[1]罗维,李显,王博发,等.共培养体系下小鼠巨噬细胞RAW264.7与骨骼肌细胞C2C12间的相互作用[J].第三军医大学学报,2019,41(20):1939-1946.
 LUO Wei,LI Xian,WANG Bofa,et al.Interaction between mouse macrophages RAW264.7 and myoblasts C2C12 in a co-culture system[J].J Third Mil Med Univ,2019,41(20):1939-1946.
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共培养体系下小鼠巨噬细胞RAW264.7与骨骼肌细胞C2C12间的相互作用(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
41卷
期数:
2019年第20期
页码:
1939-1946
栏目:
基础医学
出版日期:
2019-10-30

文章信息/Info

Title:
Interaction between mouse macrophages RAW264.7 and myoblasts C2C12 in a co-culture system
作者:
罗维李显王博发艾磊周越
南京体育学院运动健康学院运动康复教研室;北京体育大学运动生理学教研室;江苏省体育科学研究所竞技体育与科研服务二中心
Author(s):
LUO Wei LI Xian WANG Bofa AI Lei ZHOU Yue

Department of Sports Rehabilitation, Nanjing Sport Institute, Nanjing, Jiangsu Province, 210014; Department of Exercise Physiology, Beijing Sport University, Beijing, 100084; Second Center of Competitive Sports Science Research, Jiangsu Provincial Research Institute of Sports Science, Nanjing, Jiangsu Province, 210033, China

关键词:
成肌细胞巨噬细胞细胞共培养成肌分化极化表型
Keywords:
myoblasts macrophages co-culture myogenic differentiation polarization phenotype
分类号:
R329.2;R329.4;R339.4
文献标志码:
A
摘要:

目的 建立巨噬细胞RAW264.7和成肌细胞C2C12共培养体系,检测该共培养体系下培养不同时长RAW264.7和C2C12间的相互作用。方法 Transwell小室内以成肌分化培养基共培养RAW264. 7和C2C12,相差显微镜观察细胞形态,共培养1、3、5 d后,CCK-8法检测细胞增殖能力,Western blot法检测Myf5、MyoD、myogenin、iNOS和Arg-1蛋白水平,ELISA法检测培养细胞上清液IL-1β和IL-10分泌量。结果①共培养对C2C12的影响:加快成肌分化进程、促进多核肌管形成,与同时间点对照组相比,共培养1 d即抑制Myf5蛋白表达(P<0.05),增加MyoD(P<0.05)和myogenin(P<0.01)蛋白表达;共培养3 d抑制Myf5蛋白表达(P<0.01),增加MyoD蛋白表达(P<0.01);共培养5 d降低细胞活性(P<0.05)并降低Myf5蛋白表达(P<0.01),增加肌管面积(P<0.01)。②共培养对RAW264.7的影响:共培养对细胞形态无明显影响,与同时间点对照组相比,共培养1 d抑制iNOS蛋白表达(P<0.01)和IL-1β分泌 (P<0.05),增加Arg-1蛋白表达(P<0.01);共培养3 d抑制iNOS蛋白表达和IL-1β分泌 (P<0.01),增加Arg-1蛋白表达(P<0.01)和IL-10分泌 (P<0.05);共培养5 d细胞簇集更加明显,促进细胞增殖(P<0.05),抑制iNOS蛋白表达和IL-1β分泌 (P<0.01),增加IL-10分泌 (P<0.05)。结论 C2C12和RAW264.7共培养促进成肌细胞的成肌分化以及巨噬细胞的增殖和M2型极化。

Abstract:

Objective To establish a co-culture system to observe the interactions between mouse macrophage RAW264.7 and myoblast C2C12 cells. MethodsRAW264.7 and C2C12 cell lines were co-cultured in a Transwell chamber containing conditioned medium for myogenic differentiation, and the changes in the cell morphology were observed under a phase-contrast microscope. On days 1, 3 and 5 of co-culture, the cell proliferation was assessed with CCK-8 assay, and the protein expressions of Myf5, MyoD, myogenin, iNOS and Arg-1 were detected using Western blotting; the concentrations of interleukin-1β (IL-1β) and IL-10 in the supernatant were detected using enzyme-linked immunosorbent assay (ELISA). ResultsCo-culture with RAW264.7 cells obviously accelerated the myogenic differentiation and promoted the formation of multinucleated myotubes in C2C12 cells. Compared with the cells cultured alone, C2C12 cells in the co-culture system showed significantly inhibited protein expression of Myf5 (P<0.05) and enhanced expressions of MyoD (P<0.05) and myogenin (P<0.01) on day 1, and inhibited expression of Myf5 (P<0.01) and increased expression of MyoD (P<0.01) on day 3; significantly lowered cell viability (P<0.05) and Myf5 expression (P<0.01) with increased area of myotubes (P<0.01) were observed on day 5. The co-culture did not produce significant effect on the morphology of RAW264.7 cells, but caused significant inhibition of iNOS expression (P<0.01) and IL-1β secretion (P<0.05) and increased Arg-1 expression (P<0.01) on day 1 of the co-culture; significantly lowered iNOS expression and IL-1β secretion (P<0.01) and increased Arg-1 expression (P<0.01) and IL-1β secretion (P<0.01) occurred in RAW264.7 cells on day 3 of the co-culture, and on day 5, the cells showed more obvious clustering with significantly enhanced proliferation (P<0.05), lowered iNOS expression and IL-1β secretion (P<0.01), and increased secretion of IL-10 (P<0.05). ConclusionCo-culturing C2C12 cells with RAW264.7 cells accelerates the myogenic differentiation of the former and promotes the proliferation and M2 polarization of the latter.

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更新日期/Last Update: 2019-10-25