發(fā)布時(shí)間：2018-03-07 09:42

  本文選題：QDPR　切入點(diǎn)：糖尿病腎病　出處：《華北理工大學(xué)》2015年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的通過(guò)研究QDPR基因過(guò)表達(dá)、敲低及K93T突變對(duì)高糖環(huán)境下腎小管上皮細(xì)胞TGF-β1、CTGF、VEGF蛋白含量的影響,探討QDPR基因活性改變?cè)贒N發(fā)生發(fā)展中的作用及其機(jī)制。方法western blot方法檢測(cè)QDPR蛋白在大鼠肝、腦、腎皮質(zhì)、腎髓質(zhì)中的表達(dá)情況。免疫組化方法檢測(cè)QDPR蛋白在大鼠和人腎臟中的定位情況。用慢病毒感染腎小管上皮細(xì)胞系NRK-52E,制作QDPR過(guò)表達(dá)、敲低及K93T細(xì)胞模型�；诖四Ｐ�,研究高糖環(huán)境下(30mmol/L)QDPR基因改變對(duì)TGF-β1、CTGF、VEGF蛋白含量的影響,實(shí)驗(yàn)采取以下分組:1 NRK-52E對(duì)照組(NRK-52E Control,NC)2 NRK-52E高糖組(NRK-52E exposed to High glucose,NHG)3空載過(guò)表達(dá)病毒對(duì)照組(overexpression lentivirus control,LV-OCON)4空載過(guò)表達(dá)病毒高糖組(overexpression lentivirus control exposed to High glucose,LV-OCONHG)5 QDPR基因過(guò)表達(dá)組(lentivirus overexpressed QDPR,LV-QDPR)6 QDPR基因過(guò)表達(dá)高糖組(lentivirus overexpressed QDPR exposed to High glucose,LVQDPR-HG)7 QDPR蛋白K93T突變組(lentivirus overexpressed K93T QDPR,K93T-QDPR)8 K93T突變過(guò)表達(dá)高糖環(huán)境組(lentivirus overexpressed K93T QDPR exposed to High glucose,K93T-QDPR-HG)9敲低對(duì)照組(LV-SHCON)10敲低對(duì)照高糖組(LV-SHCON-HG)11 QDPR基因敲低組(LV-SHQDPR)12 QDPR基因敲低對(duì)照組(LV-SHQDPR-HG)。每組細(xì)胞重復(fù)3皿,測(cè)定高糖環(huán)境下QDPR及K93T突變對(duì)其酶活性的影響;對(duì)各組用western-blot法檢測(cè)高糖環(huán)境下QDPR表達(dá)改變及K93T突變對(duì)TGF-β1、CTGF和VEGF蛋白含量的影響。結(jié)果1 QDPR蛋白在大鼠肝、腦、腎皮質(zhì)、腎髓質(zhì)中高表達(dá),胰腺中等量表達(dá)。QDPR蛋白在大鼠和人的腎臟,腎小管,尤其是近曲小管上皮細(xì)胞高表達(dá)。2通過(guò)慢病毒轉(zhuǎn)染成功構(gòu)建了QDPR基因過(guò)表達(dá)、敲低及K93T突變腎小管上皮細(xì)胞模型。3正常糖環(huán)境下K93T-QDPR組QDPR酶活性低于LV-QDPR組(P0.05),高糖環(huán)境下LV-QDPR-HG組酶活性低于LV-QDPR組(P0.05)。4高糖環(huán)境下NHG組TGF-β1蛋白含量高于NC組(P0.01)。正常糖環(huán)境下空載病毒LV-OCON組TGF-β1表達(dá)水平無(wú)明顯改變;而高糖環(huán)境對(duì)TGF-β1含量影響不明顯。過(guò)表達(dá)QDPR基因LV-QDPR組與LV-OCON組相比TGF-β1蛋白含量增高(P0.05);而高糖環(huán)境下LV-QDPR-HG與LV-QDPR組相比TGF-β1表達(dá)量顯著增高(P0.01),與LV-OCON-HG組相比顯著增高(P0.001)。正常糖環(huán)境下敲低對(duì)照組LV-SHCON與NC組相比TGF-β1表達(dá)量降低;而高糖環(huán)境下TGF-β1表達(dá)量無(wú)明顯變化。敲低QDPR基因組LV-SHQDPR在正常和高糖環(huán)境下對(duì)TGF-β1表達(dá)量無(wú)明顯影響。正常糖環(huán)境下K93T突變組與LV-QDPR組相比TGF-β1含量降低,在高糖環(huán)境下能使TGF-β1表達(dá)量增高,但低于LV-QDPR-HG組(P0.05)。5高糖環(huán)境下NHG組CTGF蛋白含量高于NC組(P0.05)。正常糖環(huán)境下空載病毒LV-OCON組與NC組相比CTGF表達(dá)含量無(wú)明顯改變;而高糖環(huán)境下CTGF無(wú)明顯改變。正常糖環(huán)境下過(guò)表達(dá)QDPR基因LV-QDPR組與LVOCON組相比CTGF表達(dá)含量無(wú)明顯改變;而高糖環(huán)境下CTGF表達(dá)量與LVQDPR組相比增高(P0.01),與LV-OCON-HG組相比顯著增高(P0.01)。正常糖環(huán)境下敲低對(duì)照組LV-SHCON與NC組相比CTGF表達(dá)量有降低的趨勢(shì);而高糖環(huán)境下LV-SHQDPR組CTGF表達(dá)量無(wú)明顯改變。正常糖環(huán)境下K93T突變與LV-QDPR組相比CTGF表達(dá)含量增高(P0.05);而高糖環(huán)境能使CTGF表達(dá)量增高,但低于LV-QDPR-HG組(P0.05)。6高糖環(huán)境下NHG組VEGF蛋白含量低于NC組(P0.05)�？蛰d病毒LV-OCON組與NC組相比VEGF水平增高;高糖環(huán)境下與NHG相比VEGF水平增高(P0.05)。正常糖環(huán)境下過(guò)表達(dá)QDPR基因LV-QDPR組與LV-OCON組相比VEGF蛋白含量無(wú)明顯改變;高糖環(huán)境下VEGF表達(dá)量與LV-QDPR組相比降低(P0.05),也顯著低于LVOCON-HG組(P0.05)。正常糖環(huán)境下敲低對(duì)照組LV-SHCON組與NC組相比VEGF表達(dá)量增高(P0.05);而高糖環(huán)境下VEGF表達(dá)量顯著降低(P0.01)。敲低QDPR基因組LV-SHQDPR與LV-SHCON組相比VEGF表達(dá)量降低(P0.01);而高糖環(huán)境能使VEGF有減少的趨勢(shì)。在正常糖環(huán)境下K93T突變組與LV-OCON、LV-QDPR組相比VEGF表達(dá)量變化不大;而在高糖環(huán)境下能使VEGF表達(dá)量降低,但高于LV-QDPR-HG組(P0.05)。結(jié)論1 QDPR基因能影響TGF-β1、CTGF及VEGF蛋白含量,提示過(guò)表達(dá)QDPR基因在一定程度上促進(jìn)DN的發(fā)生發(fā)展。2敲低QDPR基因及K93T突變導(dǎo)致QDPR酶活性降低,在一定程度上延緩DN的進(jìn)展。
