Effect Of Lipopolysaccharide On Nitric Oxide Expression In Human Periodontal Ligament Stem Cells

Objective:To investigate the immunological effects of human periodontal ligament stem cells(hPDLSCs) on NO expression in lipopolysaccharides(LPS)/and L-NMMA.Methods:Fifty health premolars from patients between 12 to 18 years old were extracted for orthodontic reasons, periodontal tissues were isolated and cultured in vitro to obtain hPDLSCs. Cells in 4th-8th passage cells were used for the following experiment. Several groups was devided as follow:control group with none LPS, LPS groups(lng/ml,10ng/ml,100ng/ml,1000ng/ml,10000ng/ml)and L-NMMA group(10ngLPS+100μmol/L L-NMMA).Cells were cultured at 37℃in 5% CO2 for 3hours,6 hours,12 hours,24 hours and 28 hours. The release of NO was measured by nitric acid deoxidize enzyme in the medium.Then the amounts of IP-10 in the supernatant were determined by an enzyme-linked immunosorbent assay (ELISA) respectively. The expression of iNOS, nNOSand eNOS was determined with immunohistochemistry. The mRNA of iNOS were analyzed by RT-PCR assay.Results:1. hPDLSCs were obtained in vitro successfully by tissue culture, hPDLSCs can espress STRO-1 and CD146. hPDLSCs had the potential of osteogenesis and adipogenic under some appropriate culture situation.2. The release of NO increased along with the concentration of LPS. The release of NO increased with time, arrived the peak level at 24h, and then decreased at 28h.The release of NO in L-NMMA group decreased compared with the LPS group(10μg/ml) absolutely(P<0.05).3. The expression of nNOSand eNOS was obvious and there is no significant difference before and after LPS stimulation. The expression of iNOS was observed with LPS stimulated. The L-NMMA group also express iNOS.4. The mRNA of iNOS increased 3h after LPS stimulated, before the result of IHC.5. By ELISA, The release of IP-10 increased with time, arrived the mountain level at 48h, but decreased along with the concentration of LPS.Conclusions:1. The ability of NO production in hPDLSCs increased with LPS stimulated in 24 hours.2. The release of NO in hPDLSCs was due to the expression of inducible NOS with LPS stimulated.3. L-NMMA could not inhibit it's mRNA expression, but might inhibited the activity of iNOS.4. hPDLSCs stimulated by LPS could release IP-10, and IP-10 increased with time.