UVB对Jurkat T细胞自噬和UVRAG表达的影响

Effects of UVB on the Autophagy and Expression of UVRAG in Jurkat T Cells

目的观察UVB对人急性T淋巴细胞白血病细胞系细胞(Jurkat T细胞)自噬和紫外线抵抗相关基因(UVRAG)表达的影响。方法 UVB照射Jurkat T细胞,MTT法观察UVB照射对细胞的存活力的影响;Western blotting法检测UVB照射后细胞LC3Ⅱ/Ⅰ的变化;转染GFP-LC3质粒标记细胞,使用激光共聚焦显微镜观测照射后细胞LC3的分布和表达。qRT-PCR的方法检测照射后UVRAG和Beclin 1mRNA的表达。结果 UVB对Jurkat T细胞增殖有一定影响,呈现剂量、时间效应关系。UVB照射后,LC3Ⅱ/Ⅰ的水平随着观察时间的延长而增高。照射后12h,LC3Ⅱ/Ⅰ的水平随着照射强度增加而升高(P均<0.05);照射后24h,50mJ/cm2UVB照射组LC3Ⅱ/Ⅰ达到最大(P均<0.05)。UVB照射后细胞质有明显的GFP-LC3绿色荧光致密斑形成,成散点状分布。50mJ/cm2UVB照射组,随着观察时间的延长,UVRAG的mRNA表达明显增加,各组之间差异有统计学意义(P均<0.05);照射后12h,24h,100mJ/cm2作用后UVRAG的表达均低于50mJ/cm2(P均<0.05),高于25mJ/cm2(P均<0.05)。照射后不同时间组(12h,24h),100 mJ/cm2UVB照射后Beclin 1的表达均高于50mJ/cm2,但差异无统计学意义(P均>0.05),且均大于25mJ/cm2组(P均<0.05)。结论 UVB可以诱导Jukat T细胞产生自噬,UVRAG参与了UVB诱导自噬发生的过程。

Objective To study the effects of UVB on the autophagy and expression of UVRAG in Jurkat T cells. Methods The proliferation of Jurkat T after the exposure of UVB was measured by MTT assay at different times after the exposure to UVB. Following exposure to UVB （25,50 and 100mJ/cm^2） for 12 and 24hours ,the expression levels of LC3 Ⅱ/Ⅰ were detected by Western blotting. After labeling the ceils with transfected GFP- LC3 plasmid, the distribution and expression of LC3 in UVB irradiated cells were observed by a laser confocal microscopy. RT-PCR method was used to analyze the expression levels of UVRAG and beclinl mRNA after UVB irradiation. Results UVB irradiation inhibited Jurkat T cell proliferation in a dose- and time-dependent manner. The expression levels of LC3 Ⅱ/Ⅰ increased as the exposure time increased. After 12hours of UVB exposure, the expression levels of LC3 Ⅱ/Ⅰincreased as irradiation dosage increased （ P 〈 0.05 ）. The highest expression level of LC3 Ⅱ/Ⅰwas seen 24h after exposure to 50mJ/cm^2 UVB. Laser confocal microscopy showed the dense GFP LC3 - green fluorescence spots in the cytoplasm alter 24-hour exposure of 50 and 100 mJ/cm^2 UVB. UVB irradiation at a dosage of 50 mJ/cm^2 induced a time-dependent increase in UVRAG mRNA expression （ P 〈 0.05 ）. After exposure to UVB for either 12h or 24h, the expression levels of UVRAG mRNA in 100 mJ/cm^2 UVB irradiated cells were lower than that in 50 mJ/cm^2 UVB irradiated cells （ P 〈 0.05 ）, but higher than that in 25mJ/cm^2 UVB irradiated cells （ P 〈 0.05 ）. In contrast, irradiation with 100 mJ/cm^2 UVB for either 12h or 24h induced a slightly higher expression level of beclin 1 than 50 mJ/cm^2 （P 〉0.05）. In comparison to groups irradiated with 50 mJ/cm^2 and 100 mJ/cm^2 UVB, the expression level of beclin 1 was significantly lower following exposure to 25mJ/cm2 UVB （P 〈 0.05 ）. Conclusion UVB irradiation induces the autophagy in Jurkat T cells. UVRAG may play a role in the autophagy induced by UVB.