脂多糖对干燥综合征患者唇腺上皮细胞表达TLR4及CD14的影响

目的 探讨LPS-TLR4-CD14信号传导通路在干燥综合征(sjogren's syndrome,SS)发病机制中的作用。 方法 选择解放军第285医院风湿科2015年3月-2016年3月收治SS患者7例为研究组;另5例排除SS诊断,且无其他疾病的作为对照组。①取患者唇腺组织进行体外培养,一周后采用抗细胞角蛋白19的单克隆抗体PAP法对新生细胞和唇腺组织细胞进行染色,以鉴定新生细胞来源。②分别于培养成功的新生细胞PSS组和对照组加入浓度为1 μg/ml的内毒素(lipopolysaccharide,LPS),在继续培养后1、8、12、24、48、72 h时间点提取细胞总RNA,采用RT-PCR法分别检测不同时间点TLR4、CD14 mRNA的表达。③对新生细胞pSS组分别加入浓度为1、10、100、103、104、105 ng/ml的LPS,对照组不加LPS,继续培养24 h后分别检测TLR4和CD14 mRNA表达。 结果 ①结果显示新生细胞和唇腺组织中导管上皮细胞染色为阳性反应,唇腺组织中的淋巴细胞、腺泡细胞呈阴性反应,证明本次培养的新生细胞来源于唇腺组织的导管上皮细胞。②在新生细胞中加入LPS,作用1 h后TLR4和CD14 mRNA表达增强,分别于8、12 h后达高峰期,作用72 h后明显下降。③浓度为1 ng/ml的LPS即可引起PSS组新生细胞TLR4和CD14 mRNA表达显著增高,随着LPS浓度的增高,TLR4和CD14 mRNA表达持续升高,当LPS浓度达到104 ng/ml以上时,TLR4和CD14 mRNA表达呈平台期,变化不显著。 结论 经LPS刺激后,唇腺导管上皮细胞高表达TLR4和CD14 mRNA,且反应在一定范围内呈时间、浓度依赖性,由此推断LPS-TLR4-CD14信号转导通路参与了SS的发病机制,进而揭示感染与SS发病有关。 

The effect of lipopolysaccharide on the expressions of TLR4 and CD14 in salivary gland epithelial cells

Objective To study the role of LPS-TLR4-CD14 signal transduction pathway in the pathogenesis of Sjogren's syndrome. Methods ①The salivary glands cell of patients were cultured in vitro. After one week, the neonatal cells and labial gland cells were stained with anti-cytokeratin 19 monoclonal antibody PAP to identify neonatal cell sources. ②LPS was added to the cultured neonatal cell and the control group cell at the concentration of 1 μg/ml. Total RNA was extracted at 1, 8, 12, 24, 48 and 72 h after culture. The expression of TLR4 and CD14 mRNA was detected by RT-PCR. ③10, 100, 103, 104 and 105 ng/ml of LPS were added to the newborn cells pSS group respectively. The LPS was not added to the control group. After 24 h of culture, TLR4 and CD14 mRNA expression were detected respectively. Results ①The results showed that the epithelial cells in the labial glands and neonatal cells were stained;lymphocytes and acinar cells in the labial glands were negative, this proved that the cultured new cells were derived from the duct epithelial cells of the labial gland tissue. ②The expression of TLR4 and CD14 mRNA increased after 1 h of LPS addition, and reached the peak at 8 and 12 h, and significantly decreased after 72 h. ③The expression of TLR4 and CD14 mRNA in pSS neonatal cells was significantly increased by LPS at the concentration of 1ng/ml With the increase of LPS concentration, TLR4 and CD14 mRNA expression continued to increase, When the concentration of LPS reached more than 104 ng/ml, TLR4 and CD14 mRNA expression was plateau, the change was not significant. Conclusion After LPS stimulation, the expression of TLR4 and CD14 mRNA were highly increased in the labial ductal epithelium cell, the expression of TLR4 and CD14 mRNA increased with the increase of the LPS concentration and action times, within a certain range. The above study show that LPS-TLR4-CD14 signal transduction pathways is an important factor in the pathogenesis of SS, and thus reveal the infection participate the incidence of SS.