Effect Of Simvastatin On Synchronized Human Dental Pulp Cells (HDPCs) Proliferation

Backround and objectiveHuman dental pulp cells (HDPCs) is a fibroblast like pluripotent cells.In the case of tissue damage, stimulate undifferentiated mesenchymal cells change to odontoblast, secretion of adhesive quality, mineralization, and formate of reparative dentin. Many studies have shown that a series of growth factors expression has been enhanced in the proliferation and differentiation in HDPCs process, such as TGF-(31, VEGF, BMP-2, DMP-1, ON, OCN and so on. With the research of injury of dental pulp tissue repair increasing depth, more and more researchers tried to use one or multip drugs to repair the damage of dental pulp tissue.Simvastatin is a 3-hydroxy-3-methylglutaryl coenzyme A reductase (3-hydroxy-3-methylglutaryl coenzyme A, HMG-CoA) inhibitors, are widely used for clinical treatment of cardiovascular disease. In recent years, a large number of literature shows that simvastatin not only has anti-inflammatory lipid-lowering effect, but also promote the synthesis of the biological role of bone metabolism. Similarly, research about in the field of oral is also gradually expand, such as research intervention in the simvastatin between implant and bone changes in the stability of bone formation, clinical use of simvastatin on the extent of periodontal bone defects effects, and so on. But the literature about the simvastatin on the proliferation of and differentiation HDPCs is literature rarely report.Most of drug intervention trials are require to maitain synchronization of the cell cycle before the induction of durg in order to get a more objective and realistic results. Serum starvation is a method which is little interference on the cells, less cell cycle and development process and the ability to cell cycle arrest in G0/G1 phase of the synchronization method. Howere, there are no mature and effective report on the HDPCs serum starvation synchronization methods.The first part of this research project aimed at cultrue HDPCs in vitro and identificate the origin of the cell. The second part of this research,to finding the best ways to study for HDPCs serum starvation conditions of synchronization and provide a frame of reference for the late and post-test HDPCs experiments.The third part of the experiment, After the synchronization, the HDPCs were given different doses of simvastatin, observed the HDPCs process of proliferation and differentiation, to provide experimental evidence to study the mechanisms of dental pulp tissue repair.Part I Human dental pulp cells (HDPCs) culture and identificationMethod1 HDPCs collection and culturingPrimary cultures of HDPCs were obtained from healthy premolars and third molars. With enzyme digestion and tissue culture cells. the pulp tissue were cutted and digested to tissue or cell suspension culture spread in the cell culture flask.2 HDPCs observation and identification2.1 Morphological observationObservate about the cell growth, proliferation and morphological characteristics 2.2 Determination of cell growth curve2.3 Identification of cell surface markersCell cultures 2th passages were used analysis of flow cytometry to observe the surface marker CD 105,CD2,CD34,CD45 expression.Result1 HDPCs CulturingIn general, after 5 days culture, the primary cells became to adherent the plate with radioactive growth.Then the cells formation of outgrowth after lweek culturing. Adjacent cells in the tissue together after 14 days. Continuous culture of the cells showed the characteristics of overlay.2 HDPCs observation and identification2.1 Morphological observationMost of cells show a fibroblast-like morphology, such as a star or long spindle. Abundant cytoplasm, nuclei showed round and centered.,multiple nucleoli were seen in few cells.2.2 Determination of cell growth curveThe HDPCs growth curve showed a typical S-shape.2.3 Identification of cell surface markersFlow cytometry analysis showed that CD105, CD29 were (+) expressed, expression rates were 97.67%,98.33%; CD34, CD45 were (-) expressed, the expression rates were 0.13%,0.33%. Experimental results showed that the cultured source of mesenchymal cell.ConclusionThe experiment was a success in culturing Human dental pulp cells and identificated of the cell morphology and cell surface markers. Experimental results showed that cells are the source of mesenchymal and provide experimental basis for future research.Part II Study of cell cycle synchronization in human dental pulp cell by serum starvationMethod1 HDPCs CulturingMethods consistent with the part one2 Morphological observation after serum starvationCell cultures between the 3th and 5th passages were used for morphologicalchanges observed after serum starvation.3 CCK-8 cell viability assaySynchronization after the HDPCs cultured 12h,24h,48h,72h,96h. The application of CCK-8 was detected to the proliferation of cells in each group.4 FCM detection of cell cycleSynchronized culture after treatment HDPCs to Oh,24h,48h,72h, the application of flow cytometry was detected to the changes of cell cycle in each group.5 Data using statistical software SPSS13.0 of One-way ANOVA test,, the results presented as mean±SD, P<0.05 indicated significant difference.Result1 Morphological observation after serum starvationIn the early stages of starvation (24h,48h) cells did not produce significant effect on morphology. With the extension of the treatment time (72h), Changes