The Effection And Mechanism Of Glucagon-like Peptide1in Umbilical Cord-derived Mesenchymal Stem Cells Treating Ischemic Stroke

Stroke is the third cause of death in world wide,rank only second to heart disease and cancer.Ischemic stroke occupy70-80%in all stroke. Thrombolytic therapy is the most effctivetreatment, however, is limited by a narrow time window and serious side effect. Only a fewstroke patients receive this therapy. However, there is only a30–35%success rate forrecovery, even though r-tPA is administrated within3h after the initial onset of an embolicstroke. Hence,the more effctve treatment was studied by researchers and clinical doctors.With the development of neurological imaging and molecular biology technology, themechanism of ischemic stroke obtained huge progress. Cell death induced byischemic,including apoptosis and necrosis, immune responses and inflammatoryresponses,oxidatve stress and other factors interact, lead to the stroke progress. Oxidatvestress is the key point in the development of the stroke and other disease.The perfecttreatment is the one that can aim at the mechanism of ischemic stroke,without serious sideeffect and can be carry out conveniencely, also have a long time window.Accompanied with the progress of stem cells research,stem cell treatment is the mostpotential therapy for the neurologcal disease,especialy for the ischemic stroke. Celltransplantation can improve the neurlogical movement through the induction neurogenesisand vasculargenesis,secreting neurotrophic fctors, convoking endogenous precursors cellsand so on. Cell transplantation have a longer time window and intravenous adminstration.The umbilical cord-derived MSCs began to attract extensively research. Comparing with bonemarrow MSCs, the umbilical cord-derived MSCs have more superiorities,such as: withoutethical and legal constraints, a non-invasive process, stronger proliferation ability,moreprimitive.But the effection of cell transplantation is not the prospective.the cause may be the numberand activity of transpanted cell was damaged by the severe local enviroment.The solution ofthis problem is to enhance the stem cell resistance to damage and improve the localenviroment. Oxidatve stress have an important role in the ischemic stroke.So the schemewhich can reduce the damage of oxidatve stress to stem cell will be a effective therapy.Glucagon-like peptide1(GLP-1) is encoded by the proglucagon gene, which is secreted bygut L cells and some brain neurons. It exerts multiple biological effects on peripheral tissuesand the central nervous system. GLP-1stimulates glucose-dependent insulin secretion and hasbeneficial effects in the treatment of type-2diabetes. It also acts as an anorexigenic peptide, reducing food intake and hence, body weight. Strikingly, it causes an expansion of-cell massthrough the stimulation of pancreatic-cell proliferation, the induction of islet neogenesis,anddifferentiation from exocrine cells or immature islet progenitors while at the same timeproviding protection from-cell apoptosis. GLP-1, including activity in hippocampal circuits,stimulation ofneurite outgrowth, promotion of cell survival, and up-regulation of enzyme andneurotransmitter production. As such, in addition to its hormonal and neuropeptide activity,GLP-1may be considered a growth factor with actions that include regulation of mitosis, cellgrowth, and differentiation and interruption of pro-apoptotic processes, documented forpancreatic cells. Indeed,the collective data suggest that GLP-1and its analogues haveattributes that extend beyond protection against excitotoxic or oxidative damage, to broaderand unified neurotrophic properties exhibited in all GLP-1R-expressing cells.Few studies have investigated the therapeutic validityof GLP-1-based therapies in stroke. In arodent model of stroke, the middle cerebral artery occlusion (MCAO) model, which mimicsthe most common type of stroke in humans, infarct volume was reduced by50%in ratspretreated with exendin-4(ICV) compared with controls.This was accompanied by improvedshort-term functional outcome.But the research about the GLP-1improve neurogenesis andvasculargenesis can't be found.GLP-1have cell protectiive effects against oxidative stress in human umbilical venousendothelial cells,neuronal PC12cells,and cadiocyte. hBM-MSCs expressed mRNA andGLP-1receptor protein. GLP-1promotes cellular proliferation and cytoprotection andprevents cell differentiation into adipocytes. GLP-1significantly reduced cell apoptosisinduced by serum deprived. Whether hUC-MSCs express mRNA and GLP-1receptor protein,and GLP-1can protect the hUC-MSCs from the oxidative stress injury. GLP-1can improvethe effection of hUC-MSCs transplantation to the ischemic stroke. These problems shuld bestudied.The aim of our research is to isolate and culture the hUC-MSCs, identify the express ofGLP-1receptor, study the effection and mechanism of GLP-1resistant to the H2O2,andobserve the role of GLP-1in the hUC-MSCs treatment of rat MCAO model. The results willbe stated as follows:1. Isolation