氧化应激对C2C12成肌细胞增殖与凋亡的影响

Effect of oxidative stress on myoblast C2C12 proliferation and apoptosis

目的:利用过氧化氢作为外源性活性氧来源,探讨氧化应激对C2C12成肌细胞增殖与凋亡的影响。方法:实验于2005-10/2006-01在广东省组织构建与检测重点实验室完成。C2C12成肌细胞(美国ATCC,批号CRL-1722TM);过氧化氢(汕头市光华化学药厂,批号20050519)。①C2C12细胞置于含100mg/L青霉素,100mg/L链霉素和体积分数为0.1胎牛血清的DMEM-F12培养基中常规培养。②采用四甲基偶氮唑盐法测定过氧化氢对C2C12细胞增殖的影响。取体外培养的C2C12细胞,胰酶消化后制成单细胞悬液,离心重悬,血细胞计数板进行细胞计数,细胞悬液密度为3.4×106L-1,接种至96孔板,200 μL/孔。实验共分5组:过氧化氢25,50,100,300 μmol/L浓度组、空白对照组,6个平行孔/组。各组在37℃、体积分数为0.05的CO2细胞培养箱内常规培养。待细胞融合率达80%且未出现细胞分化时,过氧化氢各浓度组分别向C2C12细胞中加入含对应浓度过氧化氢的无血清培养基,空白对照组则向C2C12细胞中加入单纯无血清培养基。各组细胞分别于过氧化氢处理0,6,12,24,36,48,60h后,每孔加入2g/L的四甲基偶氮唑盐液20μL,于酶联免疫仪570nm处检测各孔吸光度值。③过氧化氢对C2C12细胞凋亡的影响:采用Hoechst 33342/PI双染检测C2C12细胞凋亡。正常细胞核Hoechst着色为淡蓝色,形态呈圆形,内有较深的蓝色颗粒;中早期凋亡细胞核Hoechst着色呈亮蓝色,或核呈分叶,碎片状,边集;坏死或晚期凋亡的细胞核PI着红色,Hoechst因细胞膜破裂而不能着色。取体外培养的C2C12细胞,制备单细胞悬液过程同上,按1.8×107L-1密度接种至6孔板,5mL/孔。实验分组及干预措施同上,3个平行孔/组。处理36h后立即进行Hoechst33342/PI凋亡双染,荧光倒置显微镜下观察,各组随机取6个不同视野进行细胞计数,计数实验重复3次,取平均值作为细胞凋亡率。结果:①过氧化氢对C2C12细胞增殖的影响:细胞处理36h后与空白对照组比较,过氧化氢25 μmol/L浓度组平均吸光度值与之相近(0.207±0.014,0.199±0.023;t=0.677,P>0.05),即促进C2C12细胞增殖;过氧化氢50,100,300 μmol/L浓度组平均吸光度值均显著下降(0.182±0.027,0.149±0.009,0.052±0.012;t=1.990,8.251,20.004,P均<0.05),即抑制C2C12细胞增殖,且呈时效和量效关系。②过氧化氢对C2C12细胞凋亡的影响:细胞处理36h后,与空白对照组比较,过氧化氢25 μmol/L浓度组细胞凋亡率明显降低[(9.09±0.85)%,(4.61±0.67)%;t=22.95,P<0.01];过氧化氢50,100,300 μmol/L浓度组细胞凋亡率则明显提高[(33.50±5.74)%,(45.95±6.82)%,(76.47±4.66)%;t=4.35,4.68,18.02,P均<0.01],且呈时效和量效关系。结论:活性氧在C2C12成肌细胞增殖与凋亡过程中扮演重要角色,低浓度可促进C2C12细胞增殖,高浓度则能够抑制其增殖并促进凋亡,且呈时效和量效关系。提示氧化应激对成肌细胞的生长具有调节作用。

