Study On Effects Of Snail Gene Transfer On Biological Characteristics Of Human Bone Marrow Mesenchymal Stem Cells

Part I Study on the amplification of Snail eukaryotic expressing plasmid and its transfer to bone mesenchymal stem cellsObjective: To investigate the transfer and expression of Snail gene in human bone mesenchymal stem cells(MSCs), and determine its effects on biologic characteristics change of the growth of MSCs, for the purpose of the feasibility study on MSCs used as accepted cells of human Snail gene.Methods: MSCs were isolated from adult human bone marrow via density gradient centifugation. Light microscopy was used to study the morphologic features and flow cytometry to examine the expression of cell surface antigens. Then the recombinant eukaryotic expression plasmid PCAGGSneo-Snail-HA or the control vector of PCAGGSneo was transferred into the cells by the liposome LipofectamineTM 2000. Then stably transfected cell colonies were obtained by G418 screening. After transfection, the marker gene coding HA and the destination gene coding Snail were observed with the fluorescent microscope and Western-blot. The growth curve of cell proliferation was also obtained based on the observation of the proliferation status of MSCs transfected by the vectors or not.Results: Bone mesenchymal stem cells in original generation were all in the long shuttle-like shape and adhered to the disk. MSCs of 3rd, 5th generation were well purified and all in the whirlpool-shape. These cultured cells showed immunore activity to CD29 but not CD34.The expression of Snail and HA in MSCs transducted with PCAGGsneo-Snail-HA (MSCS-Sna), compared with the MSCS transducted with the control vector of PCAGGsneo (MSCS-neo), was confirmed by Western-Blot and Immunofluorescence both in 48hr and 4w culture interval after transfection. Growth curve showed a little longer lag in cell doubling time of cells transfected (including MSCs-Sna and MSCs-neo) than not. However there was no significant difference of growth rate and doubling time between MSCs-Sna and MSCs-neo.Conclusion: Purified MSCs can be obtained via this protocol. Cells of 3rd, 5th generation with high proliferation are fit to gene transfer and the further experiment. The Snail gene is able to provide transient and persistent expression in MSCs,which has no significant influence on the cells'activity. This provides a further foundation for studies of effects of Snail gene expression on the survival, migration and expression of Matrix Metalloproteinase-2 in MSCs.Part II Up-regulation of Snail expression accelerates MSCs migration in vitro through PI-3K/Akt and ERK/MAPK-dependent pathwayObjective To investigate the advantage for Snail to promote MSCs migration and to test whether PI3K or other signaling pathway is implicated in Snail-mediated MSCs migration. Methods: Stably transfected cell colonies were obtained by Snail gene transfer with liposome and G418 screening. The role of Snail in MSCs motility was assessed by comparing the migration of MSCS-Sna with that of MSCs-neo in Transwell System. Migration was calculated from the number of cells found to have passed through an 8-um pore size polycarbonate membrane. Levels of PI3K and ERK/MAPK activity were detected by fluorescent microscope and Western-blot. To test further whether PI-3K or MAPK is implicated in Snail-mediated MSCs migration, we next used a variety of inhibitors in our migration assay.Results: With Western blot technique, the ratio of p-Akt to Akt or of p-ERK1/2 to total ERK1/2 in MSCs-Sna was obviously higher than that in the control group of MSCs-neo (P<0.05). Furthermore, immunofluorescence staining confirmed a considerably higher quantity of p-Akt and p-ERK present in MSCs-Sna than in MSCs-neo and confirmed the Snail-induced increase in translocation of p-Akt or p-ERK1/2 to the nucleus. MSCs-Sna showed more significantly migration than MSCs-neo in the transwell migration system (p<0.05). And suppression of PI-3K activation by the specific PI-3K inhibitor, Wortmannin, or suppression of ERK activation by the specific ERK inhibitor, PD98059 brought on a reduction in Snail-mediated MSCs migration (P<0.05). In contrast, treatment with the P38 Mitogen-activated protein kinase (MAPK) inhibitor, SB203580, did not show any significant effect on the Snail-mediated cell migration (P>0.05).Conclusion: Our results suggested that Snail expression in MSCs can increase the migration ability of MSCs. Snail-accelerated MSCS migration was mainly associated with PI-3K and ERK/MAPK, but not with P38/MAPK. Part III Study on effects of Snail gene modification on the CXCR4 expression of human bone mesenchymal stem cells and their capacity of migration to SDF-1 in vitroObjective: To investigate effects of Snail gene modification on the CXCR4 expression of human MSCs and their capacity of migration to SDF-1 in vitro, for providing the possibility for us to optimize the migration or targeting of transplanted MSCS toward damaged tissues where SDF-1 has been shown to be upregulated as part of the injury response.Methods: Stably transfected cell colonies were obtained by Snail gene transfer with liposome and G418 screening. FACS analysis and immunofluorescence staining were used to study the expression of CXCR4 on the surface of MSCs-Sna and MSCs-neo. And chemokine receptor CXCR4 mRNA was detected by RT-PCR. Chemotaxis assays were performed, using the Transwell Chamber assay and SDF-1α, a ligand for CXCR4, as a chemoattractive agent in the lower chamber, to evaluate the migratory capacity of MSCs-Sna and MSCs-neo to SDF-1 in vitro. For the blocking assay, CXCR4 bloking antibody(1μg/ml, 10μg/ml) was added into cell culture.Results: