【摘要】：目的:觀察不同濃度鈣蛋白酶抑制劑ALLN對體外Ca2+處理的C2C12成肌細(xì)胞增殖和凋亡的影響。方法:本研究中我們采用不同濃度Ca2+干預(yù)后MTT法酶標(biāo)儀檢測0、6、12、24、36、48、72 h的吸光度(Optical density,OD)值;一定濃度Ca2+和不同濃度ALLN干預(yù)后MTT法酶標(biāo)儀檢測0、6、12、24、36、48、72 h的OD值;一定濃度Ca2+和不同濃度ALLN干預(yù)后Annexin V-FITC/PI雙染法流式細(xì)胞儀分析36 h的凋亡率;一定濃度Ca2+和不同濃度ALLN干預(yù)后Giemsa染色法顯微鏡觀察36 h的細(xì)胞形態(tài),來研究不同濃度作用下不同時間點(diǎn)骨骼肌成纖維細(xì)胞增殖和凋亡情況并觀測其凋亡形態(tài)。結(jié)果:不同濃度的Ca2+的干預(yù)C2C12細(xì)胞:MTT實(shí)驗(yàn)結(jié)果表明C2C12細(xì)胞在無血清培養(yǎng)基培養(yǎng)下,6~72 h細(xì)胞開始迅速增殖,36 h的OD值與0~24 h相比差異有統(tǒng)計(jì)學(xué)意義(P0.01)細(xì)胞處于對數(shù)生長期且未出現(xiàn)分化;不同時間點(diǎn)Ca2+128 mmol/L組細(xì)胞的OD值均為最低,其次為Ca2+16 mmol/L組、Ca2+32~64 mmol/L和Ca2+0.5~8 mmol/L,Ca2+組與對照組相比OD值明顯降低(P0.05);16 mmol/L的Ca2+可以抑制C2C12細(xì)胞增殖并誘導(dǎo)其凋亡;16 mmol/L的Ca2+和不同濃度的ALLN干預(yù)C2C12細(xì)胞6、12、24、36 h后ALLN1~7組(含終濃度為16 mmol/L的Ca2+和ALLN終濃度為3.125、6.25、12.5、25、50、100、200μmol/L的無血清培養(yǎng)基)OD值顯著高于Ca2+組(干預(yù)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;干預(yù)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;干預(yù)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;干預(yù)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);干預(yù)48、72 h時ALLN組與Ca2+組差異無統(tǒng)計(jì)學(xué)意義。16 mmol/L的Ca2+和ALLN終濃度為10、50、100、200μmol/L干預(yù)C2C12細(xì)胞36 h后:流式細(xì)胞儀分析表明ALLN 10、50、100、200μmol/L組的凋亡率分別為(6.00±1.20)%、(5.02±1.13)%、(4.89±1.111)%、(2.71±1.15)%均顯著低于Ca2+組(13.70±2.30)%(均P=0.000)。16 mmol/L的Ca2+和ALLN終濃度為10、50、100、200μmol/L干預(yù)C2C12細(xì)胞36 h后:Giemsa染色顯微鏡觀察顯示Ca2+組出現(xiàn)凋亡形態(tài)學(xué)改變,ALLN組的凋亡情況明顯改善。結(jié)論:16 mmol/L的Ca2+可誘導(dǎo)C2C12細(xì)胞凋亡,ALLN可抑制細(xì)胞凋亡、促進(jìn)增殖,該作用呈時間和劑量依賴性。
[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鹵0.007,0.414鹵0.006,0.434鹵0.004,0.441鹵0.003,0.460鹵0.010,0.484鹵0.006,0.525鹵0.006姣，

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