Molecular Imaging Of Tissue Engineering Combining With Growth Factors On The Bone Marrow-derived Mesenchymal Stem Cells' Survival And Proliferation

BackgroundStem cell regenerative medicine holds a new promise therapy for many final stage diseases such as heart failure, nerve injury and liver failure etc.. Among all the ideal stem cell resources, bone marrow-derived mesenchymal stem cells (BMSCs) has been got more attentions and reseach achievements due to its advantages such as easy accessible, availability, free of immunogenicity. However, the treatment effect is impaired resulted from acute cell death and offtrack.Various cytokines and growth factors, like bFGF, IGF-1, VEGF, could promote proliferation and differentiation of stem cells via regulating cell signals. Moreover, previous studies have indicated that tissue engineering based on matrigel 3D skeletonization could improve the myocardium microenvironment, thus enhancing cell survival and differentiation. Nevertheless, whether the interaction between growth factors and matrigel have impacts on BMSCs are still not well understood.Among the molecular imaging tracking methods of transplanted stem cells, bioluminescence imaing has gained more attention with its high accuracy and reproducibility. BLI is based on the detection of light emitted by living cells expressing light-generating enzymes such as luciferases. In bioluminescent reactions, luciferases generate visible light through the oxidation of enzyme-specific substrates, termed D-luciferin. Making use of specific combination between enzyme and substrate, in vivo optical imaging technologies is assigned with high sensitivity and resolution on monitoring proliferation and survival of stem cell by bioluminescence and fluorescence, which is more accurate than traditional ways.AIMS1. In vitro:To isolate and culture BMSCs; To observe the effects of different growth factors on the proliferation and apoptotic index on BMSCs and to investigate the role of Akt signaling pathway in the growth factor mediated effects.2. In vivo:To observe the synergetic effects of matrigel and various growth factors (VEGF,bFGF,IGF-1) on the proliferation and apoptic index of BMSCs and to investigate the role of Akt signaling pathway in mediating the synergetic effects.MethodsPart 1:Isolation, culture and characterization of BMNCs and the effects of growth factors on the proliferation of BMNCs 1. Isolation,culture and purification of BMSCs derived from B-actin-lucFVB mice;2. Identification of the BMSCs with surface markers (APC-CD90(+),PE-CD44(+),PE-CD34(-)andPE-CD45(-)) by flow cytometry;3. Pretreatment of 3rd generation BMNCs with VEGF,bFGF,IGF-1 (20ng/ml,72h) respectively t;4. To explore the BMSCs proliferative ability with MTT method;5. To evaluate BMSCs apoptotic index with TUNEL method;6. Detection of Akt and p-Akt expression on different groups by Werstern-blot.Part 2:1. Isolation, culture and purification of BMSCs derived from B-actin-lucFVB mice (Same as step 1 of part 1);2. To deternine the cell number signal intensity relationship by IVIS system, standard curve were calculated;3. Subcutaneous transplantation of the mixture of BMSCs, matrigel and different growth factors to inbred line of B-Actin-luc FVB mice;4. To observe the pos-transplanting signal of each group at assumed time-points;5. To evaluate the BMSCs apoptotic indexwith TUNEL staining at 3 weeks after cell transplantion;6. to detect Akt and p-Akt expression on different groups by Werstern-blot analysis. ResultsPart1:1. BMSCs were successfully cultured and identified;2. bFGF (0.1051±0.01512% vs 0.0671±0.01745%, P<0.05), IGF-1 (0.10436±0.01252% vs 0.0671±0.01745%, P<0.05) significantly improved the proliferation of BMSCs (as compared with control group by MTT analysis;3. TUNEL staining showed that bFGF (44.0±2.0% vs 56.0±3.7%, P<0.05),IGF-1 (36.3±3.2% vs 56.0±3.7%, P<0.05) suppressed the apoptotic index of BMSCs as compared with control group;4. Western-blot analysis revealed that the expression of p-Akt in bFGF (0.47±0.0306 vs 0.28±0.037, p<0.05) and IGF-1 group was elevated remarkably (0.57±0.0200 vs 0.28±0.0378, p<0.05) as compared with control group.Part 2:The synergetic effects of growth factors and Matrigel on the proliferation of transplanted BMSCs1. Line-correlation between optic signal and cell numbers of transplanting BMSCs were demonstrated by IVIS system;2. IVIS system analysis showed that the survival rate of B+M+I group was higher than others which groups at 5th week after transplantation (B+M+I group:21%, other groups:<10%) (p<0.05)3. TUNEL staining revealed that the apoptotic rate of B+M+b group (B+M+b group(32.00±1.63% vs 42.33±2.05%, P<0.05), B+M+I group(26.66±1.69% vs 42.33±2.05%, P<0.05) were reduced as compared with control group; 4. Western blot demonstrated increased p-Akt expression in the B+M+b group(0.49±0.032 vs 0.33±0.0153, p<0.05)), B+M+I group(0.63±0.025 vs 0.33±0.0153, p<0.05) as compared with control group;Conclusions1. Compared with VEGF, bFGF and IGF-1 had more signifcant effects of pro-proliferation and anti-apoptosis on BMSCs in vitro;2,Pretreatment with IGF-1 and matrigel could promote the proliferation and surpress the apoptosis of transplanted BMSCs in vivo revealed by molecular imaging;3,The synergetic effects of IGF-1 and matrigel might relate with the activation of Akt signaling pathway.