AIM： To explore the effect of oxidative stress on the proliferation and apoptosis of myoblast C2C12 by utilizing hydrogen dioxide （H=O2） to serve as exogenous active oxygen source. METHODS： The study was completed in the Guangdong Provincial Key Laboratory of Tissue Construction and Inspection between October 2005 and January 2006. C2C12 myoblast （American ATCC, lot number： CRL-1722^TM）; H2O2 （Guanghua Chemical-pharmaceutical Factory, lot number： 20050509）. ①C2C12 cells were commonly cultured in DMEM-F12 medium that contained 100 mg/L penicillinum, 100 mg/L phytomycin and 10% fetal cattle serum （FCS）.② MTT method was used to detect the effect of H=O2 on C2C12 cell proliferation. Firstly, C2C12 cells cultured in vitro were collected by trypsinization to form monoplast suspension, and then cantdfuged to float again, counted cellular numbers using blood counting plate. Eventually, the cells were inoculated into 96-shadow mask according to 200 μL per hollow and the cell suspension density of 3.4×10^6 L^-1. The experiment was divided into five groups： H2O2 concentration groups of 25, 50, 100, 300 μmol/L, respectively and blank control group, With 6 parallel hollows per group. All cells were cultured at 37 ℃ in 5% CO2 cell incubator. While cell fusion ratio arrived 80% and cell differentiation was not generated, DMEM-F12 medium containing various kinds H2O2 was added respectively to C2C12 cell according to the H2O2 concentrations, and pure DMEM-F12 medium was added to C2C12 cell in blank control group. After every group was treated With H2O= for 0, 6, 12, 24, 36, 48, 60 hours respectively, 20 μL MTT solution of 2 g/L concentration were added to each hollow, then enzyme-linked immunizing appearance was used to detect absorbance of each hollow at 570 nm wavelength.③Effect of H2O2 on C2C12 cell apoptosis： Hoechst33342/PI double staining was applied to detect C2C12 cell apoptosis. Hoechst coloration presented light blue, round morphous, and formed fairly dark particle inside normal cell nucleus; Hoechst coloration presented sapphidne or cell nucleus presented sublobe, fragment shape, fringe collection in middle and viable apoptotic cell; Because of cell membranolysis, Hoechst coloration was not present, but PI coloration showed red cell nucleus of apoptotic cell or necrosis cell. C2C12 cells were collected to prepare monoplast suspension same as above and were inoculated into 6-shadow mask according to 5 mL per hollow and the cell suspension density of 1.8×10^7 L^-1. The experiment grouping and intervention were both the same as above, except three parallel hollows per group. After treated by H2O2 for 36 hours, C2C12 cells were managed immediately with Hoechst33342/PI double staining; whereafter, they were observed by fluorescence inverted microscope. Six different eyesights were obtained randomly from each group to count cell numbers. The cell counting experiment needed to repeat three times and then take the average value as cell apoptotic ratio. RESULTS： ①Effect of H2O2 on C2C12 cell proliferation： After the cells were treated for 36 hours, the average absorbance was not conspicuously different in 25 μmol/L concentration group of H2O2 in contrast to blank control group （0.207±0.014, 0.199±0.023; t =0.677, P 〉 0.05）, suggesting that it notably promoted C2C12 cell proliferation; However, the average absorbance were conspicuously decreased in 50, 100, 300 μmol/L concentration group of H2O2 （0.182±0.027, 0.149±0.009, 0.052±0.012; t=1.990, 8251, 20.004; P〈0.05）, so theynotablysuppressed C2C12cell proliferation and presented seasoning and dose-effect relationships. ②Effect of H2O2 on C2C12 cell apoptosis： After the calls were treated for 36 hours, cell apoptotic ratio was conspicuously decreased in 25 μmol/L concentration group of H2O2 in contrast to blank control group [（9.09±0.85）%, （4.61±0.67）%; t =22.95, P〈 0.01]; However, cell apoptotic ratio were conspicuously increased in 50, 100, 300 μmol/L concentration groups of H2O2 [（33.50±5.74）%, （45.95±6.82）%, （76.47±4.66）%; t =4.35, 4.68, 18.02; P 〈 0.01], and presented seasoning and dose-effect relationships. CONCLUSION： Active oxygen acts as an important role in the process of C2C12 cell proliferation and apoptosis. At low concentration, it can promote C2C12 cell proliferation; At high concentration, it can suppress C2C12 cell proliferation and promote C2C12 cell apoptosis; And the effects present seasoning and dose-effect relationship. Above the experimental results suggest that oxidative stress can regulate the growth of C2C12 myoblasts.