摘要
目的研究M2型巨噬细胞对放射后的骨髓间充质干细胞(BMSC)向肌成纤维细胞转化的抑制作用。方法分别培养SD大鼠来源的BMSC和由巨噬细胞极化而来的M2型巨噬细胞,通过免疫荧光鉴定其表面标志物。首先建立BMSC体外细胞辐照模型,然后依据本模型将放射后的BMSC整体单纯随机抽样分为3组,每组3个孔进行研究:实验组1(3μm组),放射后的BMSC+M2型巨噬细胞Transwell小室(3μm)共培养;实验组2(8μm组),放射后的BMSC+M2型巨噬细胞Transwell小室(8μm)共培养;对照组(NC组),BMSC单纯放射组。利用Western blot检测各组BMSC内α⁃平滑肌肌动蛋白(α⁃SMA)的含量,免疫荧光技术检测Ⅲ型胶原(ColⅢ)、活性氧(ROS)的表达水平。实验数据应用SPSS 22.0软件进行统计学分析。结果SD大鼠BMSC培养基中加入巨噬细胞集落刺激因子(M⁃CSF)30 ng/mL培养6~9 d后可获得成熟的巨噬细胞;应用20 ng/mL白细胞介素4(IL⁃4)24 h后可诱导成熟的巨噬细胞极化为M2型巨噬细胞;放射后的BMSC与M2型巨噬细胞用Transwell小室共培养48 h后,BMSC中α⁃SMA表达量明显降低,且8μm组的Transwell小室的效果(0.225±0.018)要优于3μm组的效果(0.564±0.026),差异具有统计学意义(t=18.71,P<0.001)。通过8μm Transwell小室将放射后的BMSC与M2型巨噬细胞共培养后,8μm组BMSC中ColⅢ阳性百分比[(8.2±0.5)%]明显低于NC组[(60.7±1.5)%],差异有统计学意义(t=56.47,P<0.001),且8μm组中ROS阳性百分比[(9.3±1.7)%]较NC组[(25.9±1.6)%]亦显著降低,差异也有统计学意义(t=12.4,P=0.0002)。结论M2型巨噬细胞可以减少放射后的BMSC中α⁃SMA、ColⅢ的表达并减少ROS的生成,抑制放射后的BMSC向肌成纤维细胞的转化,可能对放射性颌骨骨坏死具有一定的治疗作用。
Objective To investigate the inhibitory effect of M2 macrophages on the transformation of bone marrow mesenchymal stem cells(BMSCs)into myofibroblasts after radiation.Methods Bone marrow mesenchymal stem cells derived from SD rats and M2 macrophages polarized from macrophages were cultured separately,and their surface markers were identified by flow cytometry.Firstly,a model of irradiated BMSCs in vitro was established.According to this model,the whole BMSC after radiation was randomly sampled and divided into three groups,with three holes in each group for research:group one(3μm group),the irradiated BMSCs were co⁃cultured with M2 macrophages through the Transwell chamber(3μm);group two(8μm group),the irradiated BMSCs were co⁃cultured with M2 macrophages through the Transwell chamber(8μm);group three(NC group),irradiated BMSCs.The content ofα⁃smooth muscle actin(α⁃SMA)in BMSCs of each group was determined by western blot.Immunofluorescence technology was used to detect the expression levels of collagen type Ⅲ(Col Ⅲ)and reactive oxygen species(ROS).The data of this experiment were statistically analyzed using t⁃test and One⁃Way analysis of variance(SPSS 22.0).Results The mature macrophages can be obtained by adding macrophage colony⁃stimulating factor(M⁃CSF)30 ng/mL to the bone marrow mesenchymal stem cell culture medium of SD rats for 6⁃9 days;the mature macrophages were induced to become M2 macrophages by 20 ng/mL interleukin⁃4 for 24 h.After co⁃culture of irradiated BMSCs and M2 macrophages through the Transwell chamber for 48 h,the expression level ofα⁃SMA in BMSCs was significantly reduced,and the effect of the Transwell chamber of the 8μm group(0.225±0.018)was better than that of the 3μm group(0.564±0.026),and the difference is statistically significant(t=18.71,P<0.001).After co⁃culturing the irradiated BMSCs with M2 type macrophages through the 8μm Transwell chamber,the positive percentage of Col Ⅲ(8.2±0.5)%of BMSCs in the 8μm group was significantly lower than that of the NC group(60.7±1.5)%(t=56.47,P<0.001),and the positive percentage of reactive oxygen species(9.3±1.7)%in the 8μm group was also significantly lower than that in the NC group(25.9±1.6)%(t=12.4,P=0.0002).Conclusions M2 macrophages can reduce the production ofα⁃SMA,Col Ⅲ and ROS in BMSCs after irradiation,and inhibit the transformation of BMSCs into myofibroblasts after radiation,which may have a potential therapeutic effect on the osteoradionecrosis of jaws.
作者
赵路
张哲儒
贾骏麒
宗春琳
景莉
郭凯
田磊
Zhao Lu;Zhang Zheru;Jia Junqi;Zong Chunlin;Jing Li;Guo Kai;Tian Lei(State Key Laboratory of Military Stomatology&National Clinical Research Center for Oral Diseases&Shaanxi Clinical Research Center for Oral Diseases,Department of Oral and Maxillofacial Surgery,School of Stomatology,the Fourth Military Medical University,Xi′an 710032,China;Department of Stomatology,The Third Medical Center,Chinese PLA General Hospital,Beijing 100039,China)
出处
《中华口腔医学研究杂志（电子版）》
CAS
2021年第4期198-206,共9页
Chinese Journal of Stomatological Research(Electronic Edition)
基金
国家自然科学基金(81670964)。
关键词
骨髓细胞
间质干细胞
巨噬细胞
肌动蛋白类
胶原Ⅲ型
活性氧
Bone marrow cells
Mesenchymal stem cells
Macrophages
Actins
Collagen typeⅢ
Reactive oxygen species
