Research On The Cell Cycle And Differentiation Of Osteoblastic Cells Transfecting By PEN Loading Oligodeoxynucleotid

Oligodeoxynucleotides(ODN)can be found in the degradation products of DNA or RNA in nature.It is a single-stranded oligonucleotides which is easy to relative with their complementary strand.The number of nucleotide it contains is usually under 50.Some studies have shown that some specific sequences of ODN could regulate the expression of IL-12 through toll-like receptors(Toll like receptor 9,TLR9)thereby inhibiting osteoclast differentiation.MT01(a particular sequence of ODN)is a compound designed on the basis of human mitochondrial DNA,which contains 27 bases.According to previous research findings,MT01 can promote the differentiation of osteoblast and activate osteoblast in the process of their formation and activation.It also has effect on alveolar bone remodeling.Objective:The main way for cells to absorb MT01 was endocytosis.However,the surface of cell membrane was negative charged.ODN was also negatively charged.Therefore in the process of uptaking ODN,they are susceptible to produce electrostatic rejection.At same time ODN is not stable in internal environment,easy to be degraded by nuclease.Thus the intake rate of ODN reduces greatly,influencing the clinical application prospect.In order to solve the problems of the lower intake rate,this experiment adopts a new vector PEN to pass ODN to test the changes on the cell cycle and differentiation of osteoblast cells.Materials and methodsThis experiment tend to use three different kinds of ratio 1:2?1:4?1:6 to load MT01 and PEN forming MT01 / PEN complexes.Respectively set up PEN,MT01 and S-MT01 three groups as control group,use the flow cytometry instrument to test the impact on cell cycle of MG63.To detect the differentiation related proteins such as ALP activity,use ELISA method to detect BMP2,OCN protein expression.ResultsThe results of cell cycle: compared with the negative control group,the proportion of cells in MT01/ PEN complex in Gl and G2 phase decreased,while the proportion of cells in S phase increased(P<0.01).Compared with S-MT01 group,MT01/ PEN complex group in G2 phase reduced,as for cells in S phase and G1 phase(P<0.01),the proportion of cells increased(P<0.01).ALP Results: compared with negative control group,the activity of ALP in MT01 group increased significantly(p<0.01),compared with S-MT01 group,MT01/PEN compound with 1:4 and 1:6 ratio in 24 h,72h increased(p<0.01),and in the 48 h,three ratio of MT01/PEN cause ALP activity of MG63 increase(p<0.01).BMP2 test results: compared with the negative group,the expression of BMP2 increased and MT01,S-MT01 group,MT01/ PEN compound expression increased in 24 h.In 72 h,compared with MT01 group and S-MT01 group,the expression of 1:2 and 1:6 MT01/ PEN complex increased.The results of OCN: compared with other groups,the expression of OCN increased except in 72 h.The expression of OCN with 1:6 complex was higher than that in MT01 group(p<0.05)in 24 h and 48 h,the expression of OCN had no significant difference when different proportion of MT01/ PEN group complexes compared with MT01 group and S-MT01 group.ConclusionPEN can be used as an efficient carrier to improve the transfection efficiency of MT01.MT01/PEN composite with different proportion may have effect on the assembly of proliferation and differentiation of osteoblast cells.The proportion of 1:6 in the assembly is of the most obvious effect.MT01/PEN complex can influence the cell cycle of MG63 cells into S phase,shorten the time to promote the proliferation and the promoting effect is obviously better than that of MT01,S-MT01.MT01/PEN complexes can increase the expression of osteoblast related factors such as ALP,OCN,BMP2.And its effect is better than that of MT01,S-MT01.