压力状态下粪肠球菌培养上清液诱导人牙周膜细胞炎症反应的研究

Effects of Enterococcus faecalis supernatants on inflammatory responses of human periodontal ligament cells under pressure

目的探讨粪肠球菌培养上清液诱导人牙周膜细胞(human periodontal ligament cell,hPDLC)的炎症反应及其在压力状态下的表达变化。方法噻唑蓝(methyl thiazolyl tetrazolium,MTT)法检测不同体积分数[0%(对照组)、1%、2%、5%、10%和20%]的粪肠球菌上清液对hPDLC增殖活性的影响,采用流式细胞术检测粪肠球菌上清液刺激24 h后hPDLC表面Toll样受体2(Toll-like receptor 2,TLR-2)表达的改变;依据MTT及流式细胞术结果,将hPDLC分为不含粪肠球菌上清液的非诱导组与含5%粪肠球菌上清液的诱导组,每组均分别加载0、49及196 Pa的静压力,非诱导组和诱导组均以未加力(0 Pa)状态为对照。24 h 后观察hPDLC的形态变化,通过反转录PCR(reverse transcription-PCR,RT-PCR)和酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)法检测hPDLC肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)及白介素-1β(interleukin-1β,IL-1β)的mRNA和蛋白表达水平。结果 MTT法结果显示,培养48 h时,与对照组相比体积分数≥5%的粪肠球菌上清液能显著降低hPDLC的增殖活性(P<0.05)。流式细胞术结果显示,粪肠球菌上清液刺激hPDLC 24 h后其表面TLR-2阳性细胞率随上清液体积分数的增加而增加,0%、1%、2%、5%、10%和20%体积分数的上清液TLR-2阳性细胞率分别为(2.12±0.07)%、(2.41±0.32)%、(2.65±0.27)%、(4.76±0.46)%、(9.91±0.92)%、(12.01±1.35)%,体积分数≥5%时与对照组相比差异均有统计学意义(P<0.05)。RT-PCR结果显示,非诱导组施加49 Pa压力时,hPDLC IL-1β和TNF-α mRNA表达与对照组相比差异均无统计学意义(P>0.05),施加196 Pa压力时,hPDLC IL-1β和TNF-α mRNA表达与对照组相比差异均有统计学意义(P<0.05);而诱导组施加49和196 Pa压力时,hPDLC IL-1β和TNF-α mRNA表达均显著升高,与对照组相比差异均有统计学意义(P<0.05)。ELISA结果与PCR结果趋势一致。结论高浓度的粪肠球菌上清液能抑制hPDLC的增殖活性;粪肠球菌上清液可以促进hPDLC表面TLR-2的表达;过度的机械压力可引起hPDLC细胞的炎症反应,导致hPDLC对压力不耐受,引起炎症反应加重。

Objective To study the effect of various concentrations of Enterococcus faecalis(Ef)supernatants on human periodontal ligament cell(hPDLC)and the inflammatory response of hPDLC under static pressure.Methods The method of methyl thiazolyl tetrazolium(MTT)was used to detect the effect of various concentrations of Ef supernatants on the proliferation of hPDLCs and the flow cytometry was used to detect the expression of Toll-like receptor 2(TLR-2)on the surface of hPDLC after 24-hour-stimulation of Ef supernatant.Furthermore,the hPDLCs were divided into non inducing group without Ef supernatant and inducing group with 5%Ef supernatant,and hPDLCs in each group were loaded with 0,49 and 196 Pa static pressures respectively.The expressions of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)mRNA and protein were detected by reverse transcription-PCR(RT-PCR)and enzyme linked immunosorbent assay(ELISA)after 24 hours.Results MTT results showed that the supernatant of Ef with concentration≥5%could significantly inhibit the proliferation activity of hPDLCs at 48 hours of cell culture(P<0.05).Flow cytometry showed that the positive cell rates of TLR-2 increased with increasing volume fractions of the Ef supernatants.The values were(2.12±0.07)%,(2.41±0.32)%,(2.65±0.27)%,(4.76±0.46)%,(9.91±0.92)%and(12.01±1.35)%,respectively.The differences were statistically significant when the concentrations≥5%(P<0.05).There were no significant differences in the expressions of IL-1βand TNF-αmRNA between the non inducing group and the control group under the pressure of 49 Pa(P>0.05).However,there were significant differences in the expressions of IL-1βand TNF-αmRNA between the non inducing group and the control group under the pressure of 196 Pa(P<0.05),while the expressions of IL-1βand TNF-αin the inducing group were significantly lower than that in the control group under the pressures of 49 and 196 Pa(P<0.05).Compared with the control group,the mRNA expression was significantly increased(P<0.05).The result of ELISA was consistent with that of PCR.Conclusions High concentration of Ef supernatant could inhibit the proliferation of hPDLC.Ef supernatant might promote the expression of TLR-2 on the surface of hPDLC.Excessive mechanical pressure induced the inflammatory response of hPDLC.The presence of inflammatory mediators could lead to the intolerance of hPDLC to pressures and small pressure could aggravate the inflammatory response.