| Induced pluripotent stem cells (iPSCs) have the potential multi-potential developmentsimilar to embryonic stem cells. Compared with embryonic stem cells, iPSCs have broaderapplication prospects in biological and medical fields, devoid of many ethical and legalobstacles. It is reported that hypoxia can affect multiple functions of stem cells. Renalischemia/reperfusion injury (IRI) refers to tissue injury or organ failure due to inability of thekidney function to return to normal or even deterioration of the kidney function when renalischemia occurs and subsequent blood perfusion resumes. There is no report about whetheriPSCs have a therapeutic effect on IRI.ObjectiveThe aim of this experiment was to study the effect of hypoxia on the proliferation,migration, adhesion and apoptosis of iPSCs treated with and without HIF-1α siRNAtransfection in a renal ISI model in vivo, and determine whether iPSCs had any effect onrenal ISI.MethodsUsing retroviral vectors, four genes (Sox2, Klf4, Oct4and c-Myc) were transfected intoprimarily cultured MEF cells to induce iPSCs. There were four groups in the first part of ourstudy:①normoxia group;②hypoxia group;③hypoxia+con siRNA group; and④hypoxia+HIF-1a siRNA group. First, group③and④were transfected with con siRNAand HIF-1a siRNA respectively, and then group②③and④were placed in a hypoxicplace for12h. The proliferation, apoptosis, migration, adhesion and gene expression of iPSCs in the four groups were detected by PCR and cell culture methods. A renal IRI modelwas established by removing the right kidney and ligating the left renal artery and vein for50min. During hypoxia, PBS and the four types of treated iPSCs were injected into the renalcapsules to see whether normoxic iPSCs or hypoxic iPSCs had any effect on renal IRI.Results(1) The effect of hypoxia on the proliferation of iPSCs treated with or without HIF-1asiRNA transfection: Compared with normoxia group (1±0), the number of iPSCs in hypoxiagroup (0.86±0.04）decreased significantly to86±4%of that before hypoxia (P<0.05).Compared with hypoxia+con siRNAgroup (0.87±0.03), the number of iPSCs inhypoxia+HIF-1a siRNA group (0.77±0.02) decreased significantly (P<0.05). These dataindicate that hypoxia and reduced expression of HIF-1a inhibited the proliferation of iPSCs.(2) The effect of hypoxia on the migration of iPSCs treated with or without HIF-1a siRNAtransfection: Compared with normoxia group (1±0), the number of migrating cells in hypoxiagroup rose to3.96±0.84(P<0.05). The number of migrating cells in hypoxia HIF-1a siRNAgroup (1.22±0.04) was significantly lower than that in hypoxia con siRNA group (4.12±1.11)(P<0.05). These data indicate that hypoxia promoted the migration of iPSCs but reducedHIF-1a expression at the same time, thus inhibiting the effect of hypoxia on the migrationiPSCs.(3) The effect of hypoxia on the transendothelial migration of iPSCs treated with orwithout HIF-1a siRNA transfection: Compared with normoxia group (1±0), the number ofiPSCs via transendothelial migration in hypoxia group reduced significantly to0.56±0.10(P<0.05). The number of iPSCs via transendothelial migration in HIF-1a siRNA group (1.19±0.19) was significantly higher than that in hypoxia con of siRNA group (0.70±0.19)(P<0.05). These data indicate that hypoxia inhibited transendothelial migration of iPS cells, andreducing HIF-1a expression could promote the migration of iPSCs under a hypoxic state.(4)The effect of hypoxia on the adhesion of iPSCs treated with or without HIF-1a siRNAtransfection: Compared with1±0in normoxia group, the figure [什么figure?] in hypoxia group reduced significantly to (0.60±0.09)(P<0.05). Hypoxia in HIF-1a siRNA group (1.41±0.15) was significantly higher than that in hypoxia con siRNA group (0.75±0.10)(P<0.05). These data indicate that hypoxia inhibited the adhesion of iPSCs, and reducingHIF-1a could antagonize this effect.