[Abstract]:Objective through the study on the expression of QDPR gene knockdown and K93T mutation CTGF on renal tubular epithelial cells under high glucose condition TGF- beta 1, VEGF, protein content, activity of QDPR gene change the pathogenesis in DN. QDPR protein detection method Western blot method in rat liver, brain, renal cortex that expression in renal medulla. Immunohistochemical detection of QDPR protein localization in rat and human kidney. The slow virus infection in renal tubular epithelial cell line NRK-52E, QDPR overexpression and knockdown of K93T cell model. Based on this model, the research in high glucose environment (30mmol/L) QDPR gene CTGF the TGF- beta 1, VEGF protein content, take the following 1 groups: experimental group NRK-52E (NRK-52E Control NC) 2 NRK-52E (NRK-52E exposed to High high glucose group glucose, NHG) 3 no-load overexpression of the virus control group (overexpression lentiviru S control, LV-OCON) 4 unloaded over expression of virus (overexpression lentivirus control exposed high glucose group to High glucose, LV-OCONHG) 5 QDPR gene overexpression group (lentivirus overexpressed QDPR, LV-QDPR) 6 QDPR gene over expression of high glucose group (lentivirus overexpressed QDPR exposed to High glucose, LVQDPR-HG) 7 QDPR protein K93T mutation group (lentivirus overexpressed K93T QDPR K93T-QDPR, 8) K93T mutation expression of high glucose group (lentivirus overexpressed K93T QDPR exposed to High glucose, K93T-QDPR-HG) 9 knockdown control group (LV-SHCON) 10 knockdown control high glucose group (LV-SHCON-HG) 11 QDPR gene knockdown group (LV-SHQDPR) 12 QDPR gene knockdown in control group (LV-SHQDPR-HG). Each group was repeated 3 dish, determination and K93T QDPR in high glucose environment mutation effect on the enzyme activity of each group; with the method of Western-blot change and the expression of K9 QDPR detected in high glucose environment The 3T mutation of TGF- beta 1, CTGF and VEGF protein. Results of the 1 QDPR protein in rat liver, brain, renal cortex, high expression in renal medulla and renal.QDPR protein in rat and human kidneys, equal expression in the pancreas, especially high expression in proximal tubular epithelial cells by slow.2 a virus was successfully constructed over expression of QDPR gene knockdown and K93T mutation in the QDPR activity of K93T-QDPR group of renal tubular epithelial cell model.3 normal glucose environment is lower than that of LV-QDPR group (P0.05), high glucose group LV-QDPR-HG enzyme activity was lower than that of LV-QDPR group (P0.05.4) under high glucose group NHG TGF- beta 1 protein content higher than that of NC group (P0.01). No significant changes in normal glucose under no-load virus LV-OCON group TGF- beta 1 expression level; and the influence of high glucose on the expression of TGF- 1 was not obvious. Expression of QDPR gene in LV-QDPR group and LV-OCON group increased compared with TGF- beta 1 protein (P0.05) and LV-QDP in high glucose environment; R-HG涓嶭V-QDPR緇勭浉姣擳GF-尾1琛ㄨ揪閲忔樉钁楀楂，

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