地拉罗司对小鼠成肌细胞C2C12生物活性的影响

Effects of deferasirox on biologic activity of mouse myoblasts C2C12

目的模拟体内肌少症病理环境,观察铁螯合剂地拉罗司(deferasirox,DFS)对小鼠成肌细胞C2C12生物学活性的影响。方法构建模拟体内骨骼肌营养缺乏的细胞培养模型:体外培养小鼠成肌细胞C2C12,细胞分为3组:对照组、无血清培养基组和无血清培养基加DFS (10μmol/L)共孵育组,CCK-8法检测细胞增殖,流式细胞仪检测细胞凋亡; C2C12细胞在马血清作用下诱导分化,镜下观察细胞形态并计数骨骼肌肌管数目;构建模拟体内骨骼肌炎性损伤的细胞培养模型:C2C12细胞在马血清诱导下分化后分为3组:对照组、肿瘤坏死因子-α(tumour necrosis factor,TNF-α,10 ng/m L)干预组和TNF-α(10 ng/m L)加DFS (10μmol/L)共孵育组,丙二醛(malondialdehyde,MDA)检测试剂盒和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)试剂盒分别检测细胞MDA和GSH-Px水平,实时定量PCR法检测细胞肌球蛋白重链(myosin heavy chain,MyHC) mRNA和肌细胞生成素(myogenin,MyOG) mRNA的表达水平,镜下观察细胞形态并计数骨骼肌肌管数目。结果对照组、无血清培养基组和无血清培养基加DFS共孵育组细胞增殖最终OD值分比为2. 74±0. 01、0. 43±0. 01和0. 83±0. 01,细胞凋亡率分别为(7. 45±1. 73)%、(37. 82±2. 30)%和(25. 73±1. 42)%,肌管计数分别为9. 10±1. 45、1. 00±0. 33和3. 80±1. 14。C2C12细胞在无血清培养条件下增殖下降、凋亡率增加、细胞肌管形成减少,DFS干预后细胞增殖升高、凋亡率下降、肌管形成增加,均P<0. 05。对照组、TNF-α干预组和TNF-α加DFS共孵育组细胞内MDA含量分别为(1. 57±0. 01)、(3. 33±0. 14)和(2. 01±0. 19) nmol/L,GSH-Px含量分别为85. 44±7. 92、36. 26±7. 48和118. 50±17. 89,MyHC mRNA的表达量分别为1、0. 60±0. 14和1. 36±0. 13,MyOG mRNA的表达量分别为1、0. 88±0. 06和1. 57±0. 16,肌管计数分别为4. 30±1. 50、3. 30±0. 67和5. 30±2. 26。C2C12细胞诱导分化后,在TNF-α干预下,细胞MDA含量增加(P<0. 05)、GSH-Px含量下降(P<0. 05); DFS干预后细胞MDA含量下降(P<0. 05)、GSH-Px含量增加(P<0. 05),MyHC和MyOG mRNA表达上升(P<0. 05),肌管数目形成增加(P<0. 05)。结论 DFS可逆转C2C12细胞在营养缺乏和炎性损伤状态下的部分生物学活性指标的下降。

Objective To investigate the effects of iron chelator deferasirox (DFS) on biologic activity of mouse myoblasts C2C12 by simulating the pathological environment of sarcopenia in vivo . Methods A cell culture model simulating nutrient deficiency of skeletal muscle in vivo was construct. Mouse myoblast cells C2C12 were cultured in vitro . The cells were divided into 3 groups: control group, serum-free medium group, and serum-free medium plus DFS (10 μmol/L) group. The proliferation was evaluated by CCK-8 assay, and the apoptosis was detected by flow cytometry. After the differentiation of C2C12 cells under induction of horse serum, the cell morphology was observed under the microscope and the number of myotubes was counted. Another cell culture model simulating inflammatory injury of skeletal muscle in vivo was constructed. C2C12 cells under induction of horse serum were divided into 3 groups: control group, tumor necrosis factor alpha (TNF-α, 10 ng/mL) group, and TNF-α(10 ng/mL) plus DFS (10 μmol/L) group. The content of malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) was evaluated by MDA and GSH-Px detection kit respectively. The mRNA level of myosin heavy chain (MyHC) and myogenin (MyOG) were measured by real-time quantitative PCR. The cell morphology was observed under the microscope and the number of myotubes was counted. Results In control group, serum-free medium group and serum-free medium plus DFS group, OD value of cell proliferation was 2.74±0.01, 0.43±0.01 and 0.83±0.01, respectively;apoptosis rate was (7.45±1.73)%,(37.82±2.30)% and (25.73±1.42)%, respectively;the number of myotubes was 9.10±1.45, 1.00±0.33 and 3.80±1.14, respectively. C2C12 cells in the serum-free culture showed decreased proliferation activity (P <0.05), increased apoptosis rate (P <0.05), and decreased myosial formation (P <0.05). After DFS intervention, the proliferation activity was increased (P <0.05), and the apoptosis rate was decreased (P <0.05), and myosial formation was increased (P <0.05). In control group, TNF-α group and TNF-α plus DFS group, the content of MDA was (1.57±0.01),(3.33±0.14) and (2.01±0.19) nmol/L, respectively;the content of GSH-Px was 85.44±7.92, 36.26±7.48 and 118.50±17.89, respectively;the mRNA expression of MyHC was 1, 0.60±0.14 and 1.36±0.13, respectively;the mRNA expression of MyOG was 1, 0.88±0.06 and 1.57±0.16, respectively;the number of myotubes was 4.30±1.50, 3.30±0.67, 5.30±2.26, respectively.After differentiation under induce of horse serum, the content of MDA was increased (P <0.05), and GSH-Px was decreased in presence of TNF-α(P <0.05). After DFS intervention, the content of MDA was decreased (P <0.05), and GSH-Px was increased (P <0.05). After differentiation under induce of horse serum, the mRNA expression of MyHC and MyOG was increased (P <0.05), and the number of myotubes formed was increased (P <0.05). ConclusionDFS can reverse the decline of biological activity indicators of C2C12 cells under nutrient deficiency and inflammatory damage.