The Effects Of Dihydromyricetin And Cepharanthine Hydrochloride On The Proliferation And ALP In Human Periodontal Ligament Cell Population

Objective The periodontal ligament is a kind of dense connective tissue which packages and protect teeth, also provide nutrition. The aim of this study is to explore the potential beneficial effect of effective components contained in traditional Chinese medicine on treating periodontal disease by observing the influence of Dihydromyricetin and Cepharanthine Hydrochloride on the proliferation and differentiation of human periodontal ligament cell population(PDLP), aiming to provide the theoretical basis for this medicine in the application of periodontal disease.MethodsHuman periodontal ligament cells were primarily cultured by cover slip covering tissue culture method in vitro. Then Observing the growth conditions of PDLP under inverted phase contrast microscope. Moreover, the progenitor of human periodontal ligament cells was identified by Immunohistochemical methods and the cell growth curve was determined by MTS assay. Using MTS method draw the cell growth curve, respectively in cell 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days and eight days OD value of the periodontal ligament cells, draw the curve of MTS, to observe the shape of the curve,analysis of cell growth; Using immune histochemical method, the purpose is to determine whether the cultivated cells from mesenchymal, and no other organization cell mixed in; To cultivate cells for osteogenesis induced 21 days, using alizarin red staining, observe whether out after after the induction of cell has the mineralized nodules, used to illustrate the cultured cells can after mineralized induced osteogenesis portion; Respectively by the methods of MTS, coomassie brilliant blue staining, ALP alkaline phosphatase detection kits at different concentrations after dihydrogen arbutus pigment, stephania hydrochloric acid alkali activity of periodontal membrane of cells proliferation, the influence of the total protein concentration and alkaline phosphatase activity. By MTS method to make two kinds of different concentration respectively drugs’ effects on the periodontal ligament cells for 24 h, 48 h and 72 h, observe the cell value; Coomassie brilliant blue staining method was used with two kinds of drug concentrations used separately in the periodontal ligament cells group of 72 h, measuring whether there is a change in the periodontal ligament cells of total protein; Will different concentrations of the two drugs role in the periodontal ligament cells, respectively, using alkaline phosphatase kits, measure the drug in the periodontal ligament cells, the activity of alkaline phosphatase increased ever. Besides, the influence of Dihydromyricetin and Cepharanthine Hydrochloride on the proliferation and differentiation of human periodontal ligament cell population were investigated by MTS assay, ALP Kit, Coomassie brilliant blue assay, Q-PCR. Results1. Electronic microscope and active better organization, in the microscopic observation of visible organization block shear walls are golden, and poor activity, or not to stick wall tissue block is visible tissue is seen microscopically to block floating or black. The periodontal ligament cell population was in form normal and good condition. The growth curve model of PDLP looked like "S". Immunohistochemical analysis showed that the vimentin staining was in positive and the certain was in negative. The osteogenic induction of PDLP showed that: Cepharanthine nodule was formed and alizarin red staining was red.2. The effects of Dihydromyricetin and Cepharanthine on the proliferation of human periodontal ligament cell population.The MTS assay results showed that Dihydromyricetin and Cepharanthine Hydrochloride(100 μmol/L、80μmol/L、50μmol/L、25μmol/L、5μmol/L) could increase the proliferation of PDLP, and there were significant differences when compared with control group(P<0.05).3. The effects of Dihydromyricetin and Cepharanthine Hydrochloride on the differentiation of PDLP.The ALP Kit, Coomassie brilliant blue assay results showed that Dihydromyricetin and Cepharanthine Hydrochloride(80μmol/L、40μmol/L、20μmol/L、10μmol/L、5μmol/L) can increase the ALP, total protein of PDLP(P<0.05).