take place in cell morphology. Most of the cells became round, loss of polarity, cells appeared more fragmented, apoptosis and loss of adhesion.2 CCK-8 to detect cell proliferation activityIn the early stage of starvation (12h), The cells in each experimental group had a small amount of proliferation, groups of serum-free and gradient of serum starvation are slightly higher than the normal culture group. With the extension of the treatment time (24h), the OD values of serum-free and Gradient of serum starvation are began to steadiy decline.The result between the starvate groups was no significant difference (P>0.05).,but compared with the normal training group, there was significant difference (P<0.05).3 FCM detection of cell cycleCompared with the normal culture group, all serum starvation group was higher than the normal culture group in G0/G1 stages.5 mL/L FBS serum starvation group make most of cell cycle Remain in G0/G1 phase, S phase and G2/M phase was decreased (P<0.05).And the rate of G0/G1 stages amount for 85.95% after 48h starvation. Compared with the normal culture group, there was significant difference (P<0.05).ConclusionThe result of histological observation, examination of cell proliferation and cell cycle showed that 5 mL/L FBS serum starvation group will be get the best effect of synchronization after treatment HDPCs for 48h..Part III Effect of simvastatin on synchronized human dental pulp cells (HDPCs) proliferationMethod1 HDPCs CulturingMethods consistent with the part one2 synchronized HDPCsCell cultures between the 3th and 5th passages were used for serum starvation(5 mL/L FBS,48h).3 Solution preparation of simvastatinWeigh simvastatin for 5mg to the tube, mix well with 1.19ml of DMSO. Preparation of a 10-2 mol/L stock solution (Dark,-20℃saved 2m). Then, stock solution used to configure the ratio of 1:9 concentration of 10"3 mol/L of the working fluid. Dark,-20℃saved 1m).4 CCK-8 cell viability assayAfter the synchronization, the HDPCs were given different doses of simvastatin (0,10-8 mol/L,10-7mol/L,10-6mol/L,10-5 mol/L), observed on the HDPCs process of proliferation by CCK-8, after cultured with 1d,3d,5d.5 FCM detection of cell cycleHDPCs were given doses of 10-6 mol/L simvastatin, after the synchronization. At the same time establishment of positive control group and blank control group. After cultured 3days, the application of flow cytometry was detected to the changes of apoptotic percentages in each group.6 FCM detection of poptotic cellsHDPCs were given different doses of simvastatin (0,10"7 mol/L,10"6 mol/L,10-5 mol/L), after the synchronization. After cultured 3 days, the application of flow cytometry was detected to the changes of apoptotic percentages in each group..7 Data using statistical software SPSS13.0 of One-way ANOVA test,, the results presented as mean±SD, P<0.05 indicated significant difference.Result1 CCK-8 to detect cell proliferation activityApplication of CCK-8 concentration gradient was detected on the HDPCs growth situation. In the early cultivation (1d), each group had a small amount of cell proliferation, but no significant difference between groups (P>0.05). The OD value of low concentrations (10-8 mol/L,10-7 mol/L) and control group OD value showed a slow growth trend. At the same time, the OD value of high concentration (10-5 mol/L) were slowly declining trend, but each group no significant difference (P>0.05). After 5 days cultured, low concentration group is higher than the control group, but no significant difference between groups (P>0.05). At the same time, control group and 0 mol/L also showed no significant difference between the two group(P>0.05). Howere, OD value of the high concentration group showed a downward trend, compare with the control group and the low concentration group had significant differences (P<0.05).2 FCM detection of cell cycle10-6 mol/L Sim group and 0.03moL/L H202 group of HDPCs after the post-induction for 3 days. Compared with the control group, GO/Gl.phase cell were increased and G2/M phase cells under the appropriate. That 10-6mol/L Sim can regulate HDPCs arrested at G0/G1 phase and reduce the S and G2/M phase cells consistented with the positive control group (0.03moL/L H2O2 group).3 FCM detection of apoptotic cellsThe proportion of early apoptotic cells in roughly the same to the experimental group, With the extension of the treatment time, the high concentration (10-5 mol/L) in the proportion of early apoptotic cells was basically straight up. Howere, slightly decreased in the other groups increased slowly. High concentration group and other groups in the training to 3 days and 5 days early apoptotic cell ratio between the values significantly different, with statistical significance(P<0.05). There was no significant difference between other groups. The result showed that the high concentration group of Sim to HDPCs Sim will be enhance the proportion of the apoptotic cells.ConclusionSim in different concentrations have the different effects to synchronization HDPCs. This experiment on cultured cell proliferation and cell cycle, and compared the rate of early apoptosis. The result found that low concentrations can promote the HDPCs Sim proliferation, and high concentrations of Sim is the proliferation of HDPCs produce inhibition. Select a effective concentration of 10-7 mol/L of the Sim can promote the proliferation HDPCs. And prepare for further study to Sim on HDPCs and repair mechanisms.