CCM3基因缺陷对铅所致小鼠胚胎成纤维细胞遗传毒性影响的体外研究

Effect of CCM3 gene defect on lead-induced cell genotoxicity in mouse embryonic fibroblasts

目的探讨CCM3基因缺陷对醋酸铅所致小鼠胚胎成纤维细胞遗传毒性的影响。方法将C57雌鼠与CCM3基因敲除小鼠杂合雄鼠合笼后，取孕13．5d的胎鼠，原代分离培养胚胎成纤维细胞。基因分型后，野生型和CCM3基因缺陷型细胞分别给予6．25、12．50、25．00、50．00、100．00、200．00μmol／L醋酸铅处理，用MTS法检测细胞活性，并根据实验结果，选择6．25、25．00、100．00μmo]／L醋酸铅染毒，用双核阻滞法检测遗传毒性，用Westernblot法检测相关基因的蛋白表达情况。结果小鼠胚胎成纤维细胞经醋酸铅处理24h后，野生型细胞100．00μmol／L醋酸铅处理组（69．16±1．36）与对照组（100．00±2．33）相比，细胞活性下降了30％，杂合型细胞100．00μmo]／L醋酸铅处理组（87．16±5．50）与对照组（100±2．06）相比，细胞活性下降了13％，差异有统计学意义（F野生型=98．59，F杂合型=82．63，P值均〈0．001）。经醋酸铅处理48h后，野生型细胞100．00μmol／L醋酸铅处理组（51．99±5．62）与对照组（100．00±3．11）相比，细胞活性下降了50％，杂合型细胞100．00μmol／L醋酸铅处理组（66．33±4．06）与对照组（100．00±5．72）相比，细胞活性下降了35％，差异均有统计学意义（F野生型=82．63，F杂合型=36．86，P值均〈0．001）。双核微核实验结果显示．随着染毒剂量增加，两种基因型细胞的核分裂指数逐渐下降，双核细胞微核率呈逐渐升高趋势，野生型细胞对照组、6．25、25．00、100．00μmol／L剂量组的双核微核细胞率分别为29．6±2．2、47．3±6．6、55．5±9．1、66．8±3．5（／1000）；杂合型细胞的双核微核细胞率则分别为35．3±5．6、50．0±8．3、57．0±8．5、58．8±2．1（／1000）。Westernblot结果显示，野生型细胞100．00μmol／L醋酸铅处理组（0．70±0．03）的CCM3表达量是对照组（0．53±0．07）的1．32倍，杂合型细胞100．00μmol／L醋酸铅处理组（0．48±0．02）的CCM3表达量是对照组（0．27±0．04）的1．77倍，缺陷型细胞升高较多，差异有统计学意义（F野生型=14．77，F杂合型=25．74，P值均〈0．001）；野生型细胞100．00μmol／L醋酸铅处理组（0．69±0．03）的γ-H2AX表达量是对照组（0．65±0．07）的1．06倍，杂合型细胞100．013μmol／L醋酸铅处理组（0．99±0．04）的CCM3表达量是对照组（0．64±0．06）的1．55倍，差异有统计学意义（F野生型=7．08，P=0．012；F杂合型=13．49，P=0．002）。结论CCM3基因可能在铅所致小胚胎成纤维细胞的遗传毒性中发挥作用，在小鼠胚胎成纤维细胞毒性方面，铅暴露与CCM3基因存在交互作用。

Objective To investigate the effect of CCM3 gene defection on lead induced cell genotoxicity in mouse embryonic fibroblasts. Methods C57 female mice were mated with CCM3 gene heterozygous male mice. E13.5 embryos were taken to isolate primary mouse embryonic fibroblasts. After genotyping, wild type and heterozygous cells were treated with different doses of lead acetate. Cell viability, genotoxicity and protein expression were detected by MTS assay, CB mieronucleus method and Western blot,respectively. Results Mouse embryonic fibroblasts with lead acetate treatment for 24 h, wild-type cells 100.00 μmoL/L lead acetate-treated group （ 69. 16 ± 1.36 ） and the control group （ 100. 00± 2. 33 ） compared to cells decreased by 30% , CCM3 heterozygous type cell 100. 00 μmol/L lead acetate-treated group （87. 16 ±5.50） and the control group （100. 00 ± 2. 06） compared to cells decreased by 13%, the difference was statistically significant （ F values were 98.59,82. 63, P 〈 0. 001 ）. Lead acetate treatment after 48 h, wild-type cells 100. 00 μmol/L lead acetate-treated group （1 1.99 ± 5.62） and the control group （ 100.00± 3.11 ） compared to cells decreased by 50% , heterozygous type cells 100. 00 μmol/L lead acetate treatment group （66.33 ± 4. 06） and the control group （ 100.00 ± 5.72） compared to ceils decreased by 35% , the differences were statistically significant （F values were 82. 63,36. 86,P 〈 0. 001 ）. The results of CBMN test showed that with increased dose, micronucleus cell rate of two genotypes showed an increasing trend, in the wild-type cells, the micronucleus cell rate （/1 000） for the control group, 29.6 ± 2. 2, 6. 25 μmol/L dose group 47. 3 ± 6. 6, 25 μmol/L dose group 55.5 ± 9. 1, 100.00 μmol/L dose group 66. 8 ±3.5; heterozygous cells micronucleus cell rate （/1 000） for the control group, 35.3 ±5.6, 6. 25 μmol/L dose of 50. 0±8.5, 25.00μmol/L dose group 57.0 ±8.5, 100. 00 μmol/L dose group 58.8 ±2. 1. Micronucleus cell rates（/1 000） were significant differences, in 100. 00 μmol/L dose groups of two genotypes. Western blot results showed that wild-type cells CCM3 expression 100. 00 μmol/L lead acetate-treated group （ 0. 70 ± 0. 03 ） was 1.32 times higher than the control group （ 0. 53 ± 0. 07 ）, heterozygous ceils CCM3 expression 100.00 μmol/L lead acetate-treated group （0. 48 ±0. 02 ） was 1.77 times higher than control group that of 0. 27 ±0. 04, there was statistically significant difference （ F values were 14. 77,25.74, P 〈 0. 001 ） ; wild-type cells 3,-H2AX expression 100.00 μmol/L lead acetate-treated group （0.69 ± 0.03 ） was 1.06 times higher than the control group （ 0.65 ± 0.07 ）, heterozygous ceils γ-H2AX expression 100. 00 μmol/L lead acetate-treated group （0. 99± 0. 04） was 1.55 times higher than the control group CCM3 expression levels （ 0. 64 ± 0.06 ）, there was statistically significant difference （wild-type cells：F =7.08,P =0. 012,heterozygous type cell：F = 13.49,P = 0. 002）. Conclusion CCM3 gene may play a role in lead-induced genetic toxicity of mouse embryonic fibroblasts, CCM3 gene-lead interactions effects on mouse embryonic fibroblasts cell toxicity.