The Effects Of Developing Apical Complex Conditioned Medium And Lithium Chloride On The Proliferation And Differentiation Of Adipose Tissue-derived Stem Cells

AIM To separate and culture the developing apical complex (DAC) cells,collect the supernatant of the primary DAC cells and prepare different conditionedmediums to culture adipose tissue-derived stem cells (ADSCs). To discuss the effects ofdifferent DAC conditioned mediums (DACCM) and the effects of Licl (lithium chloride)at different concentratons on the proliferation and differentiation of ADSCs tentatively.To find the optimal concentration of Licl on proliferation and differentiation of ADSCs.METHODS DAC cells, separated from the postnatal20d Spraguee Dawley（SD）rat,were cultured by tissue block method combined with enzyme digestion method. DACtissue was identified by eosin Staining (HE) and DAC cells was identified byimmumohistochemical staining. The supernatant of the primary DAC cells wascollected and prepared to different conditioned medium by filtered and unfilteredmethods (DACCM-F and DACCM-UF). The proliferation ability of ADSCs wasdetected by MTT. The protein activity and RNA expression of alkaline phosphatase(ALP) was detected by ALP detection Kit and reverse transcription-polymerase chainreaction (RT-PCR). Meanwhile, the effect of Licl on the proliferation was detected byRT-PCR at different concentrations, the protein activity of ALP was detected by ALPdetection Kit and the RNA expression of ALP and bone sialoprotein (BSP) weredetected by RT-PCR. SPASS13.0was used for data analysis.RESULTS DAC cells were successfully separated from the developing roots of the SDrat and cultured in this research, which were CK-14and vimentin positive cells. MTT showed the proliferation ability of ADSCs cultured in DACC-UF perior to the cellscultured in α-MEM and DACCM-F as well as the protein activity and RNA expressionof ALP（P<0.05）. MTT shows that Licl at the concentration of5mM significantlypromote proliferation of ADSCs obviously higher than the other concentrations（P<0.05）and Licl at the concentration of40mM significantly inhibit the proliferationof ADSCs（P<0.05）, Detection of the activity of the ALP shows that Licl at theconcentration of5mM significantly promote differentiation of ADSCsapparently higher than the other concentrations as well as the results of RT-PCR（P<0.05）and Licl at the concentration of40mM significantly inhibits differentiation ofADSCs.CONCLUSIONS: The DAC cells cultured by tissue block combined with enzymedigestion method were composed of epithelial cells and fibroblast-like cells; theunfiltered supernatant of the primary DAC cells prepared to conditioned medium wascontributed to the proliferation and differentiation of ADSCs and the negative influenceof harmful metabolites on ADSCs was eliminated. Meanwhile the preparation procedureof conditioned medium was simplified. Meanwhile, the activator of Wnt signalingpathway Licl can promote proliferation and differentiation of ADSCs at appropriateconcentrations and It will be a promising candidate for tissue regeneration especially thebone and periodontal tissues regeneration.