The Efficacy Study Of The HAECs In The Model Of MCAO Injected Through The Carotid Artery

Backgroud:Stroke is a kind of disease that is harm to people’s health seriously,and it can lead to disability and death,of which ischemic stroke accounted for 80%.The neurons will apoptosis after staying in the ischemic anoxia environment 24 to 48 hours.Emergency window period is 3 to 6 hours,however,the vast majority of patients have lost the optimal timing of surgery and thrombolysis after onset.At present,they often use stem cells transplantation therapy to promote the recovery of neural function in patients with ischemic stroke all over the world,among them,human amniotic epithelial cells is a kind of new treatment to cure ischemic stroke.But there are sorts of problems of traditional stem cell therapy,such as cell source,security,immune rejection,ethics and many other problems that limits the development of stem cell therapy.Amniotic epithelial cells(AECs)from the amniotic membrane single permutation grow in the amniotic fluid side.The blastocyst cells that are zygote development to the eighth day differentiate to AECs;therefore they retain the characteristics of the embryonic stem cells,and are more efficient,differentiation capacity and strong plasticity.At the same time,AECs have paracrine function,can express a variety of cytokines and nutritional factions,and it can promote the growth of nerve cell,and it has good curative effect for nerve injury repair.The telomeres enzyme activity of AECs is weak,so their proliferation ability is very limited;in addition,AECs have no tumorigenicity,allograft immunogenicity and they don’t express HLA.At the same time,this kind of cells can secrete various anti-inflammatory factors and other related proteins.It has the potential for the treatment of inflammation and fibrosis.The amniotic cells from neonatal postpartum waste can easy be obtained,and there are not ethics problems.In summary,human amniotic epithelial cells have the following a bit: 1.No immunogenicity and no need for transplantation zygosity.2.Can secrete a variety of neutrotransmitters and neurotrophic factor.3.Have potential to differentiate into three layers.4.No tumorigenicity.5.Sources widely and no ethical problems.As early as 1996 Sakuragawa propose that human amniotic epithelial cells can be used as the progenitor cells of neurons and glial cells,because it can express the molecular marker of these two kind of cells.At present,there are more and more results show that human amniotic epithelial cells can be induced to nerve cells easily in vitro.Therefore,AECs have more potential to differentiate into nerve cells in the treatment of nervous system disease and nerve injury.As mentioned above,AECs have the capacity of differentiating to nervous,migrating to damaged areas and repairing injury under the microenvironment in vivo,such as injecting through ventricle,brain tissue and spinal subarachnoid.Amniotic epithelial calls as a typical example of trophoblast applied to clinical is maintaining the growth of limbal stem cells(LSCs).In the 1990 s,people mainly used surgical transplantation of amniotic membrane or living corneal tissue to treat ocular surface disease.Some researches found that injecting amniotic epithelial cells through lateral ventricle could promote the nerve function deficient recovery in mouse.Although human AECs(hAECs)attract the attention in the field of basic research,most of the results have not been applied to clinical.But it is expected to become the candidate cell of treatment of certain diseases,such as neurological disease,heart disease,and liver disease,used for the cellular therapy and tissue regeneration.From the above,we assume that SD rat’s ethology score has improved after transplanted amnion epithelial cells when compared to the control group in the animal and experiments.We found that amniotic epithelial cells can be gathered to target organs and directional differentiate to neural cells in brain tissue,and the cerebral infarction area and inflammation factors in the cerebrospinal fluid were reduced significantly.This study will make a significant sense to clinical in the future.On the one hand,there is no research about animal model with ischemic stroke transplanting ACEs through carotid artery.On the other hand,hAECs has a high clinical value used as a kind of new treatment method.Part I: To construct rat middle cerebral artery obstruction model and to analysis the behavior change of injecting the human amniotic epithelial cells through the carotid arteryObjective: To establish the rat model of middle cerebral artery obstruction and to observe the preliminary results of injecting human amniotic epithelial cells through the carotid artery.Methods: 1).the SD rats were randomly divided into MCAO(control group),MCAO+ hAECs group(experimental group),then we set up the MCAO models.2).We Separate the human amniotic epithelial cells in vitro.3).We inject hAECs in the rats of the experimental group through the carotid artery,and the same amount of saline for the control.4)We record the behavior change at 1,3,7,14,28 day after the injection with Longa score,mNSS score and beam walking test.5)We weigh the rats in both group at 1,3,7,14,28 day after the injection.Result: The rats of MCAO have a significant nerve dysfunction for the cerebral ischemia;The behavior score of the hAECs group has improved significantly than the control group.Conclusion: The injection of hAECs through the carotid artery can improve the nerve function of the MCAO model.Part Ⅱ:The study of mechanism about the improvement of nerve function by injecting hAECs through the carotid artery in MCAOObjective: To study the mechanism about the improvement of nerve function by injecting hAECs through the carotid artery in MCAOMeyhods: 1)To observe the change of cerebral infarction area by TTC staining.2)Use virus-GFP(Green Fluorescent Protein)to infect the hAECs,to observe whether hAECs cross the blood-brain barrier to the ischemic area by Fluorescence microscope.3)To observe the neural cell apoptosis by TUNEL.4)Use ELISA to test the changes of inflammatory factors and neurotrophic factor in cerebrospinal fluid.Result: TTC staining prompted the infarction area of the MCAO rats was significantly reduced in experimental group.We could find the hAECs(mark with GFP)in the frozen section of ischemic cerebral tissue.The number of the apoptotic neurons decreased in experimental group compared with control group detected by TUNEL.The level of IL-6 in cerebrospinal fluid decreased in experimental group,and the content of IL-10 and BDNF was increased.Conclusion: After injection of hAECs through the carotid artery of MCAO rat model,hAECs partly crossed the blood-brain barrier,promoting the amounts of anti-inflammatory factor and nerve growth factor,inhibiting the release of inflammatory cytokines,to reduce neuronal apoptosis and ischemic cerebral area.