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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。
Objective To investigate the effect of dihydropteridine reductase (QDPR) on autophagy in HEK293T cells. Methods Construction of wild-type QDPR and mutant QDPR recombinant plasmids were transfected HEK293T cells, and set empty vector control group. The expression of autophagy-related proteins LC3 and Beclin 1 in empty vector group, wild-type QDPR group and mutant QDPR group were detected by RT-PCR and Western blot. Results 1) Sequencing results confirmed that the cDNA sequence encoding normal QDPR was correctly amplified by PCR and the mutant cDNA was also mutated at the correct position. 2) After transfection with HEK293T cells by calcium phosphate co-precipitation method, wild-type QDPR and mutant QDPR fusion protein The expression of LC3 gene in wild-type QDPR group was significantly higher than that in control group (P <0.05). The LC3 gene level in mutant QDPR group was not significantly different from that of control group Compared with the control group, there was no significant difference in Beclin1 gene expression between wild type and mutant group.4) Western blot results showed that protein expression of LC3-II and Beclin1 in wild type QDPR group was significantly increased compared with control group (P < 0.05). However, there was no significant difference in LC3-I protein expression between the mutant QDPR group and the control group. There was no significant difference in the expression levels of LC3-I, II and Beclin1 between the two groups (P> 0.05). Conclusions Dihydrobiopterin reductase can enhance the expression of autophagy-related genes LC3 and Beclin 1 in HEK293T cells, suggesting that they may have the function of activating autophagy. The mutation of amino acid 93 of dihydrogen biopterin reductase The regulation of cell autophagy reduces the gene expression of autophagy marker molecules LC3-I and Beclin1.