Proteomic Analysis Of Human Umbilical Cord Mesenchymal Stem Cell Transplantation In Experimental Stroke

Background:Stroke is one of the leading cause of death and disability in china as well as western countries. Unfortunately, even though much attention was paid on it, there is still no useful treatment to cure stroke patients. Cell-based therapy was studied in great number recently, and showed some promising results. Treating ischemic stroke with Mesenchymal Stem Cell (MSCs) have acquired some positive results both in preclinical and clinical research. After transplantation of MSCs in animal model, the neurological behavior and infarct volume was improved significantly. However, the mechanism under it is obscure. Human umbilical cord mesenchymal stem cells (hUC-MSCs) is a kind of MSCs derived from human umbilical cord. hUC-MSCs has close relationship to embryonic stem cells(ESCs) in ontogenetic and proliferate faster than adult MSCs. In our previous work, hUC-MSCs was intravenous injected to middle cerebral artery occlusion (MCAO) animal model within 30min after reperfusion. In the result, hUC-MSC could significantly improve neurological function and reduce infarct volume. Besides of that, we found hUC-MSCs could reduce the expression of pro-inflammatory cytokines includingIL-1, TNF-a, IL-23, IL-17 and increase the expression of anti-inflammatory factor IL-10. And these effects could partly be inhibited by TGF-(3 neutralizing antibody. We hypothesized that hUC-MSC maybe exert protective function through TGF-β, which shifts the Thl7/Treg differentiation from Naive CD4+ T cells and regulates peripheral immune response. However, the further mechanism under it is still unclear.Purpose:we used iTRAQ (isobaric tags for relative and absolute quantitation), which is one of the major quantification tools used in differential proteomic research, to analyze the difference of protein expression between stroke and control, and difference between whether or not intravenous injection of hUC-MSCs.Methods:The male mice of C57BL/6 were randomly designated into 4 groups:The mcao-24h vs sham group, the mcao-48h vs sham group, the mcao-48h vs mcao-24h group and the mcao-48h+MSCs vs mcao-48h group. MCAO ischemia-reperfusion (90min) was established. NS or hUC-MSCs were administrated via caudal vein 30min after reperfusion. The mice were sacrificed at indicated time point (all in ischemia reperfusion after 24 h,48 h) and separated their cortical peri-infarct areas for sample to be demonstrated:1. The protein from sample be used the iTRAQ labeling coupled with 2D-nanoLC-MS/MS to do the analysis. The ProteinPilotTM Software 3.0 was used to analyse the differential expressed protein.2. Using the Software DAVID coupled with wego, we got the figure of GO term analysis about differential expressed protein of each group.3. the Software STRING (V9.1) were used to get the figure of network with crosstalk key molecular and network with Transcription factors.5.The differential expressed protein be detected the mRNA level by qPCR and the protein level by ELISA.Results:1.There were 16 different expressed proteins in these four groups; 2.An analysis of Gene Ontology about 16 different expressed proteins from the four groups; 3.After analysis of relationship between key molecular with neuron-glial crosstalk (CD200-CD200R1, CCL21-CXCR3) and different expressed proteins of these four groups, we found the different expressed proteins of the mcao-24h vs sham group were almost opposite to the mcao-48h vs mcao-24h group; 4. We found that were complicated connections between different expressed proteins and these transcription factors; 5. Further verification of the proteomic results.Conclusions:In the present study, we analyzed the protein profile of stroke and treatment with hUC-MSCs using iTRAQ. There were 16 proteins be identified. And there were complicated connections between them and Transcription factors or other protein. Proteomic validation results showed that Abca13 and Grb2 are of interest and could be further pursued. Many of them didn’t be reported in stroke researches before. We believe the present study could guide researchers to do researches about these proteins in stroke, which could help us understanding stroke and treatment with hUC-MSCs further.