转染VMAT_2基因的CHO细胞抵抗多巴胺毒性作用的研究

The VMAT_2 transgenic CHO cells resist dopamine toxicity

目的：探讨囊泡单胺转运体（vesicular monamine transporter-2，VMAT2）对多巴胺（dopamine，DA）毒性的抵抗作用。方法：采用细胞免疫荧光染色法测定VMAT2基因在转染的CHO细胞（VMAT2-CHO）中的表达：MTF比色法检测不同浓度DA作用下的野生型CHO（WT-CHO）和VMAT2-CHO细胞存活率；应用ELISA法DA检测试剂盒测定细胞内DA的水平；采用活性氧检测试剂盒测定细胞内活性氧的水平。结果：VMAT2-CHO细胞内有较强的特异性荧光表达，主要定位在核周区域，胞浆中也有散在的荧光；0．25～0．4mmol／L DA作用72h，VMAT2-CHO细胞存活率显著高于WT-CHO细胞（P〈0．05）；0．4mmol／L DA作用于WT-CHO和VMAT2-CHO细胞24h，VMAT2-CHO细胞内DA为（199．33±15．155）ng／mi，显著高于WT-CHO细胞（141．4±8．784）ng／ml（P〈0．01）；而细胞内活性氧（ROS）水平则由（144．490±5．295）U／106 cells增加至WT-CHO细胞的（217．895±15．885）U／10^6cells（P〈0．01）。结论：转染VMAT2的CHO细胞对DA引起的毒性有显著的抵抗作用。

Objective： To study the resistance of VMAT2 transgenic CHO cells to the toxicity of dopamine. was used to detect the expression of VMAT2 ; MTr assay was used to detect the viability of wild type china hamster ovary cells （WT-CHOs） and the VMAT2 transgeneic CHO cells （VMAT2-CHOs） with different concentrations of dopamine; meanwhile ELISA assay and ROS kit were respectively used to measure the dopamine level and reactive oxygen species of the cytoplasm. Result： The result indicated that there was an apparent green fluorescence at the circumference of the nucleus in VMAT2-CHO, but there was no fluorescence in WT-CHO. Being acted upon by dopamine for 72 h with the scope of 0.25-0.4 mmol/L, the viability of VMAT2-CHO was higher than that of WT-CHO （ P 〈 0.05） ; the dopamine level of VMAT2-CHO（ 199.33± 15. 155 ng/ml） was higher than that of WT-CHO（ 141.4± 8.784 ng/ml） （ P 〈 0.01 ） at 24 hours after being affected by dopamine, while the ROS decreased from 217. 895 ±15. 885 U/10^6 cells in WT-CHO to 144. 490 ± 5. 295 U/10^6 cells in VMAT2-CHO （P 〈 0.01 ）. Conclusion： VMAT2 transgenic CHO cells can resist the toxicity of dopamine.