二氢生物喋呤还原酶参与调控HEK293T细胞自噬作用的初步研究

目的探讨二氢生物喋呤还原酶（dihydropteridine reductase,QDPR）对HEK293T细胞自噬作用的影响。方法构建野生型QDPR和突变型QDPR重组质粒分别转染HEK293T细胞,并设空载体对照组。采用RT-PCR及Western blot方法检测空载体组,野生型QDPR组和突变型QDPR组自噬相关蛋白LC3和Beclin 1的表达量变化。结果 1）测序结果证实PCR扩增得到编码正常QDPR的cDNA序列正确以及突变的cDNA也在正确的位置突变;2）磷酸钙共沉淀法转染HEK293T细胞后,野生型QDPR和突变型QDPR融合蛋白成功表达;3）RT-PCR结果显示,与对照组相比,野生型QDPR组LC3基因水平明显上调（P〈0.05）,突变型QDPR组LC3基因水平与对照组相比无统计学差异;与对照组相比,野生型和突变型组Beclin1基因水平无统计学差异;4）Western blot结果显示,与对照组相比,野生型QDPR组LC3-II和Beclin1的蛋白表达量明显上调（P〈0.05）,但LC3-I的蛋白表达量无统计学差异,突变型QDPR组与对照组相比LC3-I,II和Beclin1的蛋白表达量均没有统计学差异（P〉0.05）。结论二氢生物喋呤还原酶能增强HEK293T细胞自噬相关基因LC3和Beclin 1的表达,提示其可能具有激活自噬作用的功能;二氢生物喋呤还原酶93位氨基酸的突变影响了其对细胞自噬作用的调控,降低了自噬标志分子LC3-I和Beclin1的基因表达。

Effects of QDPR on Regulating the Autophagy of HEK293T Cells

Objective To investigate the effects of QDPR on regulating the autophagy of HEK293T cells. Methods HEK293T cells were transiently transfected with recombinant plasmid DNA rQDPRwt and recombinant plasmid DNA rQDPRmut by calcium phosphate. After 72 h, the expression of rat QDPR in 293T cells was detected by Western Blot. Then, the effects of QDPR on autophagy related gene （including LC3 and Beclinl ） were analyzed by RT-PCR, and Western blot was used to monitor the changes in autophagy associated protein level. Results 1） The recombinant plasmid DNA rQDPRwt and recombinant plasmid DNA rQDPRmut were successfully constructed. 2）The fusion protein can express in HEK293T cell. 3） Compared with control vector group, the mRNA expression of LC3 was significantly up-regulated in rQDPRwt group （ P 〈 O. 05） , and the mRNA expression of Beclinl showed no significant difference among the 3 groups （ P 〉 0.05 ） ; 4） The Western blot analysis revealed that LC3-1I and Beclinl increased in rQDPRwt group when compared with control group, and there were no difference of protein levels among the 3 groups. The protein expression of LC3-I,II and Beclinl showed no significant difference between rQDPRmut group and control group. Conclusion QDPR may activate the autophagy of HEK293T cells by increasing the expression of autophagy associated genes of HEK293T ceils.