U251细胞MCT1、4在氧糖剥夺后的表达变化及其意义

Expression pattern of monocarboxylate transporter 1,4 in U251 glioma cells during oxygen-glucose deprivation and their significance

目的:观察氧糖剥夺(oxygen-glucose deprivation,OGD)后,神经胶质瘤细胞U251的乳酸转运和单羧酸转运蛋白(monocarboxylate transporter,MCT)1、4的表达变化,以探讨胶质瘤增殖生长的分子机制。方法:U251细胞随机分为对照组和模型组(OGD组),模型组的干预时间点为1、3、6 h。四甲基偶氮唑盐比色法(methyl thiazolyl tetrazolium,MTT)检测细胞增殖生长情况,HE染色观察细胞的形态变化,乳酸试剂盒检测细胞培养液中的乳酸含量,免疫荧光和免疫印迹观察MCT1和MCT4的表达变化情况。结果:与对照组相比,随着氧糖剥夺时间延长,模型组细胞增殖能力下降(对照组、OGD 1 h、OGD 3 h、OGD 6 h组的光密度值分别为0.584±0.031、0.537±0.046、0.441±0.079和0.200±0.011),细胞培养液中的乳酸浓度增高(对照组、OGD 1 h、OGD 3 h、OGD 6 h组乳酸浓度值分别为0.150±0.029、0.707±0.022、1.167±0.024和1.325±0.023)。MCT1、4的表达上调,并随氧糖剥夺时间延长其表达逐渐增强(MCT1:F=46.256,P=0.000;MCT4:F=247.400,P=0.000)。细胞培养液中加入MCTs的抑制剂4-CIN后,其乳酸浓度降低(对照组、150μmol/L组、425μmol/L组的乳酸浓度值分别为0.249±0.012、0.222±0.016、0.167±0.117)。结论:U251细胞氧糖剥夺后,随着细胞培养液中乳酸浓度增加,MCT1、4亦表达上调,而细胞的增殖生长能力下降;抑制MCT的表达,细胞培养液中的乳酸浓度降低,提示MCT1、4可能通过参与神经胶质瘤缺血缺氧微环境中乳酸的转运,进而调控细胞的增殖生长过程。

Objective：To examine expression patterns of monocarboxylate transporter 1,4 and lactate transporting in U251 malignant glioma cells with oxygen-glucose deprivation （OGD） so as to explore the possible the role of MCT 1,4 proliferating, aggressiveness in human glioma cells. Methods：U251 cells were randomly divided into two groups, namely, control group and OGD group. The cultures were incubated for certain periods（ 1,3,6 hours） with oxygen-glucose deprivation. Cell survival rate was tested by MTT assay,cell morphology was observed by HE staining. The lactic acid concentration was measured by lactate assay kit. The expression patterns of MCT1,4 were detected by immunofluorescence and Western blot. Results：Compared with those of control group, cell viability de- creased（the OD values of control group and OGD 1 hour, 3 hours, 6 hours were 0.584 ± 0.031,0.537 ± 0.046,0.441 ± 0.079, 0.200 ± 0.011 respectively） and lactate content increased in OGD group with oxygen-glucose deprivation time （the lactate content of control,OGD 1 h,3 h,6 h were 0.150 ±0.029,0.707 ± 0.022,1.167 ± 0.024, 1.325 ±0.023 respectively） ,and lactic acid content de- creased by adding 4-C IN, the inhibitor of MCTs （the lactate content of Control, 150 μmol/L, 425 μmol/L were 0.249 ± 0.012,0.222 ± 0.016,0.167 ±0.117）. The MCTI,d expression increased in the OGD group than in control group and continuously increased with the extension of oxygen-glucose deprivation time. Conclusion ： MCT1,4 increases and cell viability decreases in U251 glioma cells withthe up-regulation of lactic acid content after OGD. The lactic acid content decreases by adding 4-CIN. The above results suggest that MCT1,4 may be contributed to lactate transport of malig- nant glioma microenvironment, and regulate its proliferation and survival.