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中华口腔医学研究杂志(电子版) ›› 2021, Vol. 15 ›› Issue (04) : 198 -206. doi: 10.3877/cma.j.issn.1674-1366.2021.04.002

基础研究

M2型巨噬细胞抑制放射后骨髓间充质干细胞向肌成纤维细胞转化的实验研究
赵路1, 张哲儒1, 贾骏麒2, 宗春琳1, 景莉1, 郭凯1, 田磊1,()   
  1. 1. 军事口腔医学国家重点实验室,口腔疾病国家临床医学研究中心,陕西省口腔疾病临床医学研究中心,第四军医大学口腔医学院口腔颌面外科,西安 710032
    2. 解放军总医院第三医学中心口腔科,北京 100039
  • 收稿日期:2021-04-16 出版日期:2021-08-01
  • 通信作者: 田磊

The experimental study of M2 macrophages inhibiting the transformation of bone marrow mesenchymal stem cells into myofibroblasts after radiation

Lu Zhao1, Zheru Zhang1, Junqi Jia2, Chunlin Zong1, Li Jing1, Kai Guo1, Lei Tian1,()   

  1. 1. 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
    2. Department of Stomatology, The Third Medical Center, Chinese PLA General Hospital, Beijing 100039, China
  • Received:2021-04-16 Published:2021-08-01
  • Corresponding author: Lei Tian
  • Supported by:
    National Natural Science Foundation of China(81670964)
引用本文:

赵路, 张哲儒, 贾骏麒, 宗春琳, 景莉, 郭凯, 田磊. M2型巨噬细胞抑制放射后骨髓间充质干细胞向肌成纤维细胞转化的实验研究[J/OL]. 中华口腔医学研究杂志(电子版), 2021, 15(04): 198-206.

Lu Zhao, Zheru Zhang, Junqi Jia, Chunlin Zong, Li Jing, Kai Guo, Lei Tian. The experimental study of M2 macrophages inhibiting the transformation of bone marrow mesenchymal stem cells into myofibroblasts after radiation[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2021, 15(04): 198-206.

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(3 μm组),放射后的BMSC+M2型巨噬细胞Transwell小室(3 μm)共培养;实验组2(8 μm组),放射后的BMSC+M2型巨噬细胞Transwell小室(8 μm)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8 μm组的Transwell小室的效果(0.225 ± 0.018)要优于3 μm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8 μm Transwell小室将放射后的BMSC与M2型巨噬细胞共培养后,8 μm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8 μm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 (3 μm group) , the irradiated BMSCs were co-cultured with M2 macrophages through the Transwell chamber (3 μm) ; group two (8 μm group) , the irradiated BMSCs were co-cultured with M2 macrophages through the Transwell chamber (8 μm) ; group three (NC group) , irradiated BMSCs. The content of α-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 of α-SMA in BMSCs was significantly reduced, and the effect of the Transwell chamber of the 8 μm group (0.225 ± 0.018) was better than that of the 3 μm 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 8 μm Transwell chamber, the positive percentage of Col III (8.2 ± 0.5) % of BMSCs in the 8 μm 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 8 μm 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 of α-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.

图3 SD大鼠成熟的巨噬细胞倒置显微镜下观察 3A:可见成熟后的巨噬细胞大部分呈贴壁状态;3B:图3A的2倍放大,可见成熟的巨噬细胞形态多样,大部分细胞有伪足
图4 SD大鼠成熟的巨噬细胞荧光显微镜鉴定 对照组有极个别细胞或者杂质呈现阳性表达;实验组CD68表达的阳性细胞着色为(Dylight 594)红色
图5 M2型巨噬细胞诱导与鉴定 A:免疫荧光鉴定,对照组有个别杂质呈阳性表达,实验组M2型巨噬细胞特异性蛋白分子CD206的阳性表达率较高;B:CD206免疫荧光染色(Dylight 488绿色)结果统计学分析,实验组与对照组比较差异有统计学意义(aP<0.001)
图6 M2型巨噬细胞与4 Gy射线放射后的SD大鼠骨髓间充质干细胞(BMSC)共培养48 h后倒置显微镜下观察 A:NC组;B:3 μm组;C:8 μm组
图7 4 Gy射线放射后的SD大鼠骨髓间充质干细胞(BMSC)与M2型巨噬细胞共培养48 h后细胞内α-平滑肌肌动蛋白(α-SMA)的表达 A:Western blot检测结果;B:结果统计学分析图,可见3 μm组与NC组比较,α-SMA表达量明显减少(t = 24.06,aP<0.001),8 μm组与NC组比较,α-SMA表达量显著降低(t = 52.28,bP<0.001),8 μm组与3 μm组比较,差异明显(t = 18.71,cP<0.001)
图8 4 Gy射线放射后的SD大鼠骨髓间充质干细胞(BMSC)与M2型巨噬细胞共培养48 h后细胞内Ⅲ型胶原(ColⅢ)的表达水平 A:ColⅢ免疫荧光染色结果图,NC组为第三代SD大鼠BMSC接受4 Gy射线放射组,8 μm组为4 Gy射线放射后的SD大鼠BMSC与M2型巨噬细胞通过8 μm Transwell小室共培养组,DAPI为细胞核染色,ColⅢ特异性染色(Dylight 594)红色,Merge为DAPI和ColⅢ染色合并图;B:ColⅢ免疫荧光染色结果统计学分析图,8 μm组与NC组比较,差异具有统计学意义(aP<0.001)
图9 4 Gy射线放射后的SD大鼠骨髓间充质干细胞(BMSC)与M2型巨噬细胞共培养48 h后细胞内活性氧(ROS)的表达水平 A:ROS免疫荧光染色结果图,NC组为第三代SD大鼠BMSC接受4 Gy射线放射组,8 μm组为4 Gy射线放射后的SD大鼠BMSC与M2型巨噬细胞通过8 μm Transwell小室共培养组,DAPI为细胞核染色,ROS特异性染色为绿色,Merge为DAPI和ROS染色合并图;B:ROS免疫荧光染色统计学分析图,8 μm组与NC组比较,差异有统计学意义(aP = 0.0002)
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