外源性一氧化碳对体外培养的SH-SY5Y细胞的影响

Effects of exogenous carbon monoxide on neurocytes：experiment in vitro

目的研究不同浓度的外源性一氧化碳（CO）对体外培养的神经细胞SH—SY5Y的影响，进一步探讨CO中毒脑损伤的机制。方法SH—SYSY细胞在含有不同浓度CO的装置中培养12h，然后用MTI＇法检测细胞存活率，DNA琼脂糖凝胶电泳和流式细胞仪检测细胞凋亡的发生，以激光共聚焦显微镜荧光染色法检测细胞内活性氧的产生和线粒体膜电位的变化，用荧光酶标仪测定caspase-3的活性，并用Western印迹的方法检测Bax蛋白的表达水平。结果1000、10000ppmCO处理SH—SY5Y细胞12h后显著降低细胞的存活率；诱导细胞发生凋亡，凋亡率分别达29．03％和41．67％；激光共聚焦显微镜结果细胞内活性氧水平及caspase-3的活性显著升高；线粒体膜电位明显降低，红／绿荧光强度的比值由正常的5．97分别降低为1．56±0．07和0．34±0．02；而且细胞中Bax蛋白表达水平显著升高。而100ppm CO对体外培养的SH—SY5Y细胞的存活率、caspase-3的活性、活性氧水平、线粒体膜电位以及Bax蛋白的表达无明显影响。结论高浓度的外源性CO（1000、10000ppm）对体外培养的SH—SY5Y细胞造成损伤，具有明显的细胞毒性；而低浓度的CO（100ppm）对SH—SY5Y细胞没有明显的损伤作用。

Objective To study the effect of CO on nenroeytes. Methods Human neuroblastoma eells of the line SH-SY5Y were cultured and exposed to CO of the concentrations of 100, 1000, and 10 000 ppm for 12 h, and some SH-SYSY cells were cultured and exposed to 5% CO2 to be used as control group. The cell viability was observed with MTT assay. The DNA content and percentage of apoptosis were measured by flow cytometry and DNA agarose gel electrophoresis. Laser scanning eonfoeal microscopy （LSCM） was used to detect the reactive oxygen species and the reduction in mitoehondrial membrane potential. The activation of easpase-3 was measured with the caspase-3 activity assay kit. The expression of Bax was deteeted with Western blotting. Results After exposure to 1000 and 10 000 ppm CO for 12 h the viability of the cells decreased to 85.6 ± 9. 2 and 63. 4±8.1 respectively （ both P 〈 0. 05 ）, the apoptotie rates of the cells were 29. 03% and 41.67% respectively, DNA agarose gel electrophoresis showed clear DNA ladder band, the caspase-3 activity increased by 129% and 443% respectively, LSCM showed that the number of the SH-SY5Y cells decreased and the fluorescence intensity inside the cells increased signifieantly （both P 〈 0. 05 ）, the red/green fluorescence ratio inside the cells decreased from the baseline level of （5. 91 ± 0. 21 ） to （ 1.56 ± 0.07 ） and （ 0. 34 ± 0.02 ） respectively, and the Bax protein expression increased greatly. However, 100 ppm CO had no obvious effects on the cell viability, reactive oxygen species, mitochondrial membrane potential, caspase-3 activity, and expression of Bax. Conclusion High concentration CO has direct cytotoxicity on neurocytes cultured in vitro, but low concentration CO does not induce marked injuries of neurocytes.