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Purpose: through observation adipose tissue of mesenchymal stem cells (ADSCs) to dopamine can neural element differentiation process wnt3a protein's expression change, explore Wnt3a in directional induction of neural stem cells to dopamine can neural element differentiation process, further research wnt signaling The pathway on the differentiation of neural stem cells for clinical cell transplantation in the treatment of the foundation of the new cell sources and cytology. Method: 1. Adipose-derived stromal cells in culture under aseptic conditions to take 2-4 weeks SD rats inguinal fat, adipose stromal cells isolated and cultured with reference Zuk PA, conventional cell culture, and medium was changed passages. Inverted phase contrast microscope cell growth and morphological changes in primary and passaged under continuous observation and photographic recording. 2 fat-derived mesenchymal stem cells to nerve cells will be the third generation of ADSCs by trypsin digestion, the to pass fourth generation of cells to the pre-preparation of cells seeded in 6-well culture plates placed in sterile coverslip, remove the culture solution, adding to the cells to grow to 80% confluency, inducing medium. Experiment was divided into five groups: A group for 8 hours induced neural induction medium (NIM), B group induced NIM group of 16 hours group C NIM induced a 24-hour group the group D induced NIM 32 hours group; control group cultured normal medium. Cells were observed under a microscope the growth and morphological changes in the inverted phase contrast and photographic records. Specific marker of nerve cells and neurotransmitter synthesis process of key enzymes detected in the specified time point using immunohistochemical method and indirect immunofluorescence detection of neural precursor cells specific marker Nestin protein (expression of Nestin), nerve cell specific markers of neural specific neuron-specific enolase (NSE), microtubule the joint protein 2 (MAP2) and dopamine synthesis process, a key enzyme tyrosine hydroxylase (TH) expression. 4 of Nurr1, TH Wnt3a protein in Real Time-PCR detection using Trizol step extraction after induction of total cellular RNA the UV spectrophotometer RNA A260/A280 value. Obtained cDNA, the mRNA from the total RNA by reverse transcription reaction and then the PCR reaction to amplify the target gene: a dopaminergic neuron precursor cell specific flag nurr1 of dopamine during the synthesis of the key enzyme tyrosine hydroxylase, TH, and Wnt initiation protein signaling pathway Wnt3a expression. PCR total reaction system of 10μl, the reaction conditions were: 94 ° C 20sec, 53 ° C 20sec, 60 ° C 40sec, 40cycles. The fluorescence curve results application Rotor-Gene 3000 real-time PCR instrument Comparative Delta-delta CT method for the analysis of each group, six samples to detect the gene expression results. HE staining of cells after inducing differentiation after induction of cell morphology to observe. 6 statistically rigorous proofreading before all data entry, statistical analysis SPSS13.0 statistical package Excel database collated. The obtained data were expressed as mean ± standard deviation. Different time points within the same group, the number of compared using one-way ANOVA, p lt; 0.05 for the difference was statistically significant. Microsoft Excel Software plotted. Results: 1. Primary and passaged cell growth and morphological changes in primary cells observed under an inverted microscope just after inoculation, rounded bright spot seen a lot of oil droplets and a small amount of connective tissue fibers, about four hours, a small number of cells adherent 24 hours was observed after the first exchange of medium, not adherent cells and impurities cells were removed, a large number of survival cells adherent, adherent cells triangular, polygonal, round or class fusiform, and gradually began to stretch, split, fusiform or polygonal, 3 ~ 5 d rapidly proliferating cells were set after 5 to 7 d colony growth, colony sizes, containing dozens to hundreds of cells ranging, and contains a small amount of round or oval round cells mixed growth. Passaged cell initial form rounded, suspended state, and soon adherent, just adherent cells were fusiform, polygonal or oval, 3 to 4 hours after the survival cells completely adherent, uniform distribution, and the original compared to the generation of cells, cell morphology more single, fusiform and proliferation significantly faster, more swirling growth area that covered the bottom of the bottle 4 to 5 days. 2 after the induction of cell growth and morphological changes Member inducing medium within 1 to 2 hours in each group on cell morphology change significantly. Cell shrinkage, cytoplasm to the nucleus to shrink to the sides extended gradually formed spherical cell body and the formation of protrusions, enhanced three-dimensional refraction increases. As time progresses, induced by NIM, ADSCs differentiate into nerve cells, gradually increases within a certain time, the number of differentiated neural cells, the formation of bipolar or multipolar cells, cell protrusions interwoven into a network. Specific marker of nerve cells and neurotransmitter synthesis process, a key enzyme in the expression (1) after the completion of the induction of differentiation, the control group did not find a positive expression of Nestin, the rest of the group were Nestin positive expression, and the highest in the Group D groups A, B, C, D, the difference was statistically significant (p lt; 0.05). (2) induction of differentiation is completed, the control group did not find a positive expression of NSE, the rest of the group were NSE positive expression, expression of the difference between the highest, A, B, C, D group and D group had a statistically significant significance (p lt; 0.05) (3) completion of the induction of differentiation, the control group did not find the positive expression of MAP2, the rest of the group were MAP2 positive expression to express the highest, A, B, C, D group, and Group D The differences were statistically significant (p lt; 0.05). (4) induced differentiation is complete, the control group was not found TH-positive expression, the rest of the group are TH positive expression, expression of the difference between the highest, A, B, C, D group and D group were statistically significant (p lt; 0.05). (5) the indirect immunofluorescence test results, in addition to the control group, the rest of the group were TH expression. HE staining results after the induction of ADSCs typical bipolar or multipolar cells visible by HE staining, the middle visible blue nucleus, cytoplasm, and projections for the red. Cell morphology presents typical neuron-like changes. Real Time-PCR detection Nurr1, TH and Wnt3a expression ADSCs induced differentiated cells of Nurr1, TH, Wnt3a mRNA expression results: Application NIM inducer ADSCs induced after the induction of the same gene mRNA in different groups between the average relative concentration of comparison, the differences were statistically significant differences (p lt; 0.05). Correlation analysis of Wnt3a, TH, Nurr1 PCR results, Wnt3a, respectively, consistent with TH, Nurr1, was positively related (p lt; 0.05 rTH = 0.957, rNurr1 = 0.979). Conclusion: 1. Induced by the NIM, ADSCs can differentiate into dopaminergic neurons, the nerve cells of each component into a positive rate compare D group was the highest, the difference between groups was statistically significant (p lt; 0.05). 2. Real Time-PCR to detect, in addition to the control group, the other groups are of Nurr1, TH and Wnt3a mRNA expression, and the expression of various markers are Group D markers group comparison, there were significant differences statistically significant (p lt; 0.05). 3. Wnt3a may promote neural stem cell differentiation, differentiated cells can express dopaminergic neuron specific markers to dopaminergic neurons.
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