發(fā)布時(shí)間：2018-05-19 06:09

  本文選題：糖尿病腎病 + 醌型二氫生物喋呤還原酶��； 參考：《華北理工大學(xué)》2017年碩士論文

【摘要】：目的探討醌型二氫生物喋呤還原酶(Quinoid dihydropteridine reductase,QDPR)基因改變對(duì)高糖環(huán)境下腎小管上皮細(xì)胞系NRK-52E葡萄糖調(diào)節(jié)蛋白75(Glucoseregulated protein 75,GRP75)的影響及其在糖尿病腎病(Diabetic nephropathy,DN)中的可能作用機(jī)制。方法Western blot檢測(cè)糖尿病大鼠OLETF及其對(duì)照鼠LETO腎皮質(zhì)內(nèi)GRP75蛋白的表達(dá)情況;利用慢病毒技術(shù)構(gòu)建過(guò)表達(dá)及低表達(dá)QDPR基因及其對(duì)照的NRK-52E模型,分別于正常糖(5.4mmol/L)和高糖(30mmol/L)環(huán)境下培養(yǎng)72h,并分組如下:1 NRK-52E對(duì)照組(NC組)2 NRK-52E對(duì)照高糖組(NHG組)3空載過(guò)表達(dá)病毒對(duì)照組(LV-OCON組)4空載過(guò)表達(dá)病毒對(duì)照高糖組(LV-OCONHG組)5 QDPR基因過(guò)表達(dá)組(LV-QDPR組)6 QDPR基因過(guò)表達(dá)高糖組(LVQDPR-HG組)7敲低隨機(jī)序列對(duì)照組(LV-SHCON組)8敲低隨機(jī)序列對(duì)照高糖組(LV-SHCON-HG組)9敲低QDPR基因組(LV-SHQDPR組)10敲低QDPR基因高糖組(LV-SHQDPR-HG組)。用Western blot檢測(cè)GRP75蛋白在細(xì)胞模型各組的表達(dá)水平,Propidium Iodide(PI)單染法檢測(cè)細(xì)胞周期。結(jié)果1 OLETF大鼠腎皮質(zhì)內(nèi)GRP75蛋白含量明顯低于對(duì)照組[(1.53±0.29)vs(0.79±0.28),P0.01],差異有統(tǒng)計(jì)學(xué)意義;2與NRK-52E對(duì)照NC組相比,NRK-52E對(duì)照高糖NHG組的GRP75蛋白含量降低[(0.62±0.04)vs(0.46±0.07),P0.05];3與NRK-52E對(duì)照NC組相比,NRK-52E對(duì)照高糖NHG組的G0/G1期細(xì)胞增多[(39.80±1.61)%vs(50.35±0.40)%,P0.01],S期細(xì)胞減少[(48.55±2.27)%vs(37.17±0.12)%,P0.05];4成功構(gòu)建了過(guò)表達(dá)QDPR基因的NRK-52E細(xì)胞模型;5成功構(gòu)建了敲低QDPR基因的NRK-52E細(xì)胞模型;6正常糖環(huán)境下,QDPR基因過(guò)表達(dá)LV-QDPR組的GRP75蛋白表達(dá)含量較空載過(guò)表達(dá)LVOCON組無(wú)明顯變化(P0.05);與QDPR基因過(guò)表達(dá)LV-QDPR組及空載過(guò)表達(dá)病毒對(duì)照高糖LV-OCON-HG組相比,QDPR基因過(guò)表達(dá)高糖LV-QDPR-HG組的GRP75蛋白含量降低[(0.95±0.10)、(0.85±0.13)vs(0.45±0.20),P0.05];7與空載過(guò)表達(dá)病毒對(duì)照高糖LV-OCON-HG組相比,QDPR基因過(guò)表達(dá)高糖LVQDPR-HG組的G0/G1期細(xì)胞增多[(43.73±0.59)%vs(61.87±0.21)%,P0.01],S期細(xì)胞減少[(42.42±0.81)%vs(25.29±0.14)%,P0.01];8與敲低隨機(jī)序列對(duì)照高糖LV-SHCON-HG組相比,敲低QDPR基因高糖LV-SHQDPR-HG組的GRP75表達(dá)量[(1.06±0.05)vs(1.00±0.11),P0.05]無(wú)明顯變化;9與敲低隨機(jī)序列對(duì)照高糖LV-SHCON-HG組相比,敲低QDPR基因高糖LV-SHQDPR-HG組的G2/M期細(xì)胞增多[(13.86±0.87)%vs(16.69±0.50)%,P0.01]、S期細(xì)胞減少[(42.78±1.46)%vs(38.59±0.16)%,P0.01],但G0/G1期細(xì)胞無(wú)明顯變化[(43.36±1.19)%vs(44.71±0.48)%,P0.05]。結(jié)論1 GRP75蛋白在DN模型中表達(dá)含量減少,提示GRP75參與了DN的發(fā)病;2高糖環(huán)境下過(guò)表達(dá)NRK-52E細(xì)胞內(nèi)QDPR基因能下調(diào)GRP75表達(dá),誘導(dǎo)細(xì)胞周期阻滯,提示QDPR可能通過(guò)GRP75影響細(xì)胞周期進(jìn)而影響DN的發(fā)生、發(fā)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 鹵0.29)vs(0.79 鹵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 鹵0.04)vs(0.46 鹵0.07 P 0.05). [0.62 鹵0.04)vs(0.46 鹵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 鹵0.40 1.61)%vs(50.35 鹵0.40] S phase cells decreased [48.55 鹵0.12 2.27)%vs(37.17 鹵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 鹵0.10 + 0.85 鹵0.13)vs(0.45 鹵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 鹵0.59)%vs(61.87 鹵0.21] S phase cells decreased [42.42 鹵0.81)%vs(25.29 鹵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緇勭殑GRP75琛ㄨ揪閲廩(1.06鹵0.05)vs(1.00鹵0.11),P0.05]鏃犳槑鏄懼彉鍖，

本文編號(hào)：1909011