The Study Of Human Amnion Membrane Mesenchymal Stem Cell Transplantation For The Treatment Of Transgenic Mice With Alzheimer's Disease

Background and Purpose:The main symptoms of Alzheimer's disease are progressive memory decline,and with varying degrees of cognitive impairment, language and personality aspects of the anomaly.Age is the most important risk factor for AD,after the age of 70,for each additional 5 years of age,the risk of AD increases 1-fold,about 50%of people is in the risk of AD after 80 years old.As life expectancy increases,the world's increasingly aging population,the incidence of senile dementia is also increased.Alzheimer's has become the the fourth killer of mankind after heart disease,cancer,stroke.At present,researchers have produced a variety of animal models of Alzheimer's disease,hoping to find an effective cure for Alzheimer's disease.The major ones are the natural aging animal model,Aβinjection animal model,hypoxia-induced animal model of chronic ischemia animal model,animal model of cholinergic damage,animal model of aluminum poisoning,but the animal model to simulate only part of the pathological changes of Alzheimer's disease or the symptoms of Alzheimer's disease, and these animal models is expensive,difficult to keep it and easy death,thus the study of Alzheimer's disease has been greatly hindered,the most obvious advantage of transgenic animal model is to simulate the characteristics of AD neuropathology, including extracellular Aβdeposition,dystrophic neuritis components,glial hyperplasia.Transgenic mice may provide a better AD animal model for analysis the mechanism of Aβdeposition and effective therapy.At present,the treatment of Alzheimer's include cholinergic drugs,brain cell metabolism activator,blood circulation promoting agents,calcium antagonists, neurotrophic factors and antioxidants,but have poor efficacy.Zhang cultivate human embryonic stem cell into neural stem cells that have differentiation potential of a neuron,astrocyte,oligodendrocyte,after transplantation into the brain of neonatal rats, we found that these neural stem cells can be integrated into the receptor in mouse brain,and can differentiate into three kinds of nerve cells,there is a literature of human neural stem cells be transplanted to the lateral ventricle of rats of 24 months old,4 weeks later these cells migrate in an orderly manner to the cerebral cortex and hippocampus,and differentiate into nerve cells and the stars shaped glial cells,some cells can set up synaptic contact with host cells,and can significantly improve cognitive function in aging rats.At present,the neural stem cell transplantation has been widely used in the experimental research of variety of nervous system degeneration,after combined treatment of Alzheimer's disease in rats with the application of vitro cultured neural stem cells and brain-derived neurotrophic factor,people found that study ability and memory of rats can be promoted.Bone marrow stromal cell transplantation for the treatment of vascular dementia has also made much effect.The smaller immunogenicity Human amniotic mesenchymal stem cell is,may be the more suitable for allogeneic transplantation in the treatment of diseases of the nervous system research,but the treatment involved in human amnion-derived mesenchymal stem cells for Alzheimer's rarely reported at home and abroad.This Experimental Study is to discuss Human amnion-derived mesenchymal stem cells transplantation for the treatment of Alzheimer's disease.Methods:①Human amniotic mesenchymal stem cell extraction,purification, culture:Placenta was obtained from a normal full-term cesarean section specimen. The amnion was separated at aseptic condition and washed with PBS,pooled in a dish and cut into the small pieces.The samples were digested at the room temperature for 60 minutes by 0.25%Trypsin,and then digested by the collagenaseⅣof 1.0 g/L at medium 37℃for 2 h,then made into single-cell suspension,stained by typan blue, count living cells,then the cells were cultivated in 75ml bottle with intensity of 2×10~5个/ml.The Medium was VDMEM:VF12=1:1 of DMEM/F12 plus 10%fetal bovine serum(FBS),bFGF(final concentration of 20ng/ml),the cell culture bottles placed in incubator of 37℃,saturated humidity,volume fraction of 5%CO2 for 48-72h,then replace culture medium,dispose inadherent cells,change culture medium totally every 3 or 4 days.When cells reached 80%-90%integration,then digested by 0.25%trypsin,and carried out subculture according to the ratio of 1:3, and recorded as P1 generation.In the course of subculture,culture medium was totally changed every 3 days,until the adherent cells integrated with each other fully, and then repeat the above operation,cultured for P2 generation,and the rest by analogy.②Identification of Human amnion-derived mesenchymal stem cells:The Human amnion-derived mesenchymal stem cells were cultured to the third generation,the Nestine immunocytochemical staining.③Flow cytometry analysis of cell surface markers:The Human amnion-derived mesenchymal stem cells were cultured to the third generation,flow cytometry analysis of cell surface markers of CD40,CD80,CD86.④Experimental animal grouping:the mice were identified by PCR technology before divided into 3 groups,respectively for transplantation group,control group, normal group,the transplantation group for 10 APP~+ mice were equally divided between male and female,to give the treatment of human amnion-derived mesenchymal stem cell transplantation through the tail vein;the control group for 10 APP~+ mice were equally divided between male and female,to give the tail vein injection of saline;normal group for the 5 female APP~- mice and 4 male APP~- mice was not given any interventions.⑤Human amnion-derived mesenchymal stem cells taged in vitro:when amnion membrane mesenchymal stem cell was cultured to the third generation,before transplantation,Brdu was added into cell culture bottles of hMSCs with 3μg/ml final concentration,then hMSCs was cultured in incubator of 37℃,5%CO2 and saturated humidity for 24 hours.Washed 3 times by saline,digested by 1 ml0.25%trypsin for 30-60s,short-term low-speed centrifugation 3 times(1000prm,5min),removing cell debris,resuspended cells with saline,adjusting the cell concentration by 1×10~6/ml.