The Relationship Between Mitochondrial Apoptosis And Autophgy In The Process Of Adipose-derived Stromal Cells Differentiation Into Astrocytes

Objectives To research the relationship between mitochondrial apoptosis and auophgy inthe process of ADSCs differentiation into astrocytes in vitro, so as to increase the numberof ADSCs-derived astrocytes. Thus, it has important significance for further research.Methods1According to the method by Ye et al, ADSCs were isolated and cultured, thenthe morphology of ADSCs were observed.2ADSCs with passages3were induced intoastrocytes for48hours and7,14,21days by induction medium containing IBMX. Themorphology of cells waere observed.3Immunocytochemistry and Western-blotting wereused to analyze the expression of GFAP, Bcl-2, Bax, Cyt-c, Caspase-3and LC3.4Flowcytometry Annexin Ⅴ/PI double staining assay was used for quantification of the numberof apoptotic cells.5TEM was used to observe the ultrastructure of mitochondria, apoptosisand autophgy in the process of ADSCs differentiation into astrocytes for14days.6MTTassay was used to detect the survival state of cells in the process of ADSCs differentiationinto astrocytes.7All experimental data were compiled with Microsoft Excel2003.Statistical analyses were performed with SPSS software (version17.0). Measurement datawere expressed as Mean±standard deviation (SD). Intragroup differences were comparedwith one-way analysis of variance. Values of P <0.05were considered to be statisticallysignificant.Results1Primary ADSCs adherent in24h. There were a large number of long spindlecells with whorled arrangement when cultured for7-10days.2When induced for48h, partsof the cell bodies stretched out slender processes and multiple branches. Having beeninduced for7days, some of the cells showed typical astrocytes morphology. When inducedfor14days, the cell morphology had no significant changes compared with the7th day.Induced for21days, the number of cells was significantly reduced and cell protrusions wereshorter and less.3There was no positive expression of GFAP in uninduced ADSCs, butafter induction for48h and7,14and21days GFAP showed positive expression. Positiveparts were mainly located in cell bodies and protrusions. It was increased gradually with theinduction time extending and reached a peak on the7th day (P <0.05). Bcl-2, Bax, Cyt-c,Caspase-3and LC3in uninduced and induced cells were all positive expression and positiveparts were mainly located in the cytoplasm surrounding the nucleus, protrusions were not obvious. The expression of Bcl-2between uninduced ADSCs and induced for48h and7,14,21days were significant differences (P <0.01) and decreased with the induction time (P <0.05). The expression of Bax, Cyt-c, Caspase-3and LC3were all increased gradually withthe induction time extending and reached a peak on the14th day (P <0.05; P <0.05; P <0.05; P <0.05).4Western-nlotting showed there was no expression of GFAP in uninducedADSCs. It was increased gradually with the induction time extending and reachaed a peakon the7th day (P <0.05). The expression of Bcl-2between uninduced ADSCs and inducedfor each time point was significant difference (P <0.01) and decreased with the inductiontime (P <0.05). The expression of LC3Ⅰwas decreased gradually with the induction timeextending (P <0.01). The expression of Bax, Cyt-c, Caspase-3, LC3Ⅱ was increasedgradually with the induction time extending and reached a peak on the14th day(P <0.05; P<0.05; P <0.05; P <0.05).5When ADSCs differentiation into astrocytes for14days, thephenomenon of apoptosis and autophgy existed in some cells.6The survival ratiodecreased gradually, the rate of early apoptosis, and the rate of late apoptosis or necrosis allincreased gradually with the induction time extending.7MTT showed that the survivalquantity of cells decreased gradually.Conclusions1The induction and differentiation of ADSCs to astrocytes by the inductionapproach mainly containing IBMX could be terminated when induced for7~14days, andthe differentiated cells could be isolated and stored for further application and research.2Apoptosis and autophagy generated by mitochondrial signaling pathways was weak inADSCs.3In the process of ADSCs differentiation into astrocytes, the mitochondrialapoptosis and autophagy were significantly enhanced, but this selective autophagy couldnot clear all the damage mitochondria leading to mitochondrial apoptosis.