摘要
目的探讨miR-124修饰的大鼠牙髓干细胞(rDPSCs)来源外泌体对巨噬细胞极化的影响。方法从SD大鼠牙髓中分离出rDPSCs,流式细胞术鉴定rDPSCs表面标志物CD14、CD34、CD45、CD90、CD105、CD146的阳性比率;油红O染色、茜素红S染色观察rDPSCs成脂成骨能力。利用细胞转染技术将miR-124模拟物(miR-124 mimics)及其阴性对照(miR-NC)转染于rDPSCs,分组为空白组、miR-NC组、miR-124组,并提取转染后rDPSCs的外泌体,分别命名为Exosome组(Exo组)、Exo/miR-NC组、Exo/miR-124组,透射电镜观察外泌体形态;Western blot检测外泌体标志蛋白CD63和CD81表达水平;qRT-PCR检测rDPSCs及rDPSCs外泌体中miR-124表达水平。将25只大鼠随机分为sham组、model组、rDPSCs Exo组、rDPSCs Exo/miR-NC组和rDPSCs Exo/miR-124组,每组5只,构建皮肤缺损模型,分别于皮肤缺损相应区域注射30μl对应的外泌体进行干预,qRT-PCR检测大鼠创伤组织中miR-124表达水平;ELISA法检测大鼠血清中诱导型一氧化氮合酶(iNOS)、甘露糖受体(CD206)含量;Western blot检测大鼠创伤组织中肿瘤坏死因子(TNF)-α、TNF-1β、白细胞介素(IL)-6、IL-4、IL-10、IL-13蛋白表达。结果rDPSCs表面标志物CD14、CD34、CD45的阳性比率均低于CD90、CD105、CD146;rDPSCs具有成骨成脂能力;透射电镜可观察到外泌体为大小均一,直径50~100 nm,呈圆形或椭圆形的囊泡结构;CD63和CD81蛋白在rDPSCs外泌体中高表达;与空白组和miR-NC组比较,miR-124组rDPSCs中miR-124表达水平显著升高,且Exo/miR-124组rDPSCs外泌体中miR-124表达水平显著高于Exo组和Exo/miR-NC组(P<0.05);与sham组比较,model组大鼠血清中iNOS含量、创伤组织中TNF-α、TNF-1β、IL-6蛋白表达显著升高,CD206含量、IL-4、IL-10、IL-13蛋白表达显著降低(P<0.05);与rDPSCs Exo组和rDPSCs Exo/miR-NC组比较,rDPSCs Exo/miR-124组中iNOS含量、TNF-α、TNF-1β、IL-6蛋白表达显著降低,CD206含量、IL-4、IL-10、IL-13蛋白表达显著升高(P<0.05)。结论miR-124过表达修饰的rDPSCs外泌体可通过抑制M1型巨噬细胞极化、促进M2型巨噬细胞极化来调控炎性反应。
Objective To investigate the effects of miR-124-modified rat dental pulp stem cells(rDPSCs)-derived exosomes on the polarization of macrophages.Methods The rDPSCs were isolated from the dental pulp of SD rats,and flow cytometry was used to detect the positive rates of rDPSCs surface markers CD14,CD34,CD45,CD90,CD105,and CD146,and oil red O staining and alizarin red S staining were used to observe the osteogenic and adipogenic abilities of rDPSCs.MiR-124 mimics(miR-124 mimics)and its negative control(miR-NC)were transfected into rDPSCs using cell transfection technology,and the rDPSCs were divided into blank group,miR-NC group,miR-124 group,and the exosomes of transfected rDPSCs were extracted and named as Exosome group(Exo group),Exo/miR-NC group,Exo/miR-124 group respectively,the morphology of exosomes was observed with transmission electron microscope;the expression levels of exosomal marker proteins CD63 and CD81 were detected with Western blot;the expression level of miR-124 in rDPSCs and rDPSCs exosomes was detected with qRT-PCR.Twenty-five rats were randomly divided into sham group,model group,rDPSCs Exo group,rDPSCs Exo/miR-NC group and rDPSCs Exo/miR-124 group,with five rats in each group.A skin defect model was established,and injected with 30μl of corresponding exosomes into the corresponding areas of the skin defect to intervene,and qRT-PCR was used to detect the expression levels of miR-124 in rat traumatic tissues;ELISA method was used to detect the serum levels of inducible nitric oxide synthase(iNOS)and mannose receptor(CD206),Western Blot was used to detect the protein expression levels of tumor necrosis factor(TNF)-α,TNF-1β,interleukin(IL)-6,IL-4,IL-10,and IL-13 in rat trauma tissues.Results The positive rates of rDPSCs surface markers-CD14,CD34,and CD45 were lower than those of CD90,CD105,and CD146,and rDPSCs had osteogenic and adipogenic abilities.The transmission electron microscopy showed that exosomes were uniform in size,with a diameter of 50-100nm,and a round or elliptical vesicle structure,and CD63 and CD81 proteins were highly expressed in rDPSCs exosomes.Compared with those in blank group and miR-NC group,the expression levels of miR-124 in rDPSCs in miR-124 group were significantly increased,amoreover,which in Exo/miR-124 group were significantly higher than those in in Exo group and Exo/miR-NC group(P<0.05).Compared with those in sham group,the serum levels of iNOS,TNF-α,TNF-1βand IL-6 in wound tissue of model group were significantly increased,however,the expression levels of CD206,IL-4,IL-10,and IL-13 proteins were significantly decreased(P<0.05).Compared with those in rDPSCs Exo group and rDPSCs Exo/miR-NC group,the expression levels of iNOS,TNF-α,TNF-1βand IL-6 in rDPSCs Exo/miR-124 group were significantly decreased,however,the expression levels of CD206,IL-4,IL-10,and IL-13 were significantly increased(P<0.05).Conclusion The miR-124 overexpression-modified rDPSCs exosomes can regulate the inflammatory response by inhibiting the polarization of M1 macrophages and promoting the polarization of M2 macrophages.
作者
刘兰宁
唐焕珍
李晓媛
LIU Lanning;TANG Huanzhen;LI Xiaoyuan(Department of Stomatology,Hospital of Integrated Traditional Chinese and Western Medicine of Southern Medical University,Guangdong,Guangzhou 510315,China)
出处
《河北医药》
CAS
2022年第12期1769-1774,共6页
Hebei Medical Journal
