The Nerve Nutrition And Glutamate Clearance Function Of Adipose Stromal Cell-Derived Astrocytes

Objectives To explore whether ADSC-derived astrocytes qualify the nerve nutrition and glutamate clearance function,which are both unique to mature astrocytes.Methods 1 According to the method described by Zuk PA and Ye CQ,ADSC were extracted from subcutaneous adipose tissue of healthy adult and then cultured.2 When the third-generation ADSC grew well,they were added with DE-1 induction medium for 48 h,7,14 and 21 days,respectively.The morphology of the cells in each group were observed using inverted phase contrast microscope.3 Immunocytochemistry assay,immunofluorescence assay and Western blotting were used to detect the expression of GFAP,GDNF,GLT-1 and GS of the cells in each group.4 MTT assay was used to explore the influence of different glutamate concentration to survival rates of each group.Meanwhile,the “optimal glutamate concentration” was detected for the future use.5 The glutamate concentration of the solution was detected using Glutamate Colorimetric Assay Hit to explore whether the cell in each group have the influence of the glutamate concentration in extracellular matrix.6 All experimental data was analysed using SPSS19.0 software.P value of less than 0.05 was considered to be significantly different.Results 1 The shape of primal ADSC cultured for 24 hours were long spindle or oval.There were lots of long spindle cells which showed uniform in morphology and whorled in arrangement when cultured to the third generation.2 After induction for 48 hours,the bodies stretched out some short branches,the cytoplasm rebounded in the nucleuscentered form.When having been induced for 7 days,the cells were interconnected into a network,with its multiple slender branches stretching out from the cytoplasm.When induced for 14 days,more slender branches were observed.Induced for 21 days,the cells shape were triangular or irregular,with a few short branches stretching out from cell bodies,among which some branches were fractured.3 The results of immunocytochemistry and immunofluorescence assay indicateds that GFAP,GDNF,GLT-1,GS positive cells and GFAP/GDNF,GFAP/GLT-1,GFAP/GS co-positive cells all could not be seen in uninduction group.But the cells above could all be seen in the induction for 14 h,7d,14 d and 21 d groups and the positive parts were widely located in cells.As the inducing time extension,the positive expression ratios of GFAP,GLT-1 and GS all increased gradually and reached to the peak on the 14 th day while GDNF reached to the peak on the 7th day;there exist significant difference in each group(P<0.05,respectively).4 Western blotting indicateds that there were no expression of GFAP,GDNF,GS and GLT-1 in uninduction group,while the expression of them could all be detected during ADSC differentiation into astrocytes.With the inducing time extending,the expression levels of GFAP,GS and GLT-1 all increased gradually and reached to the peak on the 14 th day while GDNF reached to the peak on the 7th day;there exist significant difference in each group(P<0.05,respectively).5 MTT assay result indicated that the “optimal glutamate concentration” was 130?m/L.In uninduction group,the glutamate concentrations of supernatant(collected when the cells were cultured for 1,2,3 and 4h in 130?m/L glutamate solution)existed no significance compared with control concentration,respectively(P>0.05,respectively).The same went for 21 d group,respectively(P>0.05,respectively).In 48 h group,the glutamate concentrations of supernatant were significant lower compared with control concentration,respectively(P<0.05,respectively);besides,the glutamate concentrations decreased gradually as time extending.The same went for in 7d and 14 d groups(P<0.05,respectively).Moreover,the glutamate concentration decreased prominently in 14 d group when compared with the degree of decrease in other groups.Conclusions 1 When having been induced for 7-14 days,the cells were similar in morphology like mature astrocytes.2 ADSC-derived astrocytes expressed GDNF,which reached to the pink when induced into astrocytes for 7 days.3 ADSC-derived astrocytes expressed GLT-1 and GS,and had the function of clearing glutamate in extracellilar matrix,which were prominent when induced into astrocytes for 14 days.