炎症环境下敲低SHP2对人牙周膜干细胞增殖和成骨分化的影响  

Effect of SHP2 knockdown on the proliferation and osteogenic differentiation of human periodontal ligament stem cells under inflammatory environment

作　　者：张远 赵庆 吕浩东 王天丛[1] 窦昭婧 金玉琴 季骏[1] ZHANG Yuan;ZHAO Qing;LV Haodong;WANG Tiancong;DOU Zhaojing;JIN Yuqin;JI Jun(Department of Orthodontics,Nanjing Stomatological Hospital,Medical School of Nanjing University,Nanjing 210008,China)

机构地区：[1]南京大学医学院附属口腔医院·南京市口腔医院正畸科,江苏南京210008

出　　处：《口腔疾病防治》2022年第11期769-778,共10页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：国家自然科学基金项目(81700939);江苏省自然科学基金项目(BK20200150);江苏省医学青年人才基金项目(QNRC2016116);南京市医学科技发展基金资助项目(YKK16163,YKK16160);江苏省六大人才高峰项目(YY-100)。

摘　　要：目的探讨炎症环境下含有Src同源结构域2的蛋白酪氨酸磷酸酶2(Src homology-2 domain containing protein tyrosine phosphatase-2,SHP2)对人牙周膜干细胞(human periodontal ligament stem cells,hPDLSCs)增殖及成骨分化的调节作用和相关机制,为牙周炎治疗提供新的靶点。方法使用慢病毒感染构建敲低SHP2基因的hPDLSCs细胞系,通过RT-qPCR、Western blot验证SHP2的敲低水平;使用肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-1β(interleukin-1β,IL-1β)在体外模拟炎症环境;CCK-8检测正常及炎症条件下敲低SHP2对hPDLSCs增殖的影响;碱性磷酸酶(alkaline phosphatase,ALP)染色、ALP活性检测、ARS染色、RT-qPCR以及Western blot检测成骨指标;Western blot检测敲低SHP2在炎症条件下,对丝裂原活化蛋白激酶/核因子κB(mitogen-activated protein kinase/nuclear factor-κB,MAPK/NF-κB)通路蛋白的影响。结果慢病毒感染细胞72 h后在荧光显微镜下显示出明显的绿色荧光,测得SHP2 shRNA重组慢病毒的滴度为2.9×10^(8) TU/mL;Western blot及RT-qPCR检测发现,SHP2敲低组的SHP2蛋白及基因的表达显著降低(P<0.001);在正常条件下,敲低SHP2在一定程度上抑制了hPDLSCs的增殖;敲低SHP2后,hPDLSCs的早期成骨指标增高,包括ALP活性显著增高,ALP和COL-1的表达增加(P<0.05),然而敲低SHP2对hPDLSCs最终产生钙结节的量无明显影响。在TNF-α和IL-1β诱导的炎症环境下,敲低SHP2对hPDLSCs的增殖无明显影响(P>0.05),敲低SHP2后,hPDLSCs成骨相关指标均增加(P<0.05),钙结节形成的量增多(P<0.05);炎症环境下,敲低SHP2后,hPDLSCs中p65的磷酸化和IκB-α的降解降低,并且NF-κB上游MAPK通路蛋白p-p38和p-JNK的表达降低(P<0.05)。结论炎症环境下,敲低SHP2对hPDLSCs的增殖无明显影响,但可通过抑制MAPK/NFκB通路促进hPDLSCs成骨分化。Objective The purpose of this study was to clarify the regulatory effect and mechanism of Src homology2 domain containing protein tyrosine phosphatase-2(SHP2)on human periodontal ligament stem cell(hPDLSC)proliferation and osteogenic differentiation under inflammatory environment and to provide a new target for the treatment of periodontitis.Methods SHP2 was knocked down in hPDLSCs,and the transfection efficiency of SHP2 was detected by RT-qPCR and Western blot.An in vitro inflammatory environment was created using tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β).The effect of SHP2 knockdown on hPDLSC viability under normal and inflammatory conditions was detected by CCK-8,and the osteogenic capacity of hPDLSCs under normal and inflammatory conditions was detected by ALP staining,ALP activity,ARS staining,RT-qPCR and Western blot.The mechanism by which SHP2 knockdown affected the MAPK pathway and its downstream NF-κB pathway under inflammatory conditions was assessed by Western blot.Results Green fluorescence was observed after transfection for 72 h,and the titer of SHP2 shRNA recombinant lentivirus was 2.9×10^(8) TU/mL.SHP2 expression was significantly downregulated in lentivirus-transfected cells,as demonstrated by Western blot and RT–qPCR(P<0.001).SHP2 knockdown inhibited hPDLSC proliferation to a certain extent and increased the expression of early osteogenic markers under normal conditions,including increased ALP activity and increased ALP and COL-1 expression(P<0.05).However,SHP2 knockdown exerted no effect on mineralized nodule formation.In the TNF-α-and IL-1β-induced inflammatory environment,SHP2 knockdown exerted no effect on hPDLSC proliferation(P>0.05).Osteogenic markers were upregulated(P<0.05),and mineralized nodules were significantly increased(P<0.05)after SHP2 knockdown.Western blot analysis showed that p65 phosphorylation and IκB-αdegradation were reduced in SHP2-knockdown hPDLSCs in the inflammatory environment.Moreover,SHP2 knockdown significantly inhibited the expression of

关 键 词：牙周膜干细胞 增殖 牙周炎 肿瘤坏死因子-α 白细胞介素-1β 炎症环境 Src同源结构域2的蛋白质酪氨酸磷酸酶2 基因沉默 成骨分化 碱性磷酸酶 COL-1 MAPK/NF-κB信号通路 

分 类 号：R78[医药卫生—口腔医学]