and identification of hUC-MSCshUC-MSCs were isolated and cultured by tissue directly and adherent culture method.Thecells were tested by morphology, and,cell circle. Their surface markers (CD29èCD34èCD44èCD45è CD105,HLA-DR) were analysised by flow cytometry. hUC-MSCs wereinduced to differentiate into the bone and fat cells. The results showed that hUC-MSCs grewspindle-shaped whirlpool, Cell cycle analysis demonstrated that more than90%of hUC-MSCs were in G0G1..Their surface markers CD34, CD45and HLA-DR were negative,CD29, CD44, and CD105were positive.Under the special culture condition,they could makecalcium deposits and lipid droplet appeared to express osteoblast and fat cellscharacteristics.The vast majority of obtained cells were.superior hUC-MSCs. In this sectionwe established a hUC-MSCs cultured system, provided a stable and supeiror cell source forthe follow-up of stem cell research.2. Identify the GLP-1receptor of hUC-MSCsThe expression of mRNA and protein of the GLP-1R were identified in hUC-MSCs.Thepresent study tested the mRNA of GLP-1R with reverse transcript–polymerase chain reaction(RT-PCR), the protein with Western blot and immunofluorescence Human umbilical venousendothelial cells (HUVEC)was the positive control,HepG2was the negtive control.Thepositve results were showed in RT-PCR and Western blot, but they are inferior to theHUVEC. Nuclear staining was performed in blue with4,6-diamino-2-phenylindole (DAPI).GLP-1R antibody (green fluorescence), the results were detect with laser scanning confocalmicroscope. hUC-MSCs were double-positive cells. HepG2was only nuclear staining. In thispart we confirmed the mRNA and protein expression of GLP-1receptor in hUC-MSCs,established the structure base for the cell experiment.3. The protection and mechanism of GLP-1on the hUC-MSCs oxidative stress inducedby hydrogen dioxideUsing the nomal condition as the control, the oxidative stress of hUC-MSCs induced byhydrogen dioxide as different concentration (10mol/Lè20mol/L,50mol/L).The cellviability and apoptosis were detected to select the best concentration as the cell mode.Thendifferent concentration of GLP-1(10nmol/L,20nmol/L,30nmol/L) were cocultured with thecell mode. The optimum concentration were screen out according to the cell viability andapoptosis.To study the mechanism of GLP-1on the cell mode, the GLP-1receptorantagonist exendin (9-39), and the PI3kinase inhibitor LY294002respectively cocultured withGLP-1in the cell mode.The results of cell viability and apoptosis showed the protection ofGLP-1was abolished.In this part we show that GLP-1protect the hUC-MSCs against theoxidative stress. This protection appears to involve activating GLP-1receptor and PI3K/Aktsignaling pathways.4. The effect and mechanism of GLP-1in the hUC-MSCs treating the rat ischemicstroke modelMCAO was duplicated by occluding rats middle cerebral artery with nylon thread.Wistar ratswere used in this study.The rats were divided into normal control group, MCAOgroup,GLP-1treatment group (on the set of ischemic stroke Intraperitoneal Injection 90ng/100g), hUC-MSCs transplanting group(after ischemic stroke48hours), GLP-1unitinghUC-MSCs transplanting group. The group were divided into1day,3days,7days,14daysfour subgroup. The neurological function and general condition were observed on the7d and14d.We detected the GLP-1receptor on the hippocampus of normal control rats. Methods ofpathomorphologic and immunohistochemistry were employed to tested the neuronalapoptosis, neurogenesis and vasculargenesis.The markers were selected using c-fos asneuronal apoptosis, doublecortine(DCX) as neurogenesis CD31and.-SMA asvasculargenesis The results showed that the MCAO group blood glucose rised a little,whichwere thought to be stress induced by the stroke. The neurol function of all treatment groupswere improved,but the union group is the most obvious.GLP-1reduced the neuronalapoptosis after ischemic stroke. The neurogenesis and vasculargenesis of hUC-MSCstransplanting group were promoted when united with GLP-1.Conclusion1hUC-MSCs were isolated and cultured by tissue directly and adherent culture method. Thecells were tested by morphology,cell circle., surface markers, and differentiation potential.The vast majority of obtained cells were.superior hUC-MSCs. In this section we established ahUC-MSCs cultured system, provided a stable and supeiror cell source for the follow-up ofstem cell research.2The expression of mRNA and protein of the GLP-1R were identified in hUC-MSCs,established the structure base for the cell experiment.3GLP-1protect the hUC-MSCs against the oxidative stress. This protection appears toinvolve activating GLP-1receptor and PI3K/Akt signaling pathways.4GLP-1have the neuro protection on the ischemic stroke through reducing the neuronalapoptosis.5The neurogenesis and vasculargenesis of hUC-MSCs transplanting group were promotedwhen united with GLP-1. the joint use of GLP-1can enhance the therapeutic effect of thehUC-MSCs transplant. This study gives a new insight into the cells treatment of ischemicstroke.