Induced Pluripotent Stem Cells In Mice Lower Limb Ischemia-reperfusion Injury Of Protection

Induced pluripotent stem cells(i PSCs) are a novel stem cell population induced from mouse and human adult somatic cells through reprogramming by transduction of four defined transcription factors(Oct 3/4, Sox2, Klf4 and c-Myc).Recent studies have found that human i PSCs can differentiate into endothelial cells, smooth muscle cells and adventitia, and human i PSCs derived by transplantation of the cells can promote regeneration of blood vessels and muscles. The role of i PSCs against ischemic injury depends on its directional enrichment in the affected part.Objective: To study the effect of miNA on migration and adhesion of iPSCs, and see whether IPSCs had any protective effect against ischemia-reperfusion injury(IRI) of the hind limbs in an acute IRI mouse model.Methods: Based on our successful preparation of i PSCs, in vitro study was performed first by infecting i PSCs with consi RNA and mi RNA217 to observe the migration and adhesion of i PSCs. Then, an IRI model of the left hind limbs was established in 57BL/6J male mice aged 8-10 weeks, using the right hind limb as the control. 200 ul red fluorescence-stained PBS, IPSCs, MEF, i RNA+ips, mi RNA-217+ips containing 6?104 cells /mouse was injected via the tail vein to the mice on the very day and the second day of operation and observed for 72 h to collect the material.Results: 1. i PSCs were successfully prepared according to Yamanaka method. 2. Stimulation with mi RNA-217 did not produce significant effects on the migration of i PSCs. 3. The spindle-shaped i PSCs in the acute IRI mouse model of the hind limbs were able to home to the intima of the femoral artery sustaining IRI. 4. No macroscopic tumorigenesis was observed in any tissue and organ of the mice 28 days after injection of i PSCs or MEF. 5. Blood perfusion of the affected hind limb improved more markedly in mice injected with i PSCs than that in mice injected with PBS or MEF(P<0.05), and the therapeutic effect was reduced significantly in Lipo2000 and mi RNA-217 pre-treatment groups as compared with single i PSCs injection group(P<0.05). 6. HE staining of the affect limb showed that the number of muscle corpsules in i PSCs group was significantly greater than that in MEF, PBS, con-si RNA+i PSC and mi RNA-217 groups, and there was no significant difference between the latter four groups(n=9). 7. Di I-labeled i PSCs location showed that the number of positive cells in the injured tissue was significantly greater in i PSCs group than that in consi RNA+i PSC and mi RNA-217+i PSC groups(P<0.05). 8. CD31 immunofulorescence showed no significant difference in the number of positive cells in the affected part between PBS, MEF, i PSCs, con-si RNA+i PSC and mi RNA217+i PSC groups. 9.Real-time PCR showed that the tendency of gene change was identical in i PSCs and con-si RNA-i PSC groups, while IL-1?m RNA expression in i PSCs, con-si RNA-i PSC and mi R-217-i PSCs groups was significantly higher than that in PBS(1±0.24) and MEF(0.86±0.4). The IL-2m RNA expression in i PSCs group was significantly higher than that in the other four groups. The expression of TNF-? and MCP-1 in i PSCs group was significantly lower than that in the other four groups. The expression of IGF-1 in i PSCs, con-si RNA-i PSC and mi R-217-i PSC groups was significantly higher than that in PBS and MEF groups(P<0.05). There was no significant difference in other genes between the groups. 10. Teto quantitative analysis showed that the Teto content in total DNA was the highest in i PSCs group, and it was significantly higher in i PSCs and mi RNA217+i PSC groups than that in PBS and MEF groups(P<0.05).Conclusion: i PSCs were able to directionally home to the IRI site. No tumorigenesis was observed at the dose of 6?104cells/mouse/200 ul, while this dosage could reduce the infarct area of inflammation, restore blood flow and improve the function of the hind limbs. i PSCs had a targeted therapeutic and protective effect against IRI of the mouse hind limbs. Mi RNA-217 could reduce the adhesion of i PSCs and weaken the therapeutic effect of i PSCs against IRI.