The Study On The Novel System Of Stem Cells Transplantation For Ischemic Disease Of Lower Extremity

Backgroud:Ischemic disease of lower extremity is a kind of common diseases among aged people, with the highest disability rate. At present, the main treatments to ischemic disease of lower extremity are traditional drug therapy and arterial bypass, but none of the ideal therapy has been established. These years, stem cell therapy has been applied to treat the limb ischemia, which substitutes stem cells that have the capacity of differentiation and self-renewal for dead skeletal muscle cells. The effect of stem cell therapy is mainly decided by the retention rate and survival rate of cells, but the present studies showed very low retention rate and survival rate, which caused this therapy still limitated to animal and clinical tests. Thus, the most important thing is to create a new transplantation system which can solve these two problems. However, there are still no reports showing any solutions at home and abroad nowadays.Objective:To create a kind of thermo-sensitive, pH-sensitive hydrogel with optimum mechanical properties as the carrier for stem cell transplantation in order to incease the retention rate of cells; to apply oxygen release microsphere to this system to create a microenviroment which is suitable for stem cell survival in the ischemic limb in order to promote the regeneration of skeletal muscle cells, protect more muscle cells from death under hypoxic condition; to evaluate the effect of this new system with ORM; to apply bFGF which is a kind of growth factor mediating the formation of new blood vessels——a process known as angiogenesis to the new system in order to induce the stem cells differentiate to endothelium cells; to investigate the effect of this system with bFGF; to throw light on the mechanism of limb ischemic condition to the function of stem cells in order to supply the new target for improving our novel system.Method:To create a kind of novel hydrogel and establish the new transplantation systems and the animal model for ischemic disease of lower extremity. The effect of the new systems on the survival and differentiation of stem cells in vitro was evaluated by dsDNA concentration, live cell fluorescent imaging, IHC, RT-PCR. The effect of the new systems on the mice with ischemic lower limbs was evaluated by live cell fluorescent imaging, IHC, RT-PCR, western blot, H&E staining, Masson trichrome staining, Doppler bloodflow detection and running test. To study the mechanism of limb ischemic condition to the function of stem cells by tube-formation assay, adherence assay, migration assay and western blot.Results:In vitro experiment for stem cell transplantation system with ORM, the concentration of dsDNA increased in a time-dependent manner and was significantly higher than negative control group (p<0.05), which agreed with the result of live cell fluorescent imaging. The result of IHC and RT-PCR showed that there were more stem cells in ORM group differentiating into muscle cells and endothelium cells of blood vessel. In mice model of ischemic disease of lower extremity, live cell fluorescent imaging also confirmed that this system could significantly increase the survival rate of stem cells. The result of IHC and RT-PCR showed the same conclusion with the experiment in vitro. The result of H&E staining, Masson trichrome staining, Doppler bloodflow detection and running test verified the new system with ORM could effectively protect the ischemic limbs from amyotrophy and improve the faction of muscle. In vitro experiment for stem cell transplantation system with bFGF, the concentration of dsDNA increased in a time-dependent manner and was higher than negative control group (p<0.05), which agreed with the result of live cell fluorescent imaging. The result of IHC and RT-PCR showed that there were more stem cells in bFGF group differentiating into muscle cells and endothelium cells of blood vessel compared with negative control group. In mice model of ischemic disease of lower extremity, live cell fluorescent imaging also confirmed that this system could significantly increase the survival rate of stem cells. The result of IHC and RT-PCR showed the same conclusion with the experiment in vitro. The result of H&E staining, Masson trichrome staining and Doppler bloodflow detection verified the new system with bFGF could effectively lead the ischemic limbs to revascularization to protect the muscle from amyotrophy. The capacity of EPCs for adherence, migration and tube formation was significantly inhibited by PA. The result of western blot implied that palmitate-induced ceramide inhibited endothelial progenitor cells through Akt/eNOS pathway.Conclusion:The new system for stem cell transplantation could significantly increase the retentation and survival rate, effectively differentiate into celllines that we need, and grately improve the effect of cell therapy for ischemic disease. The study on the mechanism of limb ischemic condition to the function of stem cells suppled new targets for improving our novel system.