⑥Human amnion membrane mesenchymal stem cells Transplantation through tail vein:after the first round evaluation of behavior of mice by morris water maze, the third generation of amnion membrane mesenchymal stem cells were made into single cell suspension with concentration of 1×10~6 cells/ml,0.5ml cell suspension was injected into mice with a capacity of 1ml syringe though tail vein in transplantation group,injection time is longer than a minute and after the completion of injection needle 10 seconds.Control group of mice given the same volume of saline injection,normal mice not given interventions⑦Mice Determination of spatial learning ability(morris water maze)Place navigation test:Before the test,put animals in pond(not including the platform),allow them to swim freely for two minutes to become familiar with the environment of the maze.Two rounds of tests were carried out,respectively before cell transplantation and 15 days after transplantation,each round of experiment lasted a total of 8 days,testing one time every two days,a total of 4 times,each time testing each animal 4 times,respectively,Add mouse facing the wall beside the pool in the mid-point of four quadrant,4 points in random order.Record the escape latency time that mouse into the water to find and climb the platform.If the mouse did not find the platform within 120 seconds,then the experimenter lead them to the platform, recorded as 120 seconds.Allow the mice to rest on the platform for 30 seconds after they find platform then the next trial,the escape latency of Each mouse was the arithmetic average of 4 times that of mice trained.Space exploration test:to remove the platform at nineth day,put the mouse into pool facing the wall twice at two randomly point,the camera recorded mice swimming path in the water within two 2min separately,computer analyse the average time of mouse swimming at the platform quadrant and the average number of through the corresponding locations of original platform.⑧β-amyloid protein expression in mice brain were observed by stained brain slices in each group:before the first round of water maze test,model mice and normal mice was randomly taken,and one month after cell transplantation each experimental group of mice were randomly taken for sacrificed,checkβ-amyloid protein expression of mice brain in three groups by methanol Congo red staining of consecutive coronal brain tissue slices.⑨Check Brdu expression by staining mice brain slices:one month after cell transplantation,transplantation mice were sacrificed,wax-embedded block,check Brdu expression by immunohistochemical staining of mouse brain consecutive coronal brain tissue slices.⑩Check GFAP,CHAT expression in brain slices of mice in each group:one month after cell transplantation,mice of experimental groups were sacrificed,check GFAP,CHAT expression in each group by immunohistochemical staining of mouse brain consecutive coronal brain tissue slices.Results:①The amniotic-derived cells begun to attach in 24 h.When observed at 72 h,cells displayed in round,spindle,star and polygon shapes.The amniotic-derived cells reached confluence about 80%～90%in 6-7 days.When passage to the next generation,the amniotic-derived cells achieved complete confluence in 3-4 days.when passage to the third generation,cell morphology is very uniform,long-spindle and have pseudopodia.②Immunocytochemical staining shows that human amnion membrane mesenchymal stem cells express Nestine positive,with brown cytoplasm,nucleus was stained blue③Flow cytometry analysis of cell surface markers:Human amnion membrane mesenchymal stem cell surface have low expression of CD40,CD80,CD86.④In the place navigation test,before transplantation,between transplantation group and control group there is no significant difference(P＞0.05),between transplant group and the normal group the difference is significant(P＜0.05);after transplantation,between transplantation group and control group difference is significant(P＜0.05),between transplantation group and normal group there was no significant difference(P＞0.05).In explore experiment,there was no significant difference among 3 groups of mice before and after transplantation(P＞0.05).⑤Methanol Congo red stained mouse brain coronal slices shows that,before transplantation,more patch-likeβ-amyloid protein deposition can be seen in model mouse brain,and was dyed pink.Butβ-amyloid deposition is almost can not be seen in normal mouse brain.One month after cell transplantation,more patch-likeβ-amyloid protein deposition can still be seen in white matter of mice brain of the transplantation group,and were dyed pink,but the quantity had a significant decrease than the control group,area of singleβ-amyloid protein clumps significantly reduced than the control group,there is almost noβ-amyloid protein deposition in the normal mouse brain.⑥Brdu immunohistochemical staining of mouse brain coronal slices shows that there is Brdu positive cells exist in the transplantation team,nuclei was stained brown, cytoplasm was blue,positive cells gathered into a mass,there is no regularity of distribution.⑦One month after transplantation,the difference of mouse brain GFAP,CHAT expression was significant between the transplantation group and the normal group (P＜0.05);there was statistically significant difference(P＜0.05) compared with the control group.CONCLUSION:①MSCs can be isolated from amnion by trypsin and collagenaseⅣ,amniotic membrane MSCs can amplified in vitro in DMEM/F12 medium which contain 10%FBS and 20μg/L bFGF.②Human amnion membrance mesenchymal stem cells labeled in vitro by Brdu can survive and migrate to the brain when transplanted to the body of Alzheimer's disease transgenic mice via the tail vein and play a role.③Human amnion membrane mesenchymal stem cell transplantation could reduceβ-amyloid deposition in Alzheimer's disease transgenic mouse brain. ④Human amnion membrane mesenchymal stem cell transplantation can improve spatial learning and memory capacity of Alzheimer's disease transgenic mice.⑤The expression of GFAP,CHAT changes in mouse brain could be an effective empression of human amnion membrane mesenchymal stem cells transplantation for the treatment of Al zheimer's disease transgenic mice.