The Research Of CS-IGF-1C Hydrogel Promoting Angiogenesis Of Mesenchymal Stem Cells

Objective Synthetic new bioactive materials CS-IGF-1C hydrogel and human placenta between source of mesenchymal stem cells(human placenta-derived mesenchymal stem cells,h P-MSCs)co-transplantation,through local muscle injection into the hind limb ischemia animal model,can prolong the retention rates of cell,thereby promoting neovascularization of ischemic limbs,reduce the amputation rate of animal models,and explore its molecular mechanism.Methods(1)In this experiment,we build the stable expression of renal sea luciferase(renilla luciferase,Rluc)and red fluorescent protein(red fluorescent protein,RFP)double report gene h P-MSCs cell line.(2)The biocompatibility of CS hydrogel and CS-IGF-1C hydrogel with h P-MSCs and the anti-apoptosis ability of the hydrogel were tested in vitro.(3)The left femoral artery of transgenic mice was free ligated to establish the model of hind limb ischemia in mice.This experiment was divided into five groups,normal,the sham group,PBS group,h P-MSCs group,h P-MSCs+CS group,h P-MSCs+CS-IGF-1C group,and different treatment measures were given.(4)Retention of h P-MSCs after transplantation was monitored by bioluminescence imaging(BLI)on day 0,2,4,6,and 8,respectively.(5)BLI was also applied on day 3,7,10,14 and 21 to detect the angiogenesis of the hind limb ischemia model in mice.(6)HE staining and Masson staining were used to observe the general expression of the tissue structure of each group,and angiography was used to evaluate the angiogenesis of each group.(7)The mechanism of angiogenesis was explored by real-time quantitative fluorescence PCR(rt-PCR)immunofluorescence.Results(1)We successfully build stable expression renal luciferase(renilla luciferase,Rluc)and red fluorescent protein(red fluorescent protein,RFP)double report gene h PMSCs cell line.(2)In vitro experiments confirmed that CS-IGF-1C hydrogel has the effect of promoting the proliferation of h P-MSCs and anti-apoptosis(down-regulating the expression of Bax,Bad,Fas,and Fasl).(3)In vivo BLI demonstrated that CS-IGF-1C hydrogel can improve retention rate and promote angiogenesis after h P-MSCs transplantation,thus significantly reducing the amputation rate in the mouse model of hind limb ischemia.(4)Real-time quantitative fluorescence PCR(rt-PCR)results showed that CS-IGF-1C hydrogel could down-regulate the expression of apoptosis factor(Bax,Bad,caspase-3)and inflammatory factor TNF-a,up-regulate the expression of VEGF,Ang-2gene,and immunofluorescence results showed that h P-MSCs+CS-IGF-1C hydrogel could promote the expression of CD31 in the affected limb.Conclusion The co-transplantation of CS-IGF-1C hydrogel and h P-MSCs into the mouse model of hind limb ischemia can significantly improve the cell retention rate and promote angiogenesis by improving the expression of VEGF,thus reducing the amputation rate,providing a new idea for the treatment of hind limb ischemic diseases.