(5) The effect of hypoxia on endothelial adhesion ofiPSCs treated with or without HIF-1a siRNA transfection: Compared with1±0in normoxiagroup, in hypoxia group decreased significantly to0.73±0.10(P<0.05). Hypoxia in HIF-1asiRNA group increased to1.20±0.16, compared with that in hypoxia con siRNA group (0.82±0.08)(P<0.05). These data indicate that hypoxia inhibited endothelial adhesion of iPSCs,and reducing HIF-1a could antagonize this effect.(6) The effect of hypoxia on theapoptosis of iPSCs treated with or without HIF-1a siRNA transfection: Compared withnormoxia group (11.28±1.34), early apoptosis in hypoxia group increased insignificantly to14.23±2.98(P≥0.05). Compared with hypoxia+con siRNA group (19.95±4.22), earlyhypoxia in HIF-1a siRNA group decreased insignificantly to16.22±1.44(P≥0.05).Compared with normoxia group (5.35±1.09), late apoptosis in hypoxia group roseinsignificantly to6.63±0.14(P≥0.05). Compared with hypoxia con siRNA group (16.18±1.19), late apoptosis in HIF-1a siRNA group reduced significantly to14.26±0.62(P<0.05).(7) Hypoxic treatment inhibited SOD1, FGFR2, CCS, TEK, PLAU, NME4andMTA2RNA expressions of iPSCs, and reducing HIF-1a expression of iPS cells could inhibitthe effect of hypoxia on these genes.(8) The effect of hypoxia on the migration of iPSCstreated with or without HIF-1a siRNA transfection in IRI kidney: Compared with normoxiagroup, the migration distance in IRI kidney increased significantly in hypoxia group (P<0.05).The migration distance in hypoxia HIF-1a siRNA group was significantly shorter than that inhypoxia con siRNA group (P<0.05). This result was consistent with the result of iPSCs invitro.(9) Serum creatinine (SCR): Compared with normoxia group (216.66±27.87), SCR inhypoxia group decreased significantly to40.21±40.58(P<0.05). SCR in hypoxia HIF-1asiRNA group (153.71±25.26) was significantly higher than that in hypoxia con siRNA group (112.64±29.81)(P<0.05).Conclusions(1) Hypoxic treatment for12h inhibited the proliferation of iPSCs, and reducing HIF-1aexpression decreased the proliferation of iPSCs significantly.(2) Hypoxic treatment for12hpromoted the migration of iPSCs, and reducing HIF-1a expression could inhibit this effectmarkedly.(3) iPSCs could pass through endothelial cells freely, and hypoxic treatment ofiPSCs for12h inhibited this migration markedly, while reducing HIF-1a expression couldreverse this effect and promote iPSCs to migrate through endothelial cells.(4) Hypoxictreatment for12h did not seem to have significant effects on early and late apoptosis ofiPSCs, but reducing HIF expression via HIF-1a siRNA transfection could to some extentinhibit late apoptosis.(5) Hypoxic treatment for12h inhibited the adhesion and endothelialadhesion of iPSCs markedly, and reducing HIF expression via HIF-1a siRNA transfectioncould reverse this phenomenon, thus increasing the adhesion and endothelial adhesion of iPScells.(6) Hypoxic treatment inhibited SOD1, FGFR2, CCS, TEK, PLAU, NME4and MTA2RNA expressions of iPSCs, and reducing HIF-1a expression of iPS cells could inhibit theeffect of hypoxia on these genes.(7) Hypoxic treatment promoted migration of iPSCs in themouse ischemic kidney, and reducing HIF-1a expression could shorten the migrationdistance.(8) Compared with PBS and normal iPS groups, injection of hypoxia-treated iPSCsinto the renal capsules reduced the SCR level.(9) Both normoxia-and hypoxia-treated iPSCsreduced the renal ischemic area induced by IRI, and reducing HIF-1a expression ofhypoxia-treated iPSCs could inhibit the effect of hypoxic iPSCs on the IRI kidney. |
