TGF⁃β1对炎症状态下人牙周膜成纤维细胞的调控作用

Regulation of TGF⁃β1 on human periodontal fibroblasts in inflammatory state

目的探讨转化生长因子⁃β1(transforming growth factor⁃β1,TGF⁃β1)对在牙龈卟啉单胞菌来源脂多糖(lipopolysaccharide from Porphyromonas gingivalis,Pg⁃LPS)刺激模拟炎症状态下的人牙周膜成纤维细胞(human periodontal ligament fibroblasts,hPDLFs)的调控作用。方法获取hPDLFs并进行免疫组化鉴定,通过qRT⁃PCR与CCK⁃8确定Pg⁃LPS的刺激浓度。将hPDLFs分成4组:空白对照组,单纯100μg/mL Pg⁃LPS;低浓度组,1 ng/mL TGF⁃β1+100μg/mL Pg⁃LPS;中浓度组,10 ng/mL TGF⁃β1+100μg/mL Pg⁃LPS;高浓度组100 ng/mL TGF⁃β1+100μg/mL Pg⁃LPS。CCK⁃8检测细胞增殖情况;划痕实验与Transwell小室实验检测hPDLFs细胞迁移能力;流式细胞术检测hPDLFs的细胞周期;qRT⁃PCR检测hPDLFs的转录因子叉头盒p3(forkhead/winged helix transcription⁃al factor p3,Foxp3)、白细胞介素6(interleukin⁃6,IL⁃6)及EB病毒诱导基因3(Epstein⁃Barrvirus⁃induced gene 3,EBI3)mRNA表达;Western blot检测hPDLFs的Foxp3、IL⁃6及EBI3蛋白表达。结果免疫组化鉴定显示抗波形丝蛋白阳性及抗角蛋白阴性;Pg⁃LPS浓度为100μg/mL时,hPDLFs中IL⁃6 mRNA表达相比空白对照组显著上升(P<0.0001)且细胞增殖能力下降(P<0.0001),所以选择100μg/mL Pg⁃LPS用于模拟炎症状态。10、100 ng/mL TGF⁃β1能提高炎症状态下hPDLFs的增殖能力(P<0.0001);1、10、100 ng/mL TGF⁃β1能促进炎症状态下hPDLFs的迁移能力(P<0.0001);1、10、100 ng/mL TGF⁃β1可加快炎症状态下hPDLFs的细胞周期(P<0.0001);1、10、100 ng/mL TGF⁃β1可抑制炎症状态下hPDLFs的IL⁃6基因和蛋白表达量(P<0.0001),1、10 ng/mL TGF⁃β1可提高炎症状态下hPDLFs的EBI3基因及蛋白表达量(P<0.0001),1、10 ng/mL TGF⁃β1可提高炎症状态下hPDLFs的Foxp3基因表达量,10 ng/mL TGF⁃β1可提高Foxp3蛋白表达量(P<0.05)。结论TGF⁃β1可促进炎症状态下hPDLFs的增殖及迁移能力、上调EBI3表达,可能与转录因子Foxp3表达有关。

Objective To investigate the effect of transforming growth factorβ1(TGF⁃β1)on human periodontal lig⁃ament fibroblasts(hPDLFs)stimulated by lipopolysaccharide from Porphyromonas gingivalis(Pg⁃LPS).Methods hPDLFs were obtained and identified by immunohistochemistry.The stimulating concentration of Pg⁃LPS was deter⁃mined by qRT⁃PCR and CCK⁃8.The hPDLFs were divided into 4 groups:blank control group,100μg/mL pure Pg⁃LPS;low concentration group,1 ng/mL TGF⁃β1+100μg/mL Pg⁃LPS;medium concentration group,10 ng/mL TGF⁃β1+100μg/mL Pg⁃LPS;and high concentration group 100 ng/mL TGF⁃β1+100μg/mL Pg⁃LPS.Cell proliferation was measured by CCK⁃8 assay at 72 hours,cell migration was measured by scratch and Transwell chamber assays at 24 hours,and the cell cycle of the hPDLFs was measured by flow cytometry at 72 hours.The expression of Forkhead/winged helix transcrip⁃tion factor p3(Foxp3),interleukin⁃6(IL⁃6)and Epstein⁃Barr virus⁃induced gene 3(EBI3)mRNA in hPDLFs at 72 hours was measured by qRT⁃PCR,and the expression of Foxp3,IL⁃6 and EBI3 proteins in hPDLFs at 72 hours was detected by western blot.Results The immunohistochemistry results showed that anti⁃vimentin was positive and anti⁃keratin was negative.At a concentration of 100μg/mL Pg⁃LPS,the expression of IL⁃6 mRNA in hPDLFs was increased(P<0.0001),and the proliferation of hPDLFs was decreased(P<0.0001).Therefore,100μg/mL PG⁃LPS was selected to simulate the inflammatory state.10,100 ng/mL TGF⁃β1 could improve the proliferation ability of hPDLFs in inflammatory state(P<0.0001);1,10 and 100 ng/mL TGF⁃β1 could promote the migration ability of hPDLFs in inflammatory state(P<0.0001).1,10 and 100 ng/mL TGF⁃β1 could accelerate the cell cycle of hPDLFs in inflammatory state(P<0.0001).1,10 and 100 ng/mL TGF⁃β1 could inhibit the expression of IL⁃6 gene and protein in hPDLFs in inflammatory state(P<0.0001),1 and 10 ng/mL TGF⁃β1 could increase the expression of EBI3 gene and protein in hPDLFs in inflammatory state(P<0.0001).1,10 ng/mL TGF⁃β1 could increase the expression level of Foxp3 gene in hPDLFs in inflammatory state,and 10 ng/mL TGF⁃β1 could increase the expression level of Foxp3 protein(P<0.05).Conclusion TGF⁃β1 can promote the proliferation and migration of hPDLFs under inflammatory conditions,upregulate EBI3 and inhibit inflammation,which may be related to the expression of the transcription factor Foxp3.