circRNA SIPA1L1修饰外泌体对脂多糖刺激下人牙髓干细胞成骨分化的影响

Effect of circRNA SIPA1L1 modified exosomes on osteogenic differentiation of human dental pulp stem cells stimulated by lipopolysaccharide

目的:探究环状RNA(circRNA)信号诱导增殖相关蛋白1样蛋白1(signal-induced proliferation-associated 1 like 1,SIPA1L1)修饰人牙髓干细胞(human dental pulp stem cell,hDPSC)来源外泌体(exosome,Exo)对脂多糖(lipopolysaccharides,LPS)刺激下的hDPSC成骨分化能力的影响。方法:将circSIPA1L1过表达质粒载体转染至hDPSC后分离Exo,透射电镜观察形态特征,Western blot测定Exo标志蛋白CD81、Hsp70、TSG101表达,qRT-PCR检测circSIPA1L1相对表达量,用PHK26荧光标记Exo并观察hDPSC摄取情况。hDPSC分为对照组、LPS组、LPS+hDPSC Exo组、LPS+circSIPA1L1-hDPSC Exo组,CCK-8检测各组hDPSC增殖;酶联免疫吸附测定(ELISA)各组hDPSC培养上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、IL-6水平,茜素红染色检测各组hDPSC红色钙结节形成,Western blot测定各组hDPSC中碱性磷酸酶(ALP)、骨钙素(OCN)、骨桥素(OPN)、Runt相关转录因子2(Runx2)及牙本质涎磷蛋白(DSPP)蛋白表达。结果:hDPSC与转染circSIPA1L1后的hDPSC中分离得到椭圆形或圆形颗粒物,直径大多分布在约100 nm处,CD81、Hsp70及TSG101蛋白表达明显,且circSIPA1L1修饰hDPSC来源Exo中circSIPA1L1相对表达量显著高于hDPSC来源Exo(P<0.05),并观察到hDPSC来源Exo与circRNA SIPA1L1修饰hDPSC来源Exo均可被hDPSC所摄取。与LPS组比较,LPS+hDPSC Exo组和LPS+circSIPA1L1-hDPSC Exo组hDPSC增殖能力显著升高(P<0.05),hDPSC培养上清液中TNF-α、IL-1β、IL-6水平显著降低(P<0.05),红色钙结节明显增多,细胞中ALP、OCN、OPN、Runx2、DSPP蛋白相对表达量显著上调(P<0.05)。此外,与LPS+hDPSC Exo组比较,LPS+circSIPA1L1-hDPSC Exo组hDPSC增殖能力显著升高(P<0.05),hDPSC培养上清液中TNF-α、IL-1β、IL-6水平也显著降低(P<0.05),红色钙结节更多,细胞中ALP、OCN、OPN、Runx2、DSPP蛋白相对表达量也显著上调(P<0.05)。结论:circRNA SIPA1L1修饰hDPSC来源的Exo能够提高LPS刺激下hDPSC的增殖水平,抑制促炎因子释放,增强hDPSC的成骨分化能力。

Objective:To investigate the effect of circRNA signal-induced proliferation-associated 1 like 1(SIPA1L1)modification of human pulp stem cells(hDPSC)-derived exosomes(Exo)on the osteogenic differentiation of hDPSC stimulated by lipopolysaccharide(LPS).Methods:The circSIPA1L1 overexpressed plasmid vector was transfected into hDPSC and Exo was isolated,the morphological characteristics were observed by transmission electron microscopy,the expressions of Exo marker proteins CD81,Hsp70 and TSG101 were detected by Western blot,qRT-PCR was used to detect the relative expression level of circSIPA1L1,and PHK26 was used to fluorescently label Exo and observe its uptake by hDPSC.hDPSC was divided into control group,LPS group,LPS+hDPSC Exo group,LPS+circSIPA1L1-hDPSC Exo group,the proliferation of hDPSC in each group was detected by CCK-8,the levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and IL-6 in the supernatant of hDPSC culture in each group were determined by ELISA,and the formation of red calcium nodules in hDPSC was detected by alizarin red staining,the protein expressions of alkaline phosphatase(ALP),osteocalcin(OCN),osteopontin(OPN),Runt-related transcription factor 2(Runx2)and dentin sialophosphoprotein(DSPP)in hDPSC in each group were determined by Western blot.Results:Elliptic or circular particles were isolated from hDPSC and hDPSC transfected with circSIPA1L1,most of which were distributed at about 100 nm in diameter,CD81,Hsp70 and TSG101 were obviously expressed,the relative expression level of circSIPA1L1 in the Exo derived from circSIPA1L1 modified hDPSC was significantly higher than that from hDPSC(P<0.05),and it was observed that both the Exo derived from hDPSC and the Exo derived from circRNA SIPA1L1 modified hDPSC could be taken up by hDPSC.Compared with LPS group,the proliferation capacity of hDPSC in LPS+hDPSC Exo group and LPS+circSIPA1L1-hDPSC Exo group was significantly increased(P<0.05),and the levels of TNF-α,IL-1β and IL-6 in the supernatant of hDPSC culture were significantly decreased(P<0.05),red calcium nodules increased significantly,the relative expressions of ALP,OCN,OPN,Runx2 and DSPP proteins were significantly up-regulated(P<0.05).In addition,compared with LPS+hDPSC Exo group,the proliferation capacity of hDPSC in LPS+circSIPA1L1-hDPSC Exo group was significantly increased(P<0.05),and the levels of TNF-α,IL-1β and IL-6 in the supernatant of hDPSC culture were also significantly decreased(P<0.05),there were more red calcium nodules,and the relative expressions of ALP,OCN,OPN,Runx2 and DSPP proteins were significantly up-regulated(P<0.05).Conclusion:The modification of hDPSC derived Exo by circRNA SIPA1L1 can increase the proliferation level of hDPSC stimulated by LPS,inhibit the release of pro-inflammatory factors,and enhance the osteogenic differentiation ability of hDPSC.