| Backgroud and objectiveTissue engineering is widely used in the repair of bone defects today. However, large bone defects often accompanied with ischemia, acidosis, accumulation of metabolites, a series of inflammatory reaction media and pro-apoptotic factor from tissue necrosis, resulting in apoptosis and death of chemotaxis or transplanted stem cells. Bone marrow mesenchymal stem cells (BMSCs) are the main adult stem cells involved in bone formation, and are the ideal seed cells for cell therapy and tissue engineering. However, a large number of studies have shown that the survival rate of BMSCs after transplantation is disappointing. It may due to the ischemic microenvironment related cell death. Leukemia inhibitory factor (LIF) is a cytokine displays multiple biological functions. STAT3 signaling which activated by LIF can regulate cell survival, cell self-renewal as well as many related anti-apoptotic target genes. Extensive literature indicates, LIF maintains cell survival in a variety of different physiological or pathological environment. However, there is no research on the relationship between LIF and BMSCs survival. In this study, we established an in vitro ischemia model, then to observe the survival and apoptosis of BMSCs in this model. We analyzed the effect of LIF on BMSCs apoptosis and investigated the potential mechanisms and signal transduction pathways. The research has been divided into three parts:Part I Effect of hypoxia and serum-deprivation on rat bone marrow mesenchymal stem cells apoptosis1. Isolation and identification of rat bone marrow mesenchymal stem cells in vitroThe BMSCs were isolated from 4-5-weeks-old male wistar rats by whole bone marrow adherent method. After purification and amplification, the flow cytometry was performed to identify the surface antigens of BMSCs, and cells were subjected to osteogenic or adipogenic induction to identify the multi-differentiation potential.Under phase-contrast microscopy, cultured BMSCs showed adherent growth, uniform morphology, and stable proliferation rate. The third passage cells showed strongly positive expression of CD90, CD29, CD 105 and CD44 (>95%), and the hematopoietic cell marker CD45 were negative (?2%). After 6 days of osteogenic induction, cells showed positive staining of alkaline phosphatase (ALP). After 12 days of osteogenic induction, cells showed positive staining of alizarin red indicating calcium deposition. After 14 days of adipogenic induction, cells showed positive oil red O staining indicating red lipid droplets in cytoplasm. These data indicates that the cells we isolated substantially all belong to mesenchymal stem cells.2. Hypoxia and serum-deprivation treatment induces apoptosis of rat bone marrow mesenchymal stem cellsThe cellular hypoxia and serum-deprivation culture system in vitro to mimic conditions under ischemia in vivo was established. BMSCs were divided into two groups: normal culture group (control) and hypoxic & serum-free culture group (6h,12h,24h). Then compare the two groups using Hoechst 33258 staining to detect cell nuclear morphology under fluorescence microscopy; Annexin V-FITC/PI staining and flow cytometry to analyze cell apoptosis rate; Rhodamine 123 staining and flow cytometry to analyze cell mitochondrial membrane potential and real-time quantitative PCR to detect Bax, Bcl-2, Lif and Lifr gene expression.Hypoxia and serum-deprivation treatment resulted in typical apoptotic nuclear morphology changes and increased apoptosis rate of BMSCs significantly. Compared with the control group, after hypoxia and serum-deprivation treatment, the population of Annexin V+/PI- and Annexin V+/PI+ cells in BMSCs were significantly increased (P?0.05). Hypoxia and serum-deprivation treatment also resulted in the loss of mitochondrial membrane potential in a time-dependent manner (P?0.05). After 12 and 24 hours of hypoxia and serum-deprivation treatment, the expression levels of Bax was significantly increased, while the expression of Bcl-2 at each time point was significantly decreased (P?0.05), and the Bax/Bcl-2 ratio was significantly increased at each time point (P?0.01). In addition, hypoxia and serum-deprivation treatment also resulted in decreased expression of Lif and Lifr in BMSCs (P?0.01).Part ? LIF inhibits the hypoxia and serum deprivation-induced apoptosis in bone marrow mesenchymal stem