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

Objectives This study aimed to investigate the function of Fas/Fas L, TNFR1/TNF-?,DR5/TRAIL signal pathways in apoptosis during the reaction of ADSCs differentiation into astrocytes. So as to find a better environment for the growth of astrocytes, the number has increased, and the survival period, it is of epoch-making significance in clinical treatment.Methods According to the method by Ye Changqing and Zuk et al, the adipose tissue in suitable external environment on the digestion and train to become adipose stromal cells,The morphological changes of adipose derived stromal cells were recorded every day.ADSCs were induced into astrocytes for pre-induction and 48 h ? 7d ? 14 d ? 21 d by medicine. And record the characteristics of the morphological changes. Analyze the expression of GFAP?Fas ?fas L? TNFR-1? TNF? ?DR5 ?TRAIL ?caspase8 and caspase3. The early apoptosis rate of cells was detected by Annexin/PI double staining with cell flow cytometry. The survival state of astrocytes use MTT assay to detect. Using Microsoft Office Excel 2007 storage consolidation experimental data, with statistical software SPSS13.0 statistical analysis of all data. Mean ± standard deviation(SD) expresse as measurement data. with one-way analysis to compare between groups of data.Results The primary adipose derived stromal cells derived from 24 h were almost completely adherent to the wall. A variety of cells can be seen under the microscope. By the tenth day, a large number of long spindle shaped cells were seen to be arranged in a spiral shape. When induced for 48 h, parts of the cell bodies stretched out slender processes.7th day, some of the cells showed typical astrocytes morphology. When induced for 14 days, the cell morphology had no obvious changes compared with the 7th day. The number of cell protrusions were shorter and less in 21 th. Immunocytochemistry showed that different from the other group the cells all negative expression of GFAP in ADSCs. The positive expression of particles can be seen in the whole cell body and the protrusion parts.The expression of Fas/Fas L, TNFR1/TNF?, DR5/TRAIL, caspase3 and Caspase8 was observed in every point time, the positive expression sites were mainly located in cytoplasm, but there was no obvious positive expression in the process. The positive expression rate of Fas and Fas L was peaked in 7d, there was no significant difference between 7d and 14d(P > 0.05). The positive expression rates of TNFR1 and TNF? in ADSC, induced for 48 h, 7d, 14 d and 21 d were significantly different between each group(P <0.05). The positive expression rate of DR5 was peaked in 7d, while there was no significant difference between 48 h group and 21 d group(P>0.05). The positive expression rate of TRAIL was peaked in 7d, and in every group were significantly different(P<0.05).The positive expression rate of caspase8 was gradually increased with the induction time.The positive expression rate of caspase3 reached the peak in 7d. 4 Western-blotting: The expression level of GFAP reached a high peak in the 7th day. The expression level of Fas and Fas L reached the peak at 7d, 14 d and 7d were not significantly different(P>0.05).The expression level of TNFR1 and TNF? increased with the extension of the induction time, there were significant differences among the time groups(P<0.05), and reached the peak in the 21 d. The expression level of DR5 reached the peak value at 7d and was significantly higher than that in 14 d group(P<0.05). The expression level of TRAIL was significantly different among all groups and reached the peak at 7d. The expression level of caspase8 was significantly different between different time groups(P < 0.05), and gradually increased with the prolongation of the induction time. The expression level of caspase3 in 7d group was significantly higher than that in 14 d group(P<0.05). The early apoptosis rate of the cells increased with the induction time. MTT: The survival rate of cells decreased with the induction time.Conclusions Death receptor apoptosis pathway is an important cause of apoptosis, in ADSC induced differentiation into astrocytes, particularly the TNFR1/TNF? pathway.Although the Fas/Fas L and DR5/TRAIL signaling pathways were important pathways of apoptosis before induction for 7d, the signaling pathway of Fas/Fas L had a significant peak stage in 7d, the role of this pathway in the 14 d was significantly reduced. The effect of DR5/TRAIL pathway in 7d was significantly decreased and there was no obvious peak period.