【作者】 白红敏；

【导师】 刘士有； 刘大勇；

【作者基本信息】 天津医科大学 ， 口腔临床医学， 2010， 硕士

【摘要】 目的：研究人牙周膜干细胞(human periodontal ligament stem cells,hPDLSCs)在脂多糖(Lipopolysaccharides,LPS)刺激下产生一氧化氮(Nitric oxide,NO)的能力及一氧化氮合酶(Nitric Oxide Synthase,NOS),干扰素诱导蛋白10(IP-10)的表达情况,为探讨牙周膜干细胞在免疫调节中的作用机制提供前期研究基础。方法：收集临床上12-18岁因正畸拔除的健康无龋的前磨牙50例,刮取根中部的牙周膜,采用组织块直接贴壁培养法体外分离培养hPDLSCs,并进行牙周膜干细胞的鉴定。取对数生长期的4-8代细胞用于后续实验。实验分为对照组、LPS组(1ng/ml、10ng/ml、100ng/ml、1000ng/ml、10000ng/ml)及L-单甲基精氨酸(NG-Monomethyl-L-arginine,Monoacetate Salt, L-NMMA)组(10ng/ml LPS+100μmol/L L-NMMA)。细胞在37℃、5%CO2培养3h、6h、12h、24h后硝酸还原酶法检测细胞培养上清液中NO含量,ELISA法检测细胞培养上清液中IP-10的表达；免疫组化检测刺激前后牙周膜干细胞iNOS、nNOS、eNOS的表达,RT-PCR检测细胞中iNOS mRNA的表达。结果：1.组织块法培养人牙周膜细胞成功率高,表达人牙周膜干细胞标志STRO-1和CD146,具有骨向和脂肪向等多向分化的潜能,具备干细胞的特性。2.不同浓度LPS处理hPDLSCs后,hPDLSCs产生的NO的浓度随时间增加而增加,在24h时达到高峰；hPDLSCs产生的NO的量随着LPS浓度升高而增加；加入L-NMMA可使NO产生浓度降低(P<0.05)。3.免疫组化显示,不同浓度LPS处理hPDLSCs后,iNOS的中度表达,主要表达在hPDLSCs胞浆中,而eNOS、nNOS在未处理组和处理组均见中等程度表达,无显著差异。L-NMMA和LPS共处理组仍表达iNOS.4. RT-PCR结果显示LPS刺激3h时iNOS mRNA增多。5. ELISA显示,不同浓度LPS处理hPDLSCs后,上清液中IP-10的含量随时间增加而增加,在48h时达到高峰,随着LPS浓度的增加而降低。结论：1.LPS作用下,hPDLSCs产生NO的能力在一定时间内逐渐增强。2. hPDLSCs产生NO主要是因为hPDLSCs中iNOS表达增多。3. L-NMMA不抑制iNOS mRNA的表达,但可能抑制iNOS的活性。4.LPS刺激hPDLSCs产生IP-10,含量随时间增加而增加。更多还原

【Abstract】 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.更多还原