Pb-MSCs Transplantation Modulates Macrophage Polarization And Inflammation In Rat SCI Model

Objective:(1)To investigate the regulatory effect of peripheral blood mesenchymal stem cells(PB-MSCs)transplantation on local macrophages phenotype in SCI;(2)To determine the effect of PB-MSCs transplantation on local micro-environment in SCI rats.Methods:PB-MSCs were isolated from peripheral blood of rats mobilized by G-CSF by density gradient centrifugation,and the sub-cultured PB-MSCs were identified using morphological observation and flow cytometry.The rats were randomly divided into Sham group,SCI group,PBS group and PB-MSCs group.The rats SCI models belonging to SCI,PBS and PB-MSCs group were prepared through weight falling method.After surgery for1 week,PBS and PBMSCs suspension(containing 5?10~5 cells)were absorbed by gelatin sponge and then locally transplanted to the injured site so as to establish the PBS and PBMSCs groups respectively.At 3 days,1 week,2 weeks,4 weeks and 8 weeks after treatment,rats were chosen randomly for the following tests:(1)Observation of the general morphology of the spinal cord;(2)Angle of rivlin experiment and BBB score to assess the recovery of locomotor function of the rats;(3)HE staining for histopathological examination of the injured spinal cord;(4)Flow cytometry to detect the phenotype and proportion of macrophages in the injured spinal cord;(5)RT-q PCR to analyze the expression of IL-6,TNF-α,IL-10,TGF-β1 in spinal cord;(6)ELISA to examine the levels of IL-6,TNF-α,IL-10,TGF-β1 in serum.Results:(1)PB-MSCs observed under the inverted microscope were homogeneously characterized by fusiform and adherent growth,and flow cytometry revealed that these cultured cells highly expressed CD29,CD44,CD90,but were negative for CD11b,CD45,CD79;(2)The results of general appearance showed that the lesion site tissues after PB-MSCs transplantation were more tough and displayed a significant less degree of spinal cord hematoma compared to those with PBS treatment or nothing,and obvious tissue atrophy could be found at 4 and 8 weeks in the PBS and SCI groups.(3)The results of BBB score and angle of rivlin experiment both exhibited that the motor function rats in PBS,SCI and PB-MSCs groups gradually recovered,nevertheless,since the 1st week after transplantation,the rats administrated with PBMSCs had a higher BBB score as well as a larger angle of rivlin experiment at the same time point compared to rats treated with PBS or SCI.(4)HE staining showed numerous inflammatory cells and red blood cells could be found in the lesion site of SCI and PBS group at 3 days and 1 week,while the degree of inflammatory response and the formation of cavities both were significantly reduced after PB-MSCs transplantation;And the degree of inflammatory infiltration of PB-MSCs was significantly reduced at 2 weeks,4 weeks and 8 weeks;(5)Flow cytometry was used to examine changes in the M1(CD11b+CD86+)/M2(CD11b+CD206+)ratio at the injury site and the results showed that the proportion of M1 was significantly down-regulated and the proportion of M2 was significantly up-regulated after PB-MSCs transplantation at each time point;(6)RT-q PCR assay showed the expression of IL-6 and TNF-αin PB-MSCs group was significantly lower than that in SCI and PBS group at the same time point,while the expression of IL-10 and TGF-β1 was significantly increased;(7)ELISA exhibited the amount of IL-6 and TNF-αin the serum in PB-MSCs group were significantly lower than those in SCI group at 2 weeks,4 weeks and 8 weeks,and significantly lower than those in PBS group from one week onwards;the levels of IL-10 and TGF-β1 in the serum of each group also showed a gradual upward trend,and after 1 week and 3 days respectively,the amount of IL-10 and TGF-β1 in PB-MSCs group were significantly higher than those in SCI and PBS groups.Conclusion:(1)PB-MSCs transplantation after SCI can effectively skew the balance of M1/M2 macrophages polarization fulcrum towards M2 phenotype in vivo,thereby significantly increasing the proportion of M2 early on at the lesion site,which is beneficial to inhibiting the development of secondary phase post injury.(2)PB-MSCs transplantation can down-regulate the secretion and expression of pro-inflammatory cytokines,IL-6 and TNF-α,and increase the levels of anti-inflammatory cytokines,IL-10 and TGF-β1,which together not only provides a permissive micro-environment for tissue repair,but also further induce the macrophages phenotypic shift to anti-inflammatory M2 phenotype.