| Objective:(1) Isolation, culture, phenotypic characterization and in-vitro differentiation of mouse bone marrow-derived mesenchymal stem cells (MSCs);(2) Construction of PMSCV-Aktl-IRES-GFP retroviral vector and transduction of MSCs;(3) In-vitro studies of the biological characteristics of bone marrow-derived MSCs overexpressing Aktl (Aktl-MSCs);(4) Study of the effects of Aktl-MSCs on concanavalin A (ConA)-induced acute liver injury in mice.Methods:Tibia and femur were harvested from C57mice. The bone marrow cells were cultured in a medium used for BMSCs according to published protocols. The harvested mesenchymal stem cells (MSCs) were characterized by flow cytometry. Constructed Aktl-IRES-GFP retroviral vector and GFP only control retroviral vector in the laboratory, which were used to transinfect MSCs. MSCs overespressing both GFP and Aktl (Aktl-MSCs) or only GFP (GFP-MSCs) were isolated and amplified, respectively. RT-PCR and western blots were used to confirm the expression of Aktl gene, Aktl protein and phosphorylated Aktl protein in Aktl-MSCs. After treating Aktl-MSCs and GFP-MSCs cells with IFN-y at different concentrations (0,10,50, and100ng/mL), cell growth and apoptosis were analyzed by MTS assay and Annexin V/PI double staining, respectively. The PCR-array was also used to evaluate the apoptosis signaling pathways in Aktl-MSCs and GFP-MSCs. RT-PCR was used to assess the expression of Aktl, IL-4, IL-10, VEGF, HGF and PGE2in Aktl-MSCs and GFP-MSCs before and after stimulation by10ng/ml IFN-y. ELISA was used to measure IL-4, IL-10, VEGF, HGF and PGE2in Aktl-MSCs and GFP-MSCs culture media after stimulation with different concentration (0,10,50,100ng/mL) of IFN-y. Aktl-MSCs or GFP-MSCs were administered to mice receiving lethal dose ConA (40mg/kg) induced acute liver injury. Survival curves were plotted and pathological changes in the liver and spleen were examined. Aktl-MSCs and GFP-MSCs were also administered to mice receiving sub-lethal dose of ConA (20mg/kg), and the liver injury markers (AST, ALT) and histopathological changes of the liver and spleen were obtained. Serum TNF-α, IFN-γ, IL-4, IL-10, VEGF, HGF and PGE2levels were also measured by ELISA. The homing of Aktl-MSCs and GFP-MSCs in sub-lethal ConA-induced acute liver injury model was observed by direct in vivo imaging.Results:Mouse bone marrow-derived MSCs were successfully cultured and characterized. By flow cytometry; the MSCs expressed CD29, CD44, Sca-1, but not CD45, CD34, CD11b, CD31, FLK-1or MHC-II molecules. The cultured MSCs were successfully induced to differentiate into chondrocytes and adipocytes in vitro under proper induction conditions. MSCs were successfully transinfected, and Aktl-MSCs and GFP-MSCs were obtained and amplified. By RT-PCR, the Aktl gene expression in Aktl-MSCs was significantly higher than that of GFP-MSCs (P<0.01). On Western blots assays, both total Aktl protein and phosphorylated Aktl (Ser473) protein levels in Aktl-MSCs were higher than that of GFP-MSCs. Cell proliferation and apoptosis analyses showed that, under both regular culture and high concentration IFN-γ(100ng/ml) stimulation conditions, Aktl-MSCs had proliferation and survival (anti-apoptotic) advantages. On PCR-array analysis, compared to GFP-MSCs, the exogenous apoptosis pathway in Aktl-MSCs was significantly down-regulated. RT-PCR analyses showed that gene expression levels of IL-4, IL-10, VEGF, HGF and PGE-2were significantly higher in Aktl-MSCs. After stimulation IFN-γ(10ng/mL), gene expression levels of Aktl, IL-4, IL-10, VEGF, HGF and PGE-2increased in both Aktl-MSCs and GFP-MSCs; however, the increases were significantly higher in Aktl-MSCs except for VEGF. ELISA analyses were also performed on the culture media supernatants after12-hour IFN-γstimulation at various concentrations (10,50, and100ng/mL). Under10ng/ml and50ng/ml IFN-γ stimulation, cytokine IL-10concentration in the Aktl-MSCs medium was higher than GFP-MSCs; whereas under100ng/ml IFN-γ stimulation, the IL-10levels in Aktl-MSCs and GFP-MSCs media were not significantly different. Under10ng/ml,50ng/ml and100ng/ml IFN-y stimulation conditions, the concentrations of HGF and VEGF were significantly higher in Aktl-MSCs than GFP-MSCs, while the levels of cytokine PGE-2were not significantly different. The concentration levels of IL-4in the culture media were too low (<4pg/ml) in both Aktl-MSCs and GFP-MSCs to detect any difference.24hours after lethal dose of ConA (40mg/kg), no animal in the ConA40mg/kg,ConA40mg/kg+1106Aktl-MSCs, ConA40mg/kg+1106MSCs group survived;3and1survived in the ConA+5106Aktl-MSCs group and ConA+5106 GFP-MSCs group, respectively. In sublethal dose ConA (20mg/kg) induced acute liver injury mouse model, compared to GFP-MSCs, Aktl-MSCs treatment reduces mouse serum AST, ALT, TNF-a and IFN-y levels and improves liver pathology. The reduction in AST and IFN-y levels was particularly significant. In addition, Aktl-MSCs treated group had higher serum concentrations of IL-10, VEGF and HGF. Serum IL-4concentration in both Aktl-MSCs and GFP-MSCs was less than<4pg/ml. In vivo imaging showed that, on day0, hepatic fluorescence signal in ConA+Aktl-MSCs group was significantly stronger than ConA+GFP-MSCs group, whereas the signal in Aktl-MSCs and GFP-MSCs control groups was absent; on day7, focal hepatic fluorescence signal is present in ConA+Aktl-MSCs, but not in ConA+GFP-MSCs, Aktl-MSCs or GFP-MSCs group; on day14, the signal in Aktl-MSCs group was stronger than GFP-MSCs group, whereas ConA+Aktl-MSCs and ConA+GFP-MSCs groups showed no detectable fluorescence signal.Conclusions:In this study, mouse bone marrow-derived MSCs were successfully cultured and characterized. The cultured MSCs were able to differentiate into cartilage and adipocytes under proper induction conditions. PMSCV-Aktl-IRES-GFP retroviral vector and control PMSCV-IRES-GFP retroviral vector were successfully constructed. After transinfection, MSCs overexpressing Aktl and GFP (Aktl-MSCs) or GFP alone (GFP-MSCs) were isolated and propagated. Aktl-MSCs showed higher proliferation and less apoptosis activities when compared to that of the GFP-MSCs under both normal culture condition and stimulation with high concentration of IFN-y (100ng/ml). The cytokine gene expression levels were higher in Akt1-MSCs. After10ng/ml IFN-y stimulation, Aktl-MSCs showed a stronger stress capacity. After stimulation with different concentrations (10ng/ml,50ng/ml,100ng/ml) of IFN-y, Aktl-MSCs exhibited better paracrine function. Aktl-MSCs appeared to improve the survival rate in a lethal dose ConA (40mg/kg) mouse experiment. Acute liver injury induced by sub-lethal dose ConA (20mg/kg) was significantly mitigated by Akt1-MSCs treatment. Finally, in both normal mice and in ConA (20mg/kg) induced acute liver injury mouse model, Aktl-MSCs are associated with improved homing to the liver. |
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