The Effect Of Low Level Laser On The Expression Of Periostin And Type ? Collagen In Periodontal Disease Cell Model

Objectives:After cultivate human gingival fibroblast,we use Pg.LPS stimulate HGF in order to build periodontal disease cell model.To determine the impact of different energy densities of low-level lasers on the expression of type I collagen and periostin in periodontal-disease cell model,and examine its proliferation leval.Then determine the optimum parameter of low level laser,and discuss the action mechanism.Methods:1.Gingival fibroblast was cultured with tissue block method.2.Then,apply Real Time-PCR method to examine the 24-hour expressions of IL-6level of the cells that stimulated by Pg.LPS,and to detect the expression of type I collagen and periostin at different time points.3.The gingival fibroblast cells were subjected to LLLT under different energy densities with a diode laser device(650 nm,200mW,2-8J/cm~2)After 6 hours of laser irradiation,cell proliferation,gene expression(Real-time PCR)and protein synthesis(Elisa)of Type ? collagen and periostin were assessed in order to determine the optimum parameters,then join the signal inhibitor SIS3,test the concentration variation of this two proteins.Result:1.Human gingival fibroblast were cultured with tissue block method.The anti-keratin staining was negative,anti-vimentin protein staining was positive,which proved to be mesoderm-derived human gingival fibroblasts.2.After stimulated by Pg.LPS,the expression of IL-6 was significantly increased.Compared with control group,The expression of periostin was lower than the control group at 2h,4h,6h,12h(p<0.05),and there was a lowest expression level at 12h.the expression of type I collagen at 6h and12h was lower(p<0.01).Then the expression of two proteins presented ascend trend.3.MTT test showed that LLLT promoted an accelerated proliferation of gingival fibroblast compared with the control group(p<0.05).When delivering 4J/cm~2 to the cells,the proliferation rate reached the highest point(p<0.01)4.Real-Time PCR result demonstrated that LLLT accelerated the gene expression of type I collagen and periostin(p<0.05).The expression level of two proteins reached the highest point under the 4J/cm~2 energy density.(p<0.01).ELISA result showed that type I collagen concentration was improved after stimulation of LLLT(p<0.05),after join SIS3,the concentration of type I collagen and periostin decreased(p<0.05).Conclusions:1.Human gingival fibroblast can be cultivated successfully with tissue block method.And aseptic concept is the priority of experiments.2.2ug/ml Pg.LPS could stimulate HGF in order to build periodontal disease cell model.3.In the periodontal diseases cell model,the expression of type I collagen and periostin was firstly decreased and then increased,which indicated that the inflammation destruction played a dominant role in the early stage of inflammation,and over time,the tissue repairing gradually tended to be more dominant.4.Within a certain range of energy density(2-8J/cm~2,650nm),LLLT could promote gingival fibroblast cell proliferation.When the laser density is 4J/cm~2,cell proliferation reached the highest point and extracellular matrix secretion was accelerated to the utmost,SIS3 can inhibit the secretion of type I collagen and periostin through TGF-?/smad3 signal pathway.