cellsThe third passage BMSCs were seeded and grouped as follows:normal group (Control group), hypoxia and serum-deprivation group (apoptosis model group), hypoxia and serum-deprivation+LIF group, hypoxia and serum-deprivation+LIF+ LIF neutralizing antibody group. Then compare these groups using Hoechst 33258 staining to detect cell nuclear morphology; Annexin V-FITC/PI flow cytometry to analyze cell apoptosis rate; real-time quantitative PCR to detect apoptosis related genes expression; Rhodamine 123 staining and and flow cytometry to analyze cell mitochondrial membrane potential and western blotting to detect activation levels of Caspase-3.Data showed that throughout the 6 hours exposure to hypoxia and serum-deprivation, LIF exhibited a clear anti-apoptotic effect. It eased the apoptotic change of nuclear morphology significantly. The protective effect of LIF on cell survival rate was dose-dependent and peaked at 40ng/ml (P?0.01). Annexin V-FITC/PI double staining and flow cytometric analysis found that, LIF treatment could significantly reduce the population of early apoptotic cells (P?0.01). Analysis of total apoptosis rate showed:under normal culture conditions, cell apoptosis rate was (3.91 ± 0.92)%; in hypoxia and serum-deprivation culture, (17.55 ± 0.41)%; in hypoxia and serum-deprivation+LIF culture, (13.30 ± 0.56)%. It indicated that LIF treatment decreased cell apoptosis rate (P?0.01), and the protective effect of LIF can be eliminated by LIF neutralizing antibodies. Further studies revealed that LIF significantly reduced the expression of Bim and Bax/Bcl-2 ratio elevated by hypoxia and serum-deprivation treatment (P<0.05), while it had no effect on the Survivin, Mcl-1 and Bcl-xl expression. Fluorescence microscopy and flow cytometry analysis showed that LIF could inhibit the loss of mitochondrial membrane potential (P<0.05). Western blot analysis showed:normal cultured cells did not produce active fragments of Caspase-3; the hypoxia and serum-deprivation treatment triggered Caspase-3 activation of BMSCs; LIF treatment attenuated Caspase-3 activity significantly.Part ? The JAK1/STAT3 signaling pathway is involved in the LIF-mediated protective effect on hypoxia and serum deprivation-induced apoptosis in bone marrow mesenchymal stem cellsThe third passage BMSCs were seeded and grouped as follows:normal group (Control group), hypoxia and serum-deprivation group (apoptosis model group), hypoxia and serum-deprivation+LIF group, hypoxia and serum-deprivation+LIF+ JAK1/STAT3 inhibitor group, hypoxia and serum-deprivation+JAK1/STAT3 inhibitor group. Then compare these groups using real-time quantitative PCR to detect the expression of Bax and Bcl-2; Rhodamine 123 flow cytometry to analyze cell mitochondrial membrane potential; western blotting to detect the expression level of associated signaling molecules and the activation of Caspase-3; Annexin V-FITC/PI flow cytometry to analyze cell apoptosis rate.Firstly, western blot results showed that under hypoxic and serum-free environment, phosphorylation levels of JAK1 and STAT3 were significantly reduced. Exogenous LIF could significantly activate JAK1 and STAT3, but not JAK2. The use of a specific inhibitor targeting JAK1 or STAT3 is capable of blocking the LIF protective effect on maintaining mitochondrial membrane potential and regulating Bax/Bcl-2 ratio. It illustrates that the activation of JAK1 and STAT3 is involved in the LIF-mediated protective effect on mitochondria. Secondly, we also found that the activation of JAK1 and STAT3 could inhibit Caspase-3 activation. The specific inhibition of JAK1 and STAT3 could completely block the anti-apoptotic effect of LIF.Conclusion1. Hypoxia and serum-deprivation culture condition can induce apoptosis of rat bone marrow mesenchymal stem cells, and the mitochondrial apoptotic pathway is involved in this process.2. In the culture of bone marrow mesenchymal stem cells under hypoxia and serum-deprivation, leukemia inhibitory factor can protect mitochondrial function and reduce Caspase-3 activation, resulting in the inhibition of cell apoptosis.3. Leukemia inhibitory factor can activate JAK1 and STAT3 under hypoxia and serum-deprivation.The actived JAK1/STAT3 signaling pathway protects BMSCs from apoptosis through the inhibition of mitochondrial apoptotic pathways. |
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