The Expression Of Wnt3a In The Process Of Adipose Tissue-derived Stromal Cells Differentiated Into Dopaminergic Neuron

Objective:This experiment aims to study the wnt3a expression changes in the process of rat adipose tissue-derived stromal cells(ADSCs) differentiating into dopaminergic neurons, discuss the role of Wnt3a in the process the induction of neural stem cells to dopaminergic neurons; Further explore the influence of Wnt Pathway on neural stem cell differentiation.So as to provide new cells source and cellular basis for clinical cell transplantation for treatment of PD,also better guide for clinical work.Methord:1. Isolation and cultivation of ADSCs:2-4 weeks SD rat inguinal fat was acquied under sterile conditions, Isolation and cultivation were according to the methord of Zuk PA, Inverted phase contrast microscope was used to observe morphology changes of ADSCs every day.2. Induction of rat ADSCs into neural cells:The tired serial passage cells were trypsinization and cultured. When the cells reached 80% confluence,we removed DMEM and added NIM. The experiment was separated into five groups:The control Group(only DMEM),Group A (NIM 8 hours),group B(NIM 16 hours),group C(NIM 24hours),group D(NIM 32hours), Inverted phase contrast microscope was used to observe morphology changes of ADSCs after induction.3. Identification of neural-specific makers and some key enzymes in the synthesis of neurotransmitter after induction:At each specified time point after induction,we adopted the means of immunocytochemistry and immunofluorescence assay to identify the neural-specific makers:Nestin,NSE,MAP2,the key enzyme in the Dopamine synthesis,Enzyme tyrosine hydroxylase TH.4. Detection of Nurr1,TH,Wnt3a:Extract total cellular RNA, we adopted the means of Real Time-PCR to detect the expression of Dopaminergic neuron precursor cells specific markers Nurr1,the key enzyme in the Dopamine synthesis Enzyme tyrosine hydroxylase TH,Starting protein of Wnt signaling pathway Wnt3a.5. HE staining was used to identify the differentiated ADSCs after induction.6. Statistical treatment:SPSS 13.0 software was used to analyse after excel database of initial data was established.The data obtained was represented as mean±SD.There was statistically significance if p <0.05.Result:1. Morphology changes of Primary and Passage retADSCs:We used inverted phase contrast microscope to observe the morphology of primary cells, The cells were round at first, Shows a large number of oil droplets and a small amount of connective tissue fibers, 4 hours or so that a small amount cells adherent.after the first 24 hours of exchange of medium,we can observe that dead cells and impurities without cells were removed and a large number of surviving cells adherent.Adherent cells were different forms,such as triangle,polygonal,round or spindle class. Then gradually began to stretch and split,As time went on,most cells growed quickly as colony and arranged as whirlpool. The shape of the cells after increasing passage was more single than before. The initial passage cells were round highlights at first, suspension, adherent soon, compared with primary cells, even a unified form. Proliferation significantly faster, aftetr 4-5days, cover the bottom of the bottle.2. Morphology changes after neuronal induction:After1-2hours of adding induction medium,many cells displayed retraction of the cytoplasm toward the nucleus,forming compact cell bodies with cytoplasmic extensions.The cell bodies became increasingly spherical with multiple cell processes.As time went on,many ADSCs differentiate into neural cells,In a certain period of time,differentiated neural cells increased. cell processes interwoven into a network.3. Expression of neural-specific makers and some key enzymes in the synthesis of neurotransmitter after induction:(1) Nestin expressed in each group except the control Group,The number of Nestin positive cells was the highest in the group D,There was significant difference between the group A,B,C,D(p <0.05).(2) NSE expressed in each group except the control Group,The number of NSE positive cells was the highest in the groupD,There was significant difference between the group A,B,C,D(p <0.05).(3) MAP2 expressed in each group except the control Group,The number of MAP2 positive cells was the highest in the group D,There was significant difference between the group A,B,C,D(p <0.05).(4) TH expressed in each group except the control Group,The number of TH positive cells was the highest in the group D,There was significant difference between the group A,B,C,D(p <0.05).(5) Indirect immunofluorescence test results shows:Nestin,NSE,MAP2,TH expressed in each group except the control Group.4. The result of HE staining:The body of the cells after HE staining were red with one or two processes, and the cell nucleus were blue.5. The result of Real Time-PCR:To induce rat ADSCs by NIM, then assess the relative concentration of mRNA in different group by detection of RT-PCR.The average relative concentration of Nurr1,TH,Wnt3a gene were significantly statistical difference(p <0.05). Make correlation analysis of PCR result, Wnt3a, TH, Nurr1 were consistent respectively, positive correlation(p<0.05, rTH=0.957, rNurr1=0.979).Conclusion: 1.ADSCs can differentiate into neural cells after NIM induce, In a certain period of time,differentiated neural cells increased, The number of positive cells was the highest in the group D,There was significant difference between the group A,B,C,D(p <0.05).2. The result of RT-PCR showed that,Nurr1,TH,Wnt3a expressed in each group, D group was the highest,There was significant difference between the group group A,B,C,D(p <0.05).3. Wnt3a may contribute to the differentiation of neural stem cells to dopaminergic neurons,it could express dopaminergic neurons specific markers after differentiation.