| Objective:To investigate the expression and distribution of IL-17A、IL-6 and IL-1β in HPDLFs during compressive force and under the condition of inflammation,and study the combined mechanism of inflammation ande orthodotic force in the periodotal tissue.Our research was performed to investigate if the HPDLFs were seeded on six will plates and incubated with under nomal,inflammatory and biomechnical loading conditions in vitro,cells were stimulated with LPS and exposed to complex force,respectively and together.detected the gene expression of IL-17、EL-6、IL-1β and TNF-α by HPDLF,protein synthesis was analyzed by enzyme-linked immunoassay,and investigated the possible pathway and mechanism of IL-17 and other inflammatory cytokines when HPDLF treated by LPS under compressive force. We expected this research to provide certain theoretical basis and new ideas for the specific mechanism of orthodontic tooth movement in a periodotitis condition and relevant root resorption.Methods:The periodontal ligament fibroblasts were obtained from the 1/3 roots of the healthy impacted first or second premolars of adolescents who need teeth extracted for orthodontics,then primarily cultured using tissuse explant,followed by subcultrue. Cell growth and morpholgy were observed under an microscope.Immunohistochemistry staining of vimentin and keratin was used to identify the source of the cultured cells, An MTT assay was used to draw the growth curve of HPDLF.Then HPLDF were exposed to LPS and cultured under compression force (Og/cm2、1g/cm2、2g/cm2、3g/cm2、4g/cm2、 5g/cm2)for 24 h.The amounts of IL-6 and IL-17A were measured using RT-PCR. the protein expression of IL-lβ、IL-6、IL-17A by HPDLF were determined by ELISA.Results:1. The cultured cells were spindle, radial or spiral growth; and had stable biological character. MTT assay showed that the growth curve of HPDLF was "S" shape.Vimentin were positive expression.keratin were negative expression.2. MTT assay showed that the proliferation of HPDLF under Pg.LPS treatment (1～10μg/ml) was significantly increased, highest in 4 ug/ml group, slowly and gradually declined in 5～7μg/ml group. However, HPDLF proliferation activity was significantly inhibited in 8～10μg/ml group.3. RT-PCR showed that the mRNA expression of IL-17A and IL-6 of Pg.LPS treated HPDLF increased in force-relative manner, and higher than that in the compressive forces group at the same force lever.the highest expression showed respectively in 3 g/cm group and 4 g/cm2 group.4. ELISA showed that the protein expression of IL-1β、IL-6 and IL-17A increased in force-relative manner, and higher than that in the compressive forces group at the same force lever.the highest expression showed respectively in 3 g/cm2 group and 4 g/cm2 group.Conclusion:1. The HPDLF cell line was successfully established through tissue-explant method. The 4-6 generations had stable biological character to continue the next step of the experiment.2. Suitable concentration of Pg.LPS would accelerate the proliferation of HPDLF, whereas excessive concentration of Pg.LPS would inhibit HPDLF’s proliferation. Therefore,4μg/ml was the optimal concentration for HPDLF in vitro.3. Compressive forces could induce Pg.LPS treated HPDLF up-regulate the expression of the pro-inflammatory cytokine including IL-1β、IL-6 and IL-17A in a force-relative manner, and down-regulate that in compare to the group under compreesive forces only. And the highest expression show up in 3 g/cm2 group. |
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