【摘要】：目的:觀察不同濃度鈣蛋白酶抑制劑ALLN對(duì)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(cè)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(cè)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(lái)研究不同濃度作用下不同時(shí)間點(diǎn)骨骼肌成纖維細(xì)胞增殖和凋亡情況并觀測(cè)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(wú)血清培養(yǎng)基培養(yǎng)下,6~72 h細(xì)胞開(kāi)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(duì)數(shù)生長(zhǎng)期且未出現(xiàn)分化;不同時(shí)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(duì)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(wú)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(shí)ALLN組與Ca2+組差異無(wú)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(shí)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 absorbance (Optical density,OD) for 72 h with different concentrations of Ca2 by MTT method, and detected the OD value of 872 h after the intervention of certain concentration of Ca2 and different concentration of ALLN by means of the MTT method, and after the intervention of certain concentration of Ca2 and different concentration of ALLN, the MTT method was used to detect the OD value of 0: 6, 12, 24, 24, 36, 6, 6, 4, 872 h, respectively. The apoptosis rate of 36 h was analyzed by flow cytometry with Annexin V-FITC/PI double staining after certain concentration of Ca2 and different concentration of ALLN, and the cell morphology of 36 h was observed by Giemsa staining method after the intervention of certain concentration of Ca2 and different concentration of ALLN. To study the proliferation and apoptosis of skeletal muscle fibroblasts at different time points. Results: the results of different concentrations of Ca2 intervention on C2C12 cell line: the results showed that the OD value of C2C12 cells began to proliferate rapidly at 672 h in serum-free medium compared with that in 0 24 h (P0.01) cells were logarithmic (P0.01). Growth stage and no differentiation; The OD value of Ca2 128 mmol/L group was the lowest at different time points. Secondly, the OD value of Ca2 16 mmol/L group was significantly lower than that of control group (P0.05) Ca2 of 16 mmol/L could inhibit the proliferation of C2C12 cell and induce the apoptosis of C2C12 cell. Ca2 of 16 mmol/L and ALLN of different concentration interfered with C2C12 cell line 61212224h36 h later. 嫻撳害涓，

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