The Influence To Mouse Osteoblast Cells By Metformin Hydrochloride In The Different Concentration Of Glucose

Object:To observe the influence to proliferation, apoptosis and expression of functional protein of mouse osteoblast cells by the different concentration of metformin hydrochloride.And dicuss the effect of PPARγin molecular mechanism.Method:Culture mouse osteoblast cells to 3rd generation。(1)Cells was divided into 8 groups, normal glucose control team(5.5mmol/L GLU+0umol/LMET), normal glucose 25umol/L metformin hydrochloride team ( 5.5mmol/L GLU+25umol/LMET),normal glucose 50umol/L metformin hydrochloride team( 5.5mmol/L GLU+50umol/LMET ),normal glucose 100umol/L metformin hydrochloride team(5.5mmol/L GLU+100umol/LMET), high glucose control team(25mmol/L GLU+0umol/LMET),high glucose 25umol/L metformin hydrochloride team ( 25mmol/L GLU+25umol/LMET ),high glucose 50umol/L metformin hydrochloride team(25mmol/L GLU+50umol/LMET),high glucose 100umol/L metformin hydrochloride team(25mmol/L GLU+100umol/LMET),after intervention of 48h and 72h,use MTT detect the inhibition ratio of proliferation,use Annexin V-FITC/PI flow cytometry detect apoptosis ratio, prolong the time of intervention to 1w-3w,use RIA detect the level of ALP and OCN,use RT-PCR detect the mRNA level of PPARγand BMP; use western blot detect the level of expression of PPARγprotein。(2)Then cells was divided into 4 groups, control team,1umol/L Compound C team ,100umol/L metformin team,1ummol/L Compound C and 100umol/L metformin team,Use immunofluorescent detect the different level of AMPK under the effect of metformin hydrochloride and Compound C after intervention 3w。Result:1.Between the same time teams,compare to normal glucose control team,the proliferation of high glucose teams are obviously suppressed,and the proliferation is enhanced by the increasing metformin hydrochloride concentration.2. The early apoptosis ratio of high glucose teams is higher than normal glucose teams. Metformin hydrochloride has no influence to the apoptosis, the proliferation of high glucose team is redouced by the increasing metformin hydrochloride concentration.3.In the same time of invention,compared to normal glucose team the expression of ALP,OCN,BMP-2 of high glucose team is decreasing,in metformin hydrochloride team, the expression of ALP,OCN,BMP-2 is enhanced by the increasing metformin hydrochloride concentration.4. In the same time of invention,the expression of PPARγmRNA of high glucose teams are increasing than normal glucose control team,and it's decreasing by higher metformin hydrochloride concentration.5. In metformin hydrochloride team,the expression of AMPK is higher than control team,and PPARγis lower. In compound C team, the result is opposite.Conclusion 1.High glucose environment can damage osteoblast cells,cause inhibition of its proliferation,induce its apoptosis,cause reduction of ALP,OCN,BMP-2,this effect related to the induction to the expression of mRNA and protein of PPARγin high glucose environment.2.However normal glucose or high glucose, metformin hydrochloride can also enhance the proliferation, and decrease apoptosis of osteoblast cells,and rise the expression of ALP,OCN,BMP-2.This effect related to the inhibition to the expression of mRNA and protein of PPARγby metformin hydrochloride.3.As an excitomotor of AMPK,its effect that reduce the expression of PPARγmay relate to AMPK,the activation of AMPK can inhibit the expression of PPARγpartly.