不同浓度ALLN对C2C12成肌细胞增殖和凋亡的影响

发布时间：2018-09-14 16:22

【摘要】：目的:观察不同浓度钙蛋白酶抑制剂ALLN对体外Ca2+处理的C2C12成肌细胞增殖和凋亡的影响。方法:本研究中我们采用不同浓度Ca2+干预后MTT法酶标仪检测0、6、12、24、36、48、72 h的吸光度(Optical density,OD)值;一定浓度Ca2+和不同浓度ALLN干预后MTT法酶标仪检测0、6、12、24、36、48、72 h的OD值;一定浓度Ca2+和不同浓度ALLN干预后Annexin V-FITC/PI双染法流式细胞仪分析36 h的凋亡率;一定浓度Ca2+和不同浓度ALLN干预后Giemsa染色法显微镜观察36 h的细胞形态,来研究不同浓度作用下不同时间点骨骼肌成纤维细胞增殖和凋亡情况并观测其凋亡形态。结果:不同浓度的Ca2+的干预C2C12细胞:MTT实验结果表明C2C12细胞在无血清培养基培养下,6~72 h细胞开始迅速增殖,36 h的OD值与0~24 h相比差异有统计学意义(P0.01)细胞处于对数生长期且未出现分化;不同时间点Ca2+128 mmol/L组细胞的OD值均为最低,其次为Ca2+16 mmol/L组、Ca2+32~64 mmol/L和Ca2+0.5~8 mmol/L,Ca2+组与对照组相比OD值明显降低(P0.05);16 mmol/L的Ca2+可以抑制C2C12细胞增殖并诱导其凋亡;16 mmol/L的Ca2+和不同浓度的ALLN干预C2C12细胞6、12、24、36 h后ALLN1~7组(含终浓度为16 mmol/L的Ca2+和ALLN终浓度为3.125、6.25、12.5、25、50、100、200μmol/L的无血清培养基)OD值显著高于Ca2+组(干预6 h:0.449±0.024、0.472±0.022、0.513±0.008、0.540±0.014、0.588±0.016、0.607±0.030、0.700±0.020比0.355±0.012,均P=0.000;干预12 h:0.407±0.007、0.414±0.006、0.434±0.004、0.441±0.003、0.460±0.010、0.484±0.006、0.525±0.006比0.368±0.027,均P=0.000;干预24 h:0.436±0.005、0.431±0.015、0.441±0.006、0.459±0.013、0.527±0.009、0.581±0.005、0.599±0.011比0.368±0.007,均P=0.000;干预36 h:0.464±0.022、0.460±0.018、0.461±0.007、0.434±0.020、0.454±0.028、0.479±0.006、0.524±0.011比0.379±0.011,均P=0.000);干预48、72 h时ALLN组与Ca2+组差异无统计学意义。16 mmol/L的Ca2+和ALLN终浓度为10、50、100、200μmol/L干预C2C12细胞36 h后:流式细胞仪分析表明ALLN 10、50、100、200μmol/L组的凋亡率分别为(6.00±1.20)%、(5.02±1.13)%、(4.89±1.111)%、(2.71±1.15)%均显著低于Ca2+组(13.70±2.30)%(均P=0.000)。16 mmol/L的Ca2+和ALLN终浓度为10、50、100、200μmol/L干预C2C12细胞36 h后:Giemsa染色显微镜观察显示Ca2+组出现凋亡形态学改变,ALLN组的凋亡情况明显改善。结论:16 mmol/L的Ca2+可诱导C2C12细胞凋亡,ALLN可抑制细胞凋亡、促进增殖,该作用呈时间和剂量依赖性。
[Abstract]:AIM: To observe the effects of different concentrations of calpain inhibitor ALLN on proliferation and apoptosis of C2C12 myoblasts treated with Ca2+ in vitro. METHODS: In this study, we measured the Optical density (OD) at 0, 6, 12, 24, 36, 48, 72 h by MTT assay after intervention with different concentrations of Ca2+ and different concentrations of ALLN. OD value of 0,6,12,24,36,48,72 h was detected by enzyme labeling instrument; apoptosis rate of 36 h was analyzed by Annexin V-FITC/PI double staining flow cytometry after intervention with certain concentration of Ca2+ and different concentration of ALLN; the morphology of cells was observed by Giemsa staining microscopy after intervention with certain concentration of Ca2+ and different concentration of ALLN for 36 h to study the skeleton at different time points under different concentrations. The proliferation and apoptosis of myofibroblasts were observed and the morphology of apoptosis was observed. Results: Different concentrations of Ca2+ interfered with C2C12 cells: MTT results showed that C2C12 cells began to proliferate rapidly in serum-free medium for 6-72 hours, and the OD value of 36 hours was significantly different from that of 0-24 hours (P 0.01). The OD value of the cells in Ca2+128 mmol/L group was the lowest at different time points, followed by Ca2+16 mmol/L group, Ca2+32~64 mmol/L and Ca2+0.5~8 mmol/L, Ca2+ group was significantly lower than the control group (P 0.05); 16 mmol/L Ca2+ could inhibit the proliferation of C2C12 cells and induce their apoptosis; 16 mmol/L Ca2+ and different concentrations of ALLN could interfere with C2C12 fine cells; After 6, 12, 24, 24, 36 h, the OD values of ALLN1-7 group (serum-free medium containing final concentrations of Ca2+ and ALN at 16 mmol/L and final concentrations of Ca2+ and ALN at 3.125, 6.25, 12.5, 25, 25, 50, 100, 200 micromol/L) were significantly higher than those of Ca2 + group (intervention 6 h: 0.449 [0.449 [0.024,0.024,0.472 [0.472 [.472 [0.472 [0.472 2, 0.513 [.513 [.008 0.008,0.540.540 [.540.014, 0.588 [0.588 [0.0.012, P = 0.000; 12 h after intervention :0.407卤0.007,0.414卤0.006,0.434卤0.004,0.441卤0.003,0.460卤0.010,0.484卤0.006,0.525卤0.006姣，

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