炎症微环境下可溶性环氧化物酶抑制剂对人根尖牙乳头干细胞增殖及分化的影响

Effects of Soluble Epoxide Hydrolase Inhibitor on Proliferation and Osteogenic Differentiation of Stem Cells from Apical Papilla in Inflammatory Environment

目的:探讨在炎症微环境下,可溶性环氧化物酶抑制剂(TPPU)对人根尖牙乳头干细胞(hSCAPs)增殖和分化的影响。方法:采用流式细胞仪以及成骨诱导后茜素红染色鉴定hSCAPs;1 mg/mL脂多糖(LPS)处理hSCAPs模拟炎症微环境,然后用10μmol/L TPPU作用于hSCAPs,分别在第1、3、5、7天,通过CCK-8法观察TPPU对hSCAPs增殖能力的影响;在第24小时,实时定量聚合酶链式反应(qRT-PCR)检测炎症相关因子表达。在成骨诱导后的第7、12天,通过qRT-PCR检测成骨分化相关基因的表达;第8天通过碱性磷酸酶染色来分析碱性磷酸酶活性,第21天用茜素红染色观察矿化结节形成。结果:流式细胞仪结果与茜素红染色鉴定实验细胞为hSCAPs。用1 mg/mL LPS处理hSCAPs后,在第1、3、5、7天时,TPPU+LPS组、LPS组和vehicle组细胞增殖无差异(P>0.05)。在用LPS处理hSCAPs 24 h后,相对于vehicle组,LPS组白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)表达显著上调(P<0.05),LPS+TPPU组IL-1β、IL-6的表达明显低于LPS组(P<0.05)。成骨诱导液培养hSCAPs一定时间后,检测发现相对于LPS组,LPS+TPPU组碱性磷酸酶(ALP)、牙本质涎磷蛋白(DSPP)、骨钙素(OCN)、Runt相关转录因子2(RUNX2)的表达显著上调(P<0.05)。且TPPU处理组较LPS组碱性磷酸酶染色和茜素红染色均加深。结论:在体外炎症微环境下,TPPU可以抑制hSCAPs炎性因子的表达。其次,在炎症微环境下TPPU对hSCAPs的增殖可能并没有明显的促进作用,但TPPU可在一定程度上促进hSCAPs的成牙/成骨分化。

Objective:To investigate the effect of TPPU on proliferation and differentiation of hSCAPs in the inflammatory microenvironment.Methods:hSCAPs were cultured and identified by flow cytometry and alizarin red staining.The hSCAPs were treated with 1 mg/mL lipopolysaccharide(LPS)to simulate the inflammatory microenvironment,and then treated with TPPU.The CCK-8 method was used to study whether TPPU had an effect on the proliferation ability of hSCAPs on the first 1,3,5,and 7 days.After 24 hours cultivation,the expressions of inflammation-related factors were detected by qRT-PCR.On the day 7 and 12,the expression levels of genes related to osteogenic differentiation were detected by qRT-PCR.On the day 7,ALP activity was analyzed by staining.On day 21,the formation of mineralized nodules was observed by alizarin red staining.Results:hSCAPs were confirmed by flow cytometry and alizarin red staining.CCK-8 results showed that there was no significant difference in OD value between hSCAPs.The expression of IL-1βand IL-6 of hSCAPs induced by LPS was significantly up-regulated compared with the vehicle group(P<0.05).Compared with the LPS group,the expression of IL-1βand IL6 in LPS+TPPU group was significantly down-regulated(P<0.05),but the expression levels of osteogenic genes in LPS+TPPU group were significantly up-regulated(P<0.05).ALP staining and alizarin red staining showed that the TPPU treated groups had deeper staining than LPS groups.Conclusion:TPPU can inhibit the expression of inflammatory factors in inflammatory microenvironment.TPPU may not promote the proliferation,but promote the tooth/osteogenic differentiation of hSCAP in inflammatory microenvironment.