The Relationship Between Apoptosis Pathway Of Death Receptor And The Differentiation Of Adipose Derived Stromal Cells Into Neurons

Objectives To investigate the relationship between FAS/FASL, TNFR1/TNF-α, and DR5/TRAIL signaling pathways in apoptosis and the differentiation of adipose derived stromal cells(ADSC) into neurons in vitro. To improve the induction efficiency and to increase the survival cells number after the induction provide a theoretical basis, and then for the neurodegenerative disease cell transplant to provide new ideas and strategies.Methods 1 ADSC were isolated and cultured. 2 ADSC of passages 3 were induced into neurons for pre-induction and 1 h, 3 h, 5 h, 8 h by inducer buffer containing β-mercaptoethanol. Using an inverted phase contrast microscope to observe the morphological changes of the cells. 3 Immunocytochemistry method were used to detect the expression of NSE, FAS/FASL, TNFR1/TNF-α, DR5/TRAIL, Caspase8, and Caspase3 in the death receptor-mediated apoptosis pathway, during ADSC uninduction, preinduction and induction for 1 h, 3 h, 5 h, 8 h. 4 Western-blotting method were used to detect the expression of NSE, FAS/FASL, TNFR1/TNF-α, DR5/TRAIL, Caspase 8, and Caspase 3 in the death receptor-mediated apoptosis pathway, during ADSC uninduction, pre-induction and induction for 1 h, 3 h, 5 h, 8 h. 5 Cells viability detected by MTT assay. 6 Annexin V/ propidine iodide double staining assay for apoptosis state. 7 Statistical analysis: SPSS13.0 statistical software package was used for statistical analysis. All the experimental data are expressed as the mean ± SD. One-way analysis of variance was used to compare the intragroup differences. SNK-q test were used to compare the com Parisons between groups. The test level take bilateral α = 0.05. P < 0.05 was considered to be statistically differences.Results 1 Upon being separated and extracted from adipose tissue, ADSC were completely adherent at 24 h. Cells became short spindle, long spindle, round, triangular or irregular in shape. After adhering, cells began to proliferate into a large number of long spindle cells with swirl-like growth. 2 In the pre-induced group, the cells began to differentiate, projecting a small number of short projections. The cells in formal induction for 1 h that the nucleus was large and round, and the cytoplasm was slightly retracted, and the process was increased compared with that of the pre-induced group. In 3 h group, extracellular space showed obvious halos, and projections became further elongated and increased, woven into a mesh. In 5 h group, the cell refraction was further enhanced, the extracellular space showed an obvious halo, at the end of cell processes a branch of dendritic cells appeared, and the cells showed the typical morphology of neurons. In the 8 h group, portions of the cell body were retracted, the cell protrusions became shorter, the number of live cells was significantly reduced compared with the 5 h group. 3 Immunocytochemistry showed there was no positive expression of NSE in the uninduced ADSC. Expression site is in the cytoplasm and projections. The positive rate of cells that were induced for the 5 h group was higher than in the 1 h、3 h group(P <0.05) and the 8 h group was not significantly different from than the 5 h group(P>0.05). FAS, FASL, TNFR1, TNF-α, DR5, TRAIL in uninduced cells, pre-induction cells, and induced cells at 1 h, 3 h, 5 h, or 8 h were positively expressed. Expression site is in the cytoplasm and projections. With the induction of reaction time, the expression levels were significantly increased gradually, in which FAS, FASL peaked at 8 h group(P<0.05), TNFR1, TNF-α peaked at 5h group(P<0.05), DR5, TRAIL peaked at 5 h group(P<0.05). The expression of Caspase 8 gradually increased with the extension of time, peaked at 8h(P<0.05). The expression of Caspase 3 gradually increased with the extension of time, peaked at 8 h(P<0.05), and the 8 h group was not significantly different from than the 5 h group(P>0.05). 4 Westernblotting showed the cells no expressed NSE in non-induced group, and positively expressed in the other groups. The positive rate of cells that were induced for the 5 h group was higher than in the 1 h、3 h group(P<0.05) and the 8 h group was not significantly different from than the 5 h group(P>0.05). FAS, FASL, TNFR1, TNF-α, DR5, TRAIL in uninduced cells, pre-induction cells, and induced cells at 1 h, 3 h, 5 h, or 8 h were positively expressed. With the induction of reaction time, the expression levels were significantly increased gradually, in which FAS, FASL peaked at 8 h group(P<0.05), TNFR1, TNF-α peaked at 5 h group(P<0.05), DR5, TRAIL peaked at 5 h group(P<0.05). The expression of Caspase 8 gradually increased with the extension of time, peaked at 8 h(P<0.05). The expression of Caspase 3 gradually increased with the extension of time, peaked at 8 h(P<0.05), and the 8 h group was not significantly different from than the 5 h group(P>0.05). 5 The absorbance of the living cells in uninduced group, the pre-induction group, and the induced group at 1 h, 3 h, 5 h, and 8 h gradually decreased by MTT(P<0.05). And there is no statistical difference between the uninduced group and the preinduction group(P>0.05), as well as in the induced 1 h group and 3 h group(P>0.05). 6 Flow cytometry test results showed that the cells viability was decreased with the extension of induction time in the process of reaction, but the rate of early apoptotic rate and late apoptotic rate gradually increased with the extension of induction time.Conclusions Death receptor-mediated apoptosis was a central cause of cell death during the process of ADSC differentiation into neurons. Before differentiation for 5 h, the effects of the TNFR1/TNF-α and DR5/TRAIL signaling pathways was significant, while after differentiation for 5 h, the effect notably decreased. Meanwhile, the FAS/FASL apoptosis pathway was notable throughout the entirety of differentiation.