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目的 OLETF大鼠醌式二氢生物喋呤还原酶(Quinoid dihydropteridine reductase,QDPR)存在K93T点突变,QDPR催化醌型二氢生物喋呤还原成四氢生物喋呤(tetrahydrobiopterin,BH4);二氢叶酸还原酶(dihydrofolate reductase,DHFR)能将二氢喋呤还原成BH4,提示生物喋呤代谢与叶酸代谢通路之间有串扰。文中通过研究QDPR基因表达水平对肾小管上皮NRK-52E细胞DHFR表达的影响,探讨QDPR基因在糖尿病肾病(diabetic nephropathy,DN)中的作用机制。方法 Western blot检测高糖环境下NRK-52E细胞及OLETF大鼠DHFR蛋白表达情况。利用慢病毒技术感染NRK-52E细胞,制作空载过表达、过表达QDPR敲低随机序列对照和敲低QDPR模型。每组再分别给予5.4 mmol/L正常糖培养基和30 mmol/L高糖培养基培养细胞72 h,模拟DN模型。实验分组:NRK-52E对照组、NRK-52E高糖组、空载过表达病毒对照组、空载过表达病毒高糖组、QDPR基因过表达组、QDPR基因过表达高糖组、敲低随机序列对照组、敲低随机序列高糖组、QDPR基因敲低组、QDPR基因敲低高糖组。观察高糖环境QDPR基因的表达情况对DHFR蛋白表达水平的影响。结果 NRK-52E高糖组DHFR蛋白含量(0.33±0.16)低于NRK-52E对照组(0.64±0.05),差异有统计学意义(P<0.05);OLETF大鼠DHFR蛋白的表达水平(1.03±0.12)明显低于LETO大鼠的表达水平(1.56±0.16),差异有统计学意义(P<0.01)。与空载过表达病毒高糖组(0.63±0.08)相比,QDPR基因过表达高糖组DHFR蛋白含量(0.12±0.09)降低,差异有统计学意义(P<0.01);与敲低随机序列对照组(0.52±0.08)相比,QDPR基因敲低高糖组DHFR表达量(0.62±0.27)差异无统计学意义(P>0.05)。结论DHFR蛋白在DN的发生发展中可能起到重要作用,QDPR基因过表达使DHFR蛋白表达含量下降,QDPR基因过表达通过下调DHFR蛋白的表达水平进而影响DN的发展。
OBJECTIVE: The K93T point mutation was found in quinoid dihydropteridine reductase (QDPR) in OLETF rats, and the quinone dihydrobiopterin was reduced by QDPR to tetrahydrobiopterin (BH4). Dihydrofolate Dihydrofolate reductase (DHFR) can reduce dihydrotopterine to BH4, suggesting crosstalk between biopterin metabolism and the folate metabolic pathway. In this paper, we investigated the effect of QDPR gene expression on the expression of DHFR in renal tubular epithelial cell line NRK-52E and the mechanism of QDPR gene in diabetic nephropathy (DN). Methods The expression of DHFR protein in NRK-52E cells and OLETF rats under high glucose condition was detected by Western blot. NRK-52E cells were infected with lentivirus to generate empty overexpression, overexpression QDPR knockdown random sequence control and knockdown QDPR model. Each group were given 5.4 mmol / L normal glucose medium and 30 mmol / L high glucose medium cultured cells 72 h, simulated DN model. The experimental group was divided into three groups: NRK-52E control group, NRK-52E high glucose group, no-overexpression virus control group, no-overexpression high-glucose group, QDPR gene overexpression group, QDPR gene overexpression high glucose group, Sequence control group, knock-down random sequence high glucose group, QDPR gene knockdown group, QDPR gene knockdown high glucose group. To observe the effect of high glucose level QDPR gene expression on DHFR protein expression. Results The DHFR protein content in NRK-52E high glucose group (0.33 ± 0.16) was lower than that in NRK-52E control group (0.64 ± 0.05), the difference was statistically significant (P <0.05) 0.12) was significantly lower than LETO rats (1.56 ± 0.16), the difference was statistically significant (P <0.01). The content of DHFR protein in QDPR over-expressing high glucose group (0.12 ± 0.09) was lower than that in control group (0.63 ± 0.08), the difference was statistically significant (P <0.01) Compared with control group (0.52 ± 0.08), the expression of DHFR in QDPR gene knockdown hyperglycemia group (0.62 ± 0.27) showed no significant difference (P> 0.05). Conclusion DHFR protein may play an important role in the occurrence and development of DN. Overexpression of QDPR gene reduces the expression of DHFR protein, and over-expression of QDPR gene affects the development of DN by down-regulating DHFR protein expression.