Experimental Study On The Activation Of P38 Mitogen Activated Protein Kinase When Low Intensity Pulsed Ultrasound Promoted Human Periodontal Ligament Cells Osteogenic Differentiation

Objective: (1) In vitro cultured Human Periodontal Ligament Cells;(2) On irradiation of low intensity pulsed ultrasound, the number of Alkaline phosphatase,Osteocalcin,and calcium salt which was secreted by Human Periodontal Ligament Cells, When we added moderate SB203580 to the culture medium;(3) The effects of low intensity pulsed ultrasound on the activation of p38 Mitogen activated protein kinase;(4) When we added moderate SB203580 to the culture medium, the effect of low intensity pulsed ultrasound on the activation of p38 Mitogen activated protein kinase.Methods: (1)we cultured human periodontal ligament cells in vitro, observed cells'form, identified cells'origin, identified cells'purity,drew cell growth curve.(2)we used the fifth generation of human periodontal ligament cells to research.We added 10μl/L SB203580 to the culture medium of the experimental group, while adding 10μl/L dimethyl sulphoxide to the culture medium of the control group and blank group.Then, the experimental group and control group were irradiated by low intensity pulsed ultrasound, 90 mW/cm~2 ,20 minutes a day;the blank group was not irradiated.We tested the content of Alkaline phosphatase of culture solution and cell lysate at the eleventh day, we tested the content of Osteocalcin of culture solution at the fifteenth day and counted the quantity of calcium salt nodules by the method of alizarin red staining at the twenty-first day.(3) we used the fifth generation of human periodontal ligament cells to research. First,we used trypsin to digest the fifth generation of human periodontal ligament cell and inoculated them to six pore plate, 1*104/ml.When the coverage area of cells was about 60%,we used serum-free cell culture medium to cultured cells 24 hours.Then,we used low intensity pulsed ultrasound to irradiate the experimental group 0min,15 min,30 min,60 min,90 min,120 min and 6h, the control group was not irradiated.At last, we collected cells to detect the activation of p38 mitogen activated protein kinase by western blotting. (4) we used the fifth generation of human periodontal ligament cells to research. First,we used trypsin to digest the fifth generation of human periodontal ligament cell and inoculated them to six pore plate, 1*104/ml. When coverage area of cell was about 60%,we used serum-free cell culture medium to cultured cells 24 hours.Second, we added moderate 10μl/L SB203580 to the culture medium of experimental groups and added 10μl/L dimethyl sulphoxide to the culture medium of control groups.Third, we used low intensity pulsed ultrasound to irradiate experimental groups and control groups for 30 min. At last, we collected cells to detect the activation of p38 Mitogen activated protein kinase by western blotting.Results:(1) The content of Alkaline phosphatase of each group's culture solution and cell lysate was different.( Fcell lysate =1207,Fculture solution =334;P<0.05). The content of Alkaline phosphatase of the experimental groups'culture solution and cell lysate was the least , the content of Alkaline phosphatase of the blank groups'culture solution and cell lysate was less than that in the control groups'.(2)The content of osteocalcin of each group's culture solution was different.( F=28.2,P<0.05). The content of osteocalcin of the experimental groups'culture solution was the least , the content of osteocalcin of the blank groups'culture solution was less than that in the control groups'.(3) The quantity of each group's calcium salt nodules was different(F=46.458,P<0.05).We observed that there were so many red calcium salt nodules which were inequality in size in blank groups and control groups while using the method of alizarin red staining under the inverted microscope.The quantity of the experimental groups'calcium salt nodules was the least, the quantity of the blank groups'calcium salt nodules was less than that in the control groups'. (4) we used 90 mW/cm~2 low intensity pulsed ultrasound to irradiate the experimental group 0min,15 min,30 min,60 min,90 min,120 min and 6h and detected the activation of p38 Mitogen activated protein kinase by western blotting.We found that the gray values of p-p38 gradually increased from 0min to 30min,then decreased.The gray values of p-p38 was the highest at 30 min(F=170.01,p<0.05). The gray values of p38 was unchanged at each time point(F=0.748,p>0.05).(5) we used 90 mW/cm~2 low intensity pulsed ultrasound to irradiate the experimental group30 min and detected the activation of p38 mitogen activated protein kinase by western blotting( The choice of irradiation time consult'Results (4)'). We found that the gray values of p-p38 in experimental groups'was less than that in control groups'( F=2208.54 , P<0.05 ) .But the gray values of p-p38 was undifferentiated.Conclusions:(1) we used 90 mW/cm~2 low intensity pulsed ultrasound to irradiate the experimental group 0min,15 min,30 min,60 min,90 min,120 min and 6h and detect the activation of p38 Mitogen activated protein kinase by western blotting. We found that the gray values of p-p38 was the highest at 30 min(F=170.01,p<0.05). This point in time provide reference for our next experiment.(2) P38 MAPK may participat the Human Periodontal Ligament Cells osteogenic differentiation which low intensity pulsed ultrasound promoted(F=0.75,P>0.05). But we need a further study for the specific mechanism.