Expression Of TLR2 And TLR4 In Human Periodontal Ligament Cells Stimulated With Lipopolysaccharide

Objectives: Toll like receptors (TLRs) are innate immune receptor family member, mainly expressed in the surface of immune cells. TLRs are typeâ… transmembrane receptors and interleukin -1 receptor superfamily, can recognize Pathogen association molecular pattern (PAMP), including Lipopolysaccharide (LPS). Organism's inflammatory response began with TLRs associated molecular patterns of antigen recognition, causing intracellular signaling and cytokines secretion starting the inflammatory response.Many studies confirmed that TLR2 and TLR4 closely related with periodontal disease, have important clinical significance. A large number of bacteria exist in the oral environment. Periodontal disease was mixture infection mainly with Gram-negative anaerobic. LPS was a special structure of substances which was released from cell wall outer membrane after the death of Gram-negative bacteria. LPS is the main periodontal pathogens virulence factors, could cross the epithelium into the deep connective tissue. Periodontal ligament cells (PDLCs) has a strong ability of collagen synthesis and very important for the maintenance of periodontal health and the stability of the tooth. Academics were focus on whether periodontal ligament cells could be used as the local periodontal immune cells involved in immune response or not.In this study,we want to examine the expression of TLR2 and TLR4 in human Periodontal ligament cells stimulated by E coli lipopolysaccharide, and observe the expression of TLR2 and TLR4 at different time periods,learning about the role of human Periodontal ligament cells as immunocompetent cells in the development of periodontitis. Methods:1 Premolar tissue which extracted for orthodontic reasons of clinical patients of 12-26 years old was used for the study. Periodontal ligament was taken from the one-third of the root under sterile conditions.Periodontal ligament cells were cultured by using traditional method of primary culture. The third generation cells were identified through vimentin and CK Immunohistochemical staining.2 Choose the fourth generation cells which growth in the logarithmic phase, 4×10⁴/ml in the single cell was inoculated in 96 well plates, each pore volume 200ul, a total of 7 boards, each board cropping three holes, and setting the zero holes with no cells. Using the MTT assay (Methlthazoletrazolium, MTT) to draw the growth curve of periodontal ligament cells.3 Choose the fourth generation cells of growth in the logarithmic phase,inoculated in 24-well plate with the density of 5×10⁴/ml, adding appropriate amount of high glucose DMEM culture medium, cultured in 5% CO₂ incubator. Made the cells covered coverslip basically. Abandoned the original culture medium, and then add the experimental group with DMEM containing 10ug/ml LPS and cultured 4, 8, 12 and 24h, the negative control group with DMEM without LPS. Collected the coverslips with PDLCs in each group, fixed with acetone. Immunocytochemistry was used to examine the expression of TLR2 and TLR4 in the cells, observing expression of TLR2 and TLR4 in the cells with stimulation of LPS.True color multi-functional management system was used for image analysised; each five fields were randomly selected in high power (×400). Immunohistochemical score (IHS) of TLR2 and TLR4 were calculated in each group accroding to the method of Soslow RA and analyzed statistically.Results:1 Human periodontal ligament cells were successfully cultured, primary culture was radially arranged with the center of tissue block. Under inverted fluorescence microscope, the cultured cells were stellate or fusiform shape, with two or four cellular protrusions, cell body plump, cytoplasm was homogeneous, nucleus was round or oval with 1-3 nucleolus.Immunocytochemistry results showed that: in vitro cultured human periodontal ligament cells were positive for anti-vimentin staining, the positive parts were in the cytoplasm; anti-CK was negative. The results confirmed that the cells derived from mesoderm, consistent with the biological characteristics of fibroblasts.2 The fourth generation of periodontal ligament cells using MTT method showed that: the growth curve of PDLCs into a typical "S" shape, through three growth stages of incubation period, logarithmic growth phase and platforms.3 Detection of the expression of TLR2 and TLR4 in PDLCs stimulated by LPS in different time periods: Immunocytochemistry revealed that TLR2 and TLR4 were expressed only mildly in normal PDLCs, after stimulation with LPS, both TLR2 and TLR4 expressed increased significantly (P <0.05). The staining intensity was increased with the time of stimulation. Immunohistochemical staining showed that under the same stimulation time the expression of TLR2 were significantly higher than TLR4.Conclusion:1 The successful cultured of human periodontal ligament cells.2 Immunocytochemistry revealed that TLR2 and TLR4 were expressed only mildly in normal human periodontal ligament cells;stimulation of LPS could enhance the expression of TLR2 and TLR4. Detection at the same time expression of TLR2 was stronger than TLR4 significantly. Human periodontal ligament cells express TLR2 and TLR4 under LPS stimulation,indicating that it has the characteristics of immunocompetent cells.