| Objectives To research the relationship be tween Bcl-2/Bax regulatory proteins and mitochondria apoptosis in the process of ADSCs differentiation into neurons in vitro, so as to increase the number of ADSCs-derived neurons. Thus, it has important significance for further research.Methods 1 According to the method by Ye et al, ADSCs were isolated and cultured, then the morphology of ADSCs were observed through Inverted phase contrast microscope. 2 ADSCs with passages 2, 3 were induced into neurons for pre- induction and 1, 3, 5, 8 hours by induction medium containing β-mercaptoethanol. The morphology of cells were observed. 3 Immunocytochemistry and Western-blotting were used to analyze the expression of Nse, Bcl-2, Bax, Caspase-9, Cyt-c and Caspase-3. 4 Endoplasmic reticulum ultrastructure characteristics of induced ADSCs were observed by transmission electron microscope during the process of ADSCs differentiation into neurons. 5 Laser Scanning Confocal Microscope was used to detect mitochondrial fluorescent expression of the living cells of each group during the process of ADSCs differentiation into neurons. 6 Flow cytometry Annexin Ⅴ/PI double staining assay was used for quantification of the number of apoptotic cells. 7 All experimental data were compiled with Microsoft Excel 2003. Statistical ana lyses were performed with SPSS software(version 17.0). Measurement data were expressed as mean ± standard deviation(SD). Intragroup differences were compared with one-way analysis of variance. Values of P<0.05 were considered to be statistically significant.Results 1 Primary ADSCs adherent after about 24 hours, and cellular morphology diverse. There were a large number of long spindle cells with whorled arrangement when cultured for about 10 days. 2 When pre-induced for 24 h, cells differentiated, and stretched out small bumps. When induced for 1h, karyon became big and round and cytoplasm retraction, parts of the cell bodies stretched out slender processes, like neuron axon structure. When induced for 3h, cell cytoplasm refraction strengthened, processes further extended. When induced for 5h, cell cytoplasm refraction further strengthened, the cells had typical neuron morphology. When induced for 8h, cells had no significant changes compared with the 5h, the number of cells was significantly reduced and cell protrusions were shorter and less. 3 Immunocytochemistry showed the cells weakly expressed NSE in non- induced group, andpositively expressed in the other groups, the expression ratio of NSE(83.60±3.20 %)peaked at the 5th h(P<0.05), but there has no significant difference between the 5th and 8th h(P>0.05). The expression of Bcl-2 was gradually lower with extending time, and peaked in non- induced group(P<0.05). However, the expression of Bax, Caspase-9, Cyt-c and Caspase-3 were gradually higher, and peaked at the 8th h(P<0.05). 4 Western-blotting showed the cells weakly expressed NSE in non- induced group, and positively expressed in the other groups, the expression level of NSE(1.040±0.01) peaked at the 5th h(P<0.05), but there has no significant difference between the 5th and 8th h(P>0.05). The expression of Bcl-2 was gradually lower with extending time, and peaked in non- induced group(P<0.05). However, the expression of Bax, Caspase-9, Cyt-c and Caspase-3 were gradually higher, and peaked at the 8th h(P<0.05). 5 Transmission electron microscope showed cells at the 5th h karyopyknosis, chromatin edge set, mitochondria swelling and cavitation. 6 the mitochondrial fluorescent expression was obviously lowered with extending time by laser scanning confocal microscope(P<0.05). 7 The survival ratio decreased gradually, the rate of early apoptosis, and the rate of late apoptosis or necrosis all increased gradually with the induction time extending.Conclusions In the differentiation process of adipose-derived stromal cells into neurons, after the 5th h, cells differentiated into typical neurons, before the 5th h, cells showed strong Bcl-2 anti-apoptosis ability, and weak Bax apoptosis-promoting ability. Bax could effect on mitochondrial membrane, produce mitochondrial membrane potential, then C yt-c was released into cytoplasm, and triggered mitochondria apoptosis. |
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