Fluorescence-activated cell sorting(FACS) analysis showed that CXCR4 expression was higher in the Snail transfectants of MSCs(MSCs-Sna) than that in the vector control cells(MSCs-neo) (64.25%±7.22% vs. 7.45%±0.91%, P<0.05). The increase in CXCR4 expression by Snail was further supported by fluorescence confocal microscopy. Furthermore, CXCR4 mRNA was enhanced more in MSCs-Sna than in MSCs-neo, confirmed by RT-PCR(0.916±0.102 vs. 0.410±0.050,P<0.05). Chemotaxis assays showed that SDF-1αstimulated the in vitro migratory activity in MSCs-Sna more than MSCs-neo(p<0.05). Moreover, the SDF-1α-induced migration activity of MSCs-Sna was inhibited in a concentration-dependent manner by a CXCR4-blocking antibody, but not by a control IgG antibody.Conclusion: Snail enhances CXCR4 expression by MSCs, providing a plausible mechanism for Snail-mediated MSCs transmigration in vitro or metastasis to damaged tissues in vivo where SDF-1 has been shown to be upregulated as part of the injury response.PartⅣThe advantages for Snail expression to protect against serum-deprivation triggered actin cytoskeleton damage and apoptosis of bone mesenchymal stem cellsObjective: To examine whether Snail gene expression acts in the cytoskeleton stabilization and anti-apoptosis capacity of MSCs, providing the feasibility for transplanted MSCs to survive and maintain subsequently in the local microenvionment of injured tissue.Methods: Actin cytoskeleton staining by Immunofluorescence was performed to analyze the cytoskeleton of MSCs-Sna. The percentages of sub-G1 apoptotic cells and the annexin-V+PI- apoptotic cells were detected by flow cytometry.Results: After the cells were cultured in serum-free medium for 24 h, MSCs-Sna maintain the normal distribution ofβ-Actin, and there were a few bundles of stress filaments running the whole length of the cell body; however MSCs-neo displayed the disordered actin cytoskeleton, though the cellular outline still remained. Furtheremore, When cultured in in serum-free medium for 72 h, MSCs-neo even couldn't maintain the essential cellular shape, associated with damaged, depolymerized and aggregated filaments of the cytoskeletons, compared to the MSCs-Sna, which still appeared normal cellular shape with stable and well organized cytoskeletal network. Both MSCS-Sna and MSCS-neo cultured in conventional medium exhibited a low proportion of apoptotic cells as indicated by the "Sub-G1 peak"and the annexin-V+PI- cell percentage. We failed to detect the significant difference in apoptosis between MSCS-neo and MSCS-Sna, after the cells were cultured in serum-free medium for 1 day. Apoptotic percentages in both MSCS-Sna and MSCS-neo were increased after incubation in serum-free medium (3 days). However these percentages were significantly lower in MSCs-Sna (34.33%±5.51% and 12.5%±2.2%) than MSCs-neo (52%±8.19% and 30%±4.3%)(P<0.05). MSCs-neo or MSCs-Sna were examined following a 24-h cultured in complete medium with or without IL-4(20ng/ml). The apoptotic annexin-V+PI- MSCs-neo percentage was 5-fold enhanced by addition of IL-4. However the annexin-V+PI- percentage of MSCs-Sna cultured in the presence of IL-4 was just 2.6-fold enhanced, compared with controls, cultured in medium without IL-4.Conclusion: MSCs overexpressing Snail exhibited a greater resistance to the cytoskeleton damage and serum-deprivation or IL-4 triggered-apoptosis of MSCs. These support the idea that Snail may play a role in survival capacity of MSCs.Part V Snail gene modification stimulates Matrix Metalloproteinase-2 production by human bone mesenchymal stem cells through activation of ERK and PI3K pathwaysObjective: To examine whether Snail-induced expression of matix metalloproteinase-2 (MMP-2) is involved in MSCs and investigate the molecular mechanism involved in it.Methods: After both MSCs-Sna and MSCs-neo were treated with PD98059(0,10,30,50,100μmol/L)or Wortmannin(0,10,20,40,80 nmol/L) for 24h, the levels of phosphorylated Akt(p-Akt) and phosphorylated ERK1/2 (p-ERK1/2) were examined by Western blot to determine the effective concentrations for PD98059 and Wortmanin to inhibit the Snail-induced ERK and PI3K activity of cells. Then we employed these specific inhibitors with respective effective concentrations to incubate MSCs-Sna and MSCs-neo and detect the expression of MMP-2 mRNA and gelatinolytic activity by MSCs-Sna or MSCs-neo with RT-PCR and gelatin zymographic assay.Results: (1) Increased levels of p-ERKs (phospho-p42 and phospho-p44) and active AKT, but not phosphor-p38, in BMSC-Sna, were confirmed by Western-blots. Snail-induced increased activity of p-Akt and p-ERKs in MSCs-Sna could be inhibited in a concentration-dependent manner by Wortmannin and PD98059. The effective concentrations for PD98059 and Wortmanin to inhibit the Snail-induced ERK and PI3K activity of cells are 50μmol/L and 40nmol/L respectively. (2)&(3) MMP2 mRNA was up-regulated in MSCs-Sna than in MSCs-neo, which is confirmed by RT-PCR(0.680±0.104 vs. 1.803±0.301,P<0.05). The significantly decreased levels in the MMP-2 gelatinalytic activity by BMSC-Sna exposed to PD98059 (PD, 50μmol/L) or Wortmannin (WM, 40 nmol/L), compared to the MSCs-Sna untreated, were detected in zymographic assay (p<0.05). Furthermore, treatment with a combination of PD and WM induced such a decrease more (p<0.05). However MSCs-Sna exposed to the inhibitor of p38 pathway (SB203580) did not show a significant decrease of MMP-2 mRNA expression and of the gelatinolytic activity of the latent 72-kDa form of MMP-2, compared with MSCs-Sna untreated(P>0.05).Conclusion: Overexpression of Snail can induce expression and secretion of MMP-2 by MSCs through activation of ERK/MAPK and PI3K signaling pathways.