QDPR基因表达变化对高糖环境下NRK-52E细胞GRP75表达的影响

发布时间：2018-05-19 06:09

  本文选题：糖尿病肾病 + 醌型二氢生物喋呤还原酶　； 参考：《华北理工大学》2017年硕士论文

【摘要】：目的探讨醌型二氢生物喋呤还原酶(Quinoid dihydropteridine reductase,QDPR)基因改变对高糖环境下肾小管上皮细胞系NRK-52E葡萄糖调节蛋白75(Glucoseregulated protein 75,GRP75)的影响及其在糖尿病肾病(Diabetic nephropathy,DN)中的可能作用机制。方法Western blot检测糖尿病大鼠OLETF及其对照鼠LETO肾皮质内GRP75蛋白的表达情况;利用慢病毒技术构建过表达及低表达QDPR基因及其对照的NRK-52E模型,分别于正常糖(5.4mmol/L)和高糖(30mmol/L)环境下培养72h,并分组如下:1 NRK-52E对照组(NC组)2 NRK-52E对照高糖组(NHG组)3空载过表达病毒对照组(LV-OCON组)4空载过表达病毒对照高糖组(LV-OCONHG组)5 QDPR基因过表达组(LV-QDPR组)6 QDPR基因过表达高糖组(LVQDPR-HG组)7敲低随机序列对照组(LV-SHCON组)8敲低随机序列对照高糖组(LV-SHCON-HG组)9敲低QDPR基因组(LV-SHQDPR组)10敲低QDPR基因高糖组(LV-SHQDPR-HG组)。用Western blot检测GRP75蛋白在细胞模型各组的表达水平,Propidium Iodide(PI)单染法检测细胞周期。结果1 OLETF大鼠肾皮质内GRP75蛋白含量明显低于对照组[(1.53±0.29)vs(0.79±0.28),P0.01],差异有统计学意义;2与NRK-52E对照NC组相比,NRK-52E对照高糖NHG组的GRP75蛋白含量降低[(0.62±0.04)vs(0.46±0.07),P0.05];3与NRK-52E对照NC组相比,NRK-52E对照高糖NHG组的G0/G1期细胞增多[(39.80±1.61)%vs(50.35±0.40)%,P0.01],S期细胞减少[(48.55±2.27)%vs(37.17±0.12)%,P0.05];4成功构建了过表达QDPR基因的NRK-52E细胞模型;5成功构建了敲低QDPR基因的NRK-52E细胞模型;6正常糖环境下,QDPR基因过表达LV-QDPR组的GRP75蛋白表达含量较空载过表达LVOCON组无明显变化(P0.05);与QDPR基因过表达LV-QDPR组及空载过表达病毒对照高糖LV-OCON-HG组相比,QDPR基因过表达高糖LV-QDPR-HG组的GRP75蛋白含量降低[(0.95±0.10)、(0.85±0.13)vs(0.45±0.20),P0.05];7与空载过表达病毒对照高糖LV-OCON-HG组相比,QDPR基因过表达高糖LVQDPR-HG组的G0/G1期细胞增多[(43.73±0.59)%vs(61.87±0.21)%,P0.01],S期细胞减少[(42.42±0.81)%vs(25.29±0.14)%,P0.01];8与敲低随机序列对照高糖LV-SHCON-HG组相比,敲低QDPR基因高糖LV-SHQDPR-HG组的GRP75表达量[(1.06±0.05)vs(1.00±0.11),P0.05]无明显变化;9与敲低随机序列对照高糖LV-SHCON-HG组相比,敲低QDPR基因高糖LV-SHQDPR-HG组的G2/M期细胞增多[(13.86±0.87)%vs(16.69±0.50)%,P0.01]、S期细胞减少[(42.78±1.46)%vs(38.59±0.16)%,P0.01],但G0/G1期细胞无明显变化[(43.36±1.19)%vs(44.71±0.48)%,P0.05]。结论1 GRP75蛋白在DN模型中表达含量减少,提示GRP75参与了DN的发病;2高糖环境下过表达NRK-52E细胞内QDPR基因能下调GRP75表达,诱导细胞周期阻滞,提示QDPR可能通过GRP75影响细胞周期进而影响DN的发生、发展。
[Abstract]:Objective to investigate the effect of Quinoid dihydropteridine reductase (Quinoid dihydropteridine reductase) gene change on NRK-52E glucose regulatory protein (75(Glucoseregulated protein 75) and its possible mechanism in diabetic nephropathy (DN). Methods Western blot was used to detect the expression of GRP75 protein in the renal cortex of OLETF and its control rats, and a NRK-52E model of overexpression and low expression of QDPR gene and its control was constructed by using lentivirus technique. They were cultured for 72 hours in normal glucose (5.4 mmol / L) and high glucose (30 mmol / L) environments, and were divided into two groups as follows: 1 NRK-52E control group, NC group, 2 NRK-52E control group, high glucose group, nil group, no expressed virus, control group, LV-OCON group, LV-OCONHG group. LV-SHCON-HG group (LV-SHCON-HG), LV-SHCON-HG group, LV-SHCON-HG group (LV-SHCON-HG group), LV-SHQQDPR group (LV-SHQDPR-HG group), LV-SHQDPR-HG group (LV-SHQDPR-HG group). The expression level of GRP75 protein in each cell model group was detected by Western blot and the cell cycle was detected by single staining of Propidium Iodide Pi. Results 1 the content of GRP75 protein in renal cortex of OLETF rats was significantly lower than that of control group [1.53 卤0.29)vs(0.79 卤0.28, P0.01]. There was a significant difference between NRK-52E group and NRK-52E control group. The GRP75 protein content of NRK-52E group in high-glucose NHG group was significantly lower than that of NRK-52E control group (0.62 卤0.04)vs(0.46 卤0.07 P 0.05). [0.62 卤0.04)vs(0.46 卤0.07 p0.05] the content of GRP75 protein in NRK-52E group was significantly lower than that in NRK-52E control group (P 0.05). The number of G0/G1 phase cells increased [39.80 卤0.40 1.61)%vs(50.35 卤0.40] S phase cells decreased [48.55 卤0.12 2.27)%vs(37.17 卤0.12] P0. 4 A NRK-52E cell model with overexpression of QDPR gene was successfully constructed, a NRK-52E cell model with low QDPR gene knockout was constructed under normal glucose environment, GRP75 protein expression in LV-QDPR group was successfully constructed. There was no significant change in the content of GRP75 protein in the over-expressed LVOCON group compared with the over-expressed QDPR gene LV-QDPR group and the high-glucose LV-OCON-HG group of the no-load overexpression virus group, and the GRP75 protein content in the over-expressed LV-QDPR-HG group was significantly lower than that in the non-expressed LVOCON group [0.95 卤0.10 + 0.85 卤0.13)vs(0.45 卤0.20] 7. Compared with high-glucose LV-OCON-HG group, the number of G0/G1 phase cells in high-glucose LVQDPR-HG group increased [43.73 卤0.59)%vs(61.87 卤0.21] S phase cells decreased [42.42 卤0.81)%vs(25.29 卤0.14 P0.01] compared with high-glucose LV-SHCON-HG group. 鏁蹭綆QDPR鍩哄洜楂樼硸LV-SHQDPR-HG缁勭殑GRP75琛ㄨ揪閲廩(1.06卤0.05)vs(1.00卤0.11),P0.05]鏃犳槑鏄惧彉鍖，

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