Experimental Study On Mechanism Of Malignancy Reversion In Poorly Differentiated Osteosarcoma Cell

Objective: To supply a good experimental model of studying osteosarcomadifferentiation mechanism by establishment of human osteosarcoma MG-63 cell clone strain with poorly differentiated capacities. The character of benign differentiation was detected , and study the molecular mechanism of malignancy reversion in poorly differentiated osteosarcoma cell strain .Methods: Six cell clone stains derived from human osteosarcoma MG-63 cell line were isolated and established by clone technique in vitro .we compared ,analyzed and identificated their differentiation character by using cell count .rate colony formation in soft agar,cell genetics .DNA content and Alkaline phosphatase(ALP) activity analysis. We selected cell clone strain 18 with poorly differentiated capacities among 23 cell clone strains. The character of benign differentiation and the mechanism of malignancy reversion were evaluated by MTT assay,transmission electron microscope,flow cytometer,RT-PCR,western blot, cell invasion assay in vitro, DNA ladder when poorly differentiated osteosarcoma cell was treated with 1μmol/LATRA.Result:There were remarkably diversity between 5 and 18 cell strain among 23 cell clone strains. The 18th clone strain are poorly differentiation with macronucles fusiform,uncontact inhibition, high cloning rate, hyper two ploidy and inferior Alkaline phosphatase activity, chromosome model number accumulate in 70-78. After treatment with 1μmol/LATRA ,the poorly differentiated MG-63 cell proliferation was inhibited,the cell showed benign differentiation in morphology and function, cell cycle arrested in G1, The relative expression levers of CD44v6,Dnmt and PCNA mRNA decreased ,but the lever of P16mRNA increased;At the same time , the expression levers of PKC and Cyclin Dlprotein and the capacity of cell invasion decreased remarkably.At last,the cell lost the anoikis resistence. Conclusion: Human osteosarcoma MG-63 cell clone strains with distinct differentiated capacities could be established by clone technique, cell proliferation in vitro, agarose clony-formation, DNA content and chromosome analysis. ATRA can induce differentiation of human osteosarcoma cell. The poorly differentiated osteosarcoma cell emerged mature trait after treatment with ATRA. The significance of Dnmt activity was elucidated in gene lever,cell cycle regulation and cell signal conduction during the poorly differentiated osteosarcoma cell were induced in vitro. Inhibition Dnmt activity in MG-63 cells may be closed to ATRA induced differentiation.