In Vitro Study Of Peripheral Blood-derived Mesenchymal Stem Cells In Promoting Macrophage Polarization And Reprogramming

Objective: To observe the immune modification of peripheral-derived mesenchymal stem cell to macrophage and its possible mechanism.Method: Sprague Dawley rats, about 200 g, aged 8–12 weeks, were first administered with GCSF(100 μg·kg- 1) by intraperitoneal injection once daily for 5 days; on the 6th day, CXCR4 antagonist AMD3100(5 mg·kg- 1) was administrated. One hour later, 20 m L of blood was sterilely harvested with tube containing 300 U/m L heparin through the arteria cervicalis under anesthesia. Then the marrow and peripheral blood had been taken, the nucleated cell of marrow and peripheral blood had been collected with density gradient centrifugation and inoculated with a density of 106cell·ml- 1 to conduct a purified culture of MSCs to the P3 generation, and then an adipogenic and osteogenic induction had been done to MSCs.FACS analysis of surface markers(CD34、CD45、CD90、CD44、CD29、G-CSFR)was performed on cultured MSCs at P3.The adipogenic and osteogenic ability of MSCs had been tested with Oil red O, Alizarin S, the genetic expression of Runx2、SOX2、 BSP、 Wnt 5b、 PPARγ and others had been detected with QPCR, The twist1 expression of PB-MSCs/BM-MSCs had been analyzed with western blot. The separated myelomonocyte was inoculated in the cell conditioned medium containing 20% LCM and in the RIPM-1640 medium containing 10%FBS, after 6 days of culture, the purified M0 macrophage had been obtained. They were divided into: control group(M0), LPS induction group(M1), IL-4+IL-10 induction group(M2), PB-MSCs co-culture group(M P-T) and BM-MSCs co-culture group(M B-T), among which, PB-MSCs/BM-MSCs had been inoculated into the Transwell chamber and co-cultured with M0-type macrophage for 3 days. The expression of CD206、CD68、CD11b、i NOS、 Arginase 1、TNFα of macrophage had been analyzed with flow cytometry, the expression of IL-6、 IL-10、IL-1β、CCL22、TGF-β and other genes had been detected with QPCR, Part of M0 macrophages were additionally taken and induced into M1-type macrophage with LPS, they were divided into PB-MSCs co-culture group(M P-C) and BM-MSCs co-culture group(M B-C), among which, PB-MSCs/BM-MSCs(105cell·ml- 1) had been directly inoculated into the M1-type macrophage which had already been adherent to wall for co-culture, three days later, FCM had been used to detected the CD11 b positive cell. Its expression of CD206、i NOS、Arginase 1、TNFα had been analyzed with flow cytometry, the ROS expression had been analyzed with DHE staining.Result: The BM-MSCs showed a typical plastic adherence and a typical fibroblast-like morphology, the morphology of PB-MSCs was similar to BM-MSCs.The cell surface molecule had a positive expression of CD90、CD44、CD29、G-CSFR, and a negative expression of CD34、CD45; after the adipogenesis directed induction, a formation of lipid droplet can be seen in the oil red O staining, after the osteogenesis directed induction, phospheate crystal can been seen in the alizarin S staining, but in the case of same days of induction, PB-MSCs had bigger and more lipid droplets than BM-MSCs, while less phospheate crystal than BM-MSCs. It can be seen from the QPCR detection on relevant genetic expression of osteogenesis and adipogenesis that, compared with BM-MSCs, PB-MSC had a down-regulation of expression of Runx2 、 SOX2 、 BSP, and a up-regulation of expression of Wnt 5b、PPARγ. It can be seen from the QPCR detection on the inflammatory factor of two types of cell that, compared with BM-MSCs, PB-MSC had a low expression of IL-6 and high expression of IL-10, while it can be seen from the Western blot detection on protein expression of twist1 that, PB-MSC was lower than BM-MSCs.Both PB-MSC/BM-MSCs and M0-type macrophage can make the M0-type macrophage to develop into a M2-type polarization in the Transwell co-culture system, which was manifested as the up-regulation of expression of CD206、CD68、CD11b、Arginase- 1、IL-10 and down-regulation of expression of i NOS、TNFα、IL-6、IL-1β、CCL22. As compared with M B-T, M P-T had an up-regulation of expression of CD206、CD68、CD11b、Arginase 1、IL-10, and a down-regulation of expression of i NOS、TNFα、IL-6、IL-1β、CCL22, indicating that, PB-MSC had a stronger ability to induce the M0 macrophage to develop into M2 polarization than BM-MSCs.In the system of direct co-culture of PB-MSC/BM-MSCs and M1-type macrophage, it can be seen that, both PB-MSC and BM-MSCs can change the metabolic level of M1 macrophage, which was manifested as the decline of DHE staining, i NOS expression, and rise in expression of Arginase1 and CD206. PB-MSC influenced the metabolic level of M1 macrophage resulted in a reprogramming towards M2.Conclusion: PB-MSCs showed a stronger inflammatory modifying ability than BM-MSCs, which made the macrophage to transform into M2-type macrophage through expediting the M0-type macrophage to develop into M2-type polarization and reprogramming the M1-type macrophage.