Experimental Studies Of Transplantation Of Human Amnion Membrane Mesenchymal Stem Cells For Cerebral Ischemia-reperfusion Injury In Rats

BackgroundA significant cause of distress, cerebrovascular disease has greatly impaired human health and reduced patients'quality of life. Mostly, cerebrovascular disease is a group of brain dysfunctions related to disease of the blood vessels supplying the brain. The proportional morbidity rate of cerebral ischemia is 70% in total and always caused long term of neurological dysfunction due to the extensive loss of neurons and the injury of cranial nerve network. Currently, particular treatments for cerebrovascular diseases are largely palliative for the lack of our knowledge. Development of stem cells technology will therefore become increasingly more important in healing cerebrovascular diseases. It is generally thought that adult central neurons caused to be death when experienced injuries, still more did regenerate. Recently, there are many researches suggested a novel population of endogenetic nerve stem cells existed in differ areas of human brains. When the resting endogenetic nerve stem cells which stated in normal are stimulated by ischemia-hypoxia, cells that display extensive proliferation has been identified and migrated to ischemic injury regions, and then appeared to differentiate into new neurons that enhanced regeneration in injured tissues. However, the endogenetic nerve stem cells have limited abilities on the regeneration of brain likely involve multiple factors, such as lacking numbers of endogenetic nerve stem cells and negative effects of ischemic local inflammatory micro-circumstance on cell growth. Currently, most studies concerning stem cell biology focus on transplanting exogenous nerve stem cells, especially has focused on mesenchymal stem cells. These cells, termed MSCs, exhibit characteristics of adult stem cells, including clonogenicity, self-renewal and multi-lineage differentiation potential. Specially, bone marrow derived stem cells (BMSCs) have already become to a central issue and successfully avoided many contradictions on the shortage of embryonic stem cells, ethics and morality, legal restriction or the others. Traditional method that obtained BMSCs from patients has been painful to many. The number and the differentiation activities of BMSCs in bone marrow have declined with the aging process. Therefore, looking for new seed cells which could be induced to multiple lineages in specific culture system similar to BMSCs has begun to raise concerns. Amniotic tissue is an accessible and abundant source of mesenchymal stem cells and unrelated to legal restriction, moral or ethical considerations. Moreover, histopathological studies showed that amniotic mesenchymal stem cells (hAMSCs) have similar phenotypes with BMSCs presented characteristics including lower immunogenicity and higher ability of amplification. Indeed, it is preferable as the source of seed cells in regenerative medicine field.Objects:An established mouse ischemia-reperfusion model will be used to assess the actions and related mechanisms of cerebral ischemia injuries via different pathways of transplanting hAMSCs. Also, we will explore the special significances in advantages of using anti-inflammatory drugs, such as amelioration of growing conditions and the promotion of cell growth.MethodCultured adherent cells (PDACs) digested by enzyme and derived from placenta were labeled and with immunofluorescent. Flow cytometer (FCM) were used to defect the immunoflourescent stained cells to analysis phenotypes and neurobiological characteristics.hAMSCs were co-cultured with T cells. 3H-TdR was added to the culture medium and incubated for 24h.Cells were collected and cpm was read onβliquid scintillation counter. The promoting rates calculated to analysis hAMSCs immunologic properties to Tcells.Using modified suture method, the rat models of middle cerebral artery occlusion reperfusion were established. hAMSCs were transplanted by ventriculus lateralis cerebri and tail vein after 24 hours cerebral ischemia. Neurological Severity Score (NSS) was evaluated after 2 week and 4weeks cerebral ischemia. Immunofluorescent studies were carried out for detecting expression of BrdU positive cells. The volume of cerebral infarction was detected after 1 week cerebral ischemia. The results were evaluated on different transplanted pathways.Utilizing anti-adhesive effects of synthetic E-selectins, observed the changes of inflammatory cytokines (TNF-α, IL-1β) contented in brain tissues and rat serums after applying synthetic E-selectins in rats hind cerebral ischemia-reperfusion models. Both growth and expression of endogenetic/exogenous nerve stem cells were detected after ameliorated local microenvironment.ResultsCells isolated from placenta have similar formations and surface markers. After immunofluorescent labeling, flow cytometer (FCM) showed that CD29,CD44,CD105 were expressed and CD34,CD19,CD106,HLA-DR,CD105 were not. These cells were induced to express neurocyte specific markers-NSE,GFAP.hAMSCs can inhibit proliferation of lymphocytes. The concentration of AMSCs increased while the inhibition on lymphocytes enhanced ;AMSCs can also reduce the effect of Th1 cells in immunological rejection and absolutely proved that AMSCs have the property of immune regulating.hAMSCs transplanted via different pathways have been promoted on local survival and differentiation so that greatly improved animal nerve functions. The results showed transplanting pathway from ventriculus lateralis cerebri has better effects than from tail vein.The contents of TNF-αand IL-1βwere decreased in brain tissues and rat serums after applying synthetic E-selectins.Expression of exogenous nerve stem cells were enhanced. Animal nervous functions improved.ConclusionhAMSC has an accessibly abundant source which presented characteristics including lower immunogenicity and higher ability of amplification. Also, AMSC could effectively promote animal nerve functions and exactly to be considered as preferable source of seed cells in regenerative medicine field. Anti-inflammatory therapy in early stage could enhance proliferation of stem cells so that has vital significations in treating cerebrovascular diseases.