几种细胞毒性评价方法在纳米氧化铝神经细胞毒性研究中的应用比较

目的 以微米氧化铝、纳米碳做对照,通过不同的细胞活力检测方法 观察纳米氧化铝对神经细胞活力的影响.方法 体外原代培养小鼠神经细胞,将纳米氧化铝、微米氧化铝、纳米碳均按正常对照组、62.5 μmol/L、125.0 μmol/L、250.0μmoL/L、500.0 μmol/L、1.0 mmol/L、2.0 mmol/L共7个剂量组分别染毒神经细胞,用细胞计数试剂(CCK-8)、噻唑蓝(MTT)及乳酸脱氢酶试剂(LDH)等方法 检测细胞活力570 nm波长处的吸光度值(A570值)]的变化.结果 (1)CCK-8法检测结果 显示,250.0 μmol/L～2.0 mmol/L纳米氧化铝染毒组细胞活力(A570值分别为0.878±0.009、0.823±0.016、0.647±0.008、0.594 ±0.013)小于微米氧化铝染毒组(A570值分别为0.960±0.008、0.951±0.036、0.833 ±0.008、0.708±0.012)和纳米碳染毒组(A570值分别为0.977±0.003、0.973±0.002、0.924±0.006、0.891±0.023),差异具有统计学意义(同剂量组纳米氧化铝与微米氧化铝比较,t值分别为-0.082、-0.128、-0.186、-0.114,P值均<0.01;与纳米碳比较,t值分别为-0.099、-0.150、-0.277、-0.297,P值均<0.01).(2)MTT法检测结果 显示,在500.0 μmol/L、1.0 mmol/L剂量下,纳米氧化铝染毒组细胞活力(A570值分别为0.648±0.095、0.575±0.061)小于微米氧化铝染毒组(A570值分别为0.830±0.044、0.816±0.014)和纳米碳染毒组(A570值分别为0.889±0.009、0.765±0.049),差异具有统计学意义(同剂量组纳米氧化铝与微米氧化铝比较,t值分别为-0.183、-0.242,P值均<0.01;与纳米碳比较,t值分别为-0.241、-0.190,P值均<0.01).(3)LDH法检测的结果 显示,纳米氧化铝染毒组在125.0 μmol/L～2.0 mmol/L剂量下的细胞LDH释放量(A570值分别为1.862±0.102、1.905±0.066、1.930±0.037、1.946±0.033、1.967±0.068)高于纳米碳染毒组(A570值分别为1.484±0.110、1.559 ±0.039、1.663±0.014、1.732±0.076、1.765 ±0.073),差异具有统计学意义(t值分别为-0.377、0.346、0.266、0.213、0.202,P值均<0.01);在125.0 μmol/L～1.0 mmol/L剂量下的LDH释放量高于微米氧化铝染毒组(A570值分别为1.578±0.011、1.639±0.025、1.727±0.024、1.808±0.020),差异具有统计学意义(t值分别为0.284、0.266、0.202、0.172,P值均<0.01).结论 纳米氧化铝对神经细胞活力的影响比微米氧化铝和纳米碳大;LDH方法 比CCK-8和MTT法更加适合检测纳米物质作用后的细胞活力变化.

Cell toxicity assessment methodologies applied in the study of the toxicity of nano-alumina to nerve cells

Objective To observe the effect of nano-alumina on nerve cell viability through different detection kits of cell viability, using micro-alumina and nano-carbon as controls. Methods Primary culturing nerve cells of mouse in vitro, which were exposed to 7 doses of 0 μmol/L, 62.5 μmol/L,125.0 μmol/L,250. 0 μmol/L,500. 0 μmol/L, 1.0 mmol/L,2. 0 mmol/L concentrations of nano-alumina (nano-Al) ,micro alumina (micro-Al) and nano-carbon (nano-C),detecting cell viability (A570 values) with CCK-8, MTT and LDH methods. Results (1) The results of CCK-8 kit showed that, in doses of 250. 0 μmol/L-2. 0 mmol/L,the cell viability values of nano-alumina (the values of A570 were 0. 878 ±0. 009,0. 823 ±0. 016,0. 647 ± 0. 008,0. 594 ± 0. 013, respectively) were significantly lower than that of micro-Al (the values of A570 were 0. 960 ± 0. 008,0. 951 ± 0. 036,0. 833 ± 0. 008,0. 708 ± 0. 012,respectively) and nano-C (the values of A570 were 0. 977 ± 0. 003,0. 973 ± 0. 002,0. 924 ± 0. 006,0. 891 ±0. 023, respectively). While, comparing nano-Al with the same dose of micro-Al, there was significant difference (the t values were - 0. 082, - 0. 128, - 0. 186, - 0. 114, respectively, P < 0. 01), and so as to the comparison of nano-Al with the same dose of nano-C (the t values were - 0. 099, - 0. 150, - 0. 277,-0. 297, respectively, P < 0. 01). (2) MTT results showed that in the doses of 500. 0 μmol/L and 1.0 mmol/L, the cell viability of nano-Al (the values of A570 were 0. 648 ± 0. 095 and 0. 575 ± 0. 061) were lower than that of micro-Al (the values of A570 were 0. 830 ± 0. 044 and 0. 816 ± 0. 014) and nano-C (the values of A570 were 0. 889 ± 0. 009 and 0. 765 ± 0. 049), and the differences were significant (nano-Al compared with the same dose of micro-Al, the t values were -0. 183 and -0.242,P<0.01; nano-Al compared with the same dose of nano-C,the t values were -0.241 and -0. 190,P<0.01). (3) LDH results showed that in the dose from 125.0 μmol/L to 2. 0 mmol/L,the LDH release of nano-Al group(the values of A570 were 1. 862 ± 0. 102, 1. 905 ± 0. 066, 1. 930 ± 0. 037, 1. 946 ± 0. 033, 1. 967 ± 0. 068,respectively) were higher than that of nano-C (the values of A570 were 1.484 ± 0. 110, 1. 559 ± 0. 039,1. 663 ±0. 014,1. 732 ± 0. 076, 1. 765 ± 0. 073, respectively), and the differences were significant (the t values were - 0. 377,0. 346,0. 266,0. 213,0. 202 ,respectively,P <0. 01). In the dose from 125.0 μmol/Lto 1.0 mmol/L, the LDH release of nano-Al group were higher than that of micro-Al (the values of A570 were 1. 578 ± 0. 011,1. 639 ± 0. 025,1. 727 ± 0. 024, 1. 808 ± 0. 020, respectively), and the differences were significant (the t values were 0. 284, 0. 266,0. 202,0. 172, respectively, P < 0. 01). Conclusion The toxicity of nano-Al is greater than nano-C and micro-Al on the viability of nerve cells; LDH is more suitable for detecting changes of cell viability after the effect of nano-materials than CCK-8 and MTT.