Expression And Function Of Collagen Triple Helix Repeat Containing-1 In The Chronic Periodontitis

Objective: Collagen triple helix repeat containing-1(CTHRC1)is a gene which has impact on collagen matrix deposition.It was initially found in damaged adventitial tissues.The experimental objectives are as following:(1)To evaluate the expression of CTHRC1 in normal and chronic periodontitis gingival tissues.(2)To investigate expression level of CTHRC1 in human periodontal ligament fibroblasts(hPDLFs)when stimulated by various concentration of Porphyromonas gingivalis lipopolysaccharide(P.g LPS).(3)To illuminate the possible function of CTHRC1 in cell proliferation and migration,and it probably effect on the ability of synthesis of collagen matrix and osteogenesis differentiation,both in normal and inflammatory conditions.Methods:(1)normal(15 cases)and chronic periodontitis gingival tissues(30 cases)were collected,fixed with 4% paraformaldehyde for 24 h,dehydrated and embedded in paraffin.For each specimen,at least two sectioned slides were needed(4 μm in thickness),one was stained with hematoxylin and eosin(HE),the other was for immunochemistry(IHC)test.The IHC images were analyzed by Image Pro Plus 6.0.(2)hPDLFs were isolated from premolar tooth extracted for the reason of orthodontic indication.Identifying the origination of cells using immunofluorescence(IF).Stimulating cells using 0 μg/mL,1 μg/ml,5 μg/mL and 10 μg/ml P.g LPS respectively for 24 and 48 h.Then,the CCK-8 assay,enzyme linked immunosorbent assays(ELISA)and quantitative real time polymerase chain reaction(qRT-PCR)experiments were performed.(3)In the experiment part,there are four groups including control-NC group,control-si CTHRC1 group,P.g LPS-NC group and P.g LPS-si CTHRC1 group.Then CCK-8 assay,cell wound scratch assay and qRT-PCR were performed in order to uncover the possible impact on hPDLFs of CTHRC1 both on the normal and inflammation condition.Result:(1)Compared to normal gingival tissues,chronic periodontitis group showed high level expression of CTHRC1,both in epithelium and lamina propria,and mainly located in gingival fibroblasts,epithelial cell and matrix around inflammatory cells.(2)The primary hPDLFs crawled out from the edge of the tissue in 7～10 days,showing swirling growth.The result of IF showed that cells were derived from mesenchyme.Cells cultured with 1 μg/mL,5 μg/ml and 10 μg/mL P.g LPS did not influence cell proliferation activity but had an effect on the expression and secretion of CTHRC1.qRT-PCR results determined that 5 μg/mL P.g LPS was the best inducing concentration.(3)Compared to negative control group,silencing group had lower expression of CTHRC1.(1)CCK-8 assay showed that the inhibition of CTHRC1 expression significantly decreased the proliferation activity of cells in both normal and inflammatory groups(P < 0.001).(2)Cell wound scratch assay showed that under normal conditions,silencing CTHRC1 promoted cell migration,while under inflammatory stimulation,the result was the opposite(P < 0.01).(3)It showed that inhibition of CTHRC1 expression increased COL1A1 and TGF-β1 expression both on normal and inflammatory condition(4)Osteogenesis differentiation result showed that under normal conditions,inhibiting of CTHRC1 could promote the expression of COL1A1,but inhibit the expression of ALP.In the inflammatory state,silencing CTHRC1 promoted the expression of COL1A1 and ALP.Conclusion:CTHRC1 expression level is higher in chronic periodontitis gingival tissues than in the healthy tissues.P.g LPS could induce the production of CTHRC1 in hPDLFs in vitro.Down-regulating CTHRC1 has impact on cell proliferation,migration,collagen synthesis and osteogenic differentiation,indicating that CTHRC1 may play a role in the inflammatory development of chronic periodontitis.