each EAE mice respectively, and the neurological impairmentfunction score and weight were measured everyday and inflammatory cell infiltration,demyelination, axonal damage were detected by H&E, luxol fast blue and Bielschowskystaining respectively. The expression of B7-H4on spinal cord tissue was also detected byimmunohistology staining. Result1、By knocking down B7-H4on C3H10, the inhibitory effect of C3H10onspleen lymphocyte proliferation was partly revised, as well as that of apoptosis; Theexpression of chemokine receptor CXCR4was also decreased.2.、In vivo experiments showed that （1）the onset time of the attack was earlier andthe neural function deficient score was higher in C3H10-shRNA cells implanted groupcomparing with those of C3H10cells implanted group and C3H10-NC cells implantedgroup, but the onset time was later and the neural function deficient score was lower thanthat of the EAE group. The incidence rate is90%, and the mortality rate is2.5%.(2)Thesame trend could be seen in the weight detection.(3)Spinal cord pathology exam showedthat there were no obvious abnormalities in sham operated group; inflammatory cellinfiltration, demyelination and axonal damage were appeared in EAE model group, C3H10cells implanted group, C3H10-NC cells implanted group and C3H10-shRNA cellsimplanted group. The degree of inflammatory cell infiltration, demyelination and axonaldamage were more severe in C3H10-shRNA cells implanted group comparing with that ofC3H10cells implanted group and C3H10-NC cells implanted group.（4）Immunohistochemical staining showed that the number of B7-H4positive cells in C3H10cells implanted group and C3H10-NC cells implanted group were significantly higher thanthat of EAE model group and C3H10-shRNAcells implanted group.Conclusions The treatment of EAE by C3H10transplantation is safe and effective.The B7-H4molecule involved in the effect of the treatment of EAE by C3H10transplantation, which might mediate by affecting the immune regulation, cell survival,proliferation, migration and the other biologic characteristic of C3H10cell. It is provedthat the co-inhibitor molecule B7-H4expressed on C3H10cell played an important role inthe treatment of EAE.