QDPR基因过表达、敲低及K93T突变对高糖环境下肾小管上皮细胞的影响及其机制

发布时间：2018-03-07 09:42

  本文选题：QDPR　切入点：糖尿病肾病　出处：《华北理工大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的通过研究QDPR基因过表达、敲低及K93T突变对高糖环境下肾小管上皮细胞TGF-β1、CTGF、VEGF蛋白含量的影响,探讨QDPR基因活性改变在DN发生发展中的作用及其机制。方法western blot方法检测QDPR蛋白在大鼠肝、脑、肾皮质、肾髓质中的表达情况。免疫组化方法检测QDPR蛋白在大鼠和人肾脏中的定位情况。用慢病毒感染肾小管上皮细胞系NRK-52E,制作QDPR过表达、敲低及K93T细胞模型。基于此模型,研究高糖环境下(30mmol/L)QDPR基因改变对TGF-β1、CTGF、VEGF蛋白含量的影响,实验采取以下分组:1 NRK-52E对照组(NRK-52E Control,NC)2 NRK-52E高糖组(NRK-52E exposed to High glucose,NHG)3空载过表达病毒对照组(overexpression lentivirus control,LV-OCON)4空载过表达病毒高糖组(overexpression lentivirus control exposed to High glucose,LV-OCONHG)5 QDPR基因过表达组(lentivirus overexpressed QDPR,LV-QDPR)6 QDPR基因过表达高糖组(lentivirus overexpressed QDPR exposed to High glucose,LVQDPR-HG)7 QDPR蛋白K93T突变组(lentivirus overexpressed K93T QDPR,K93T-QDPR)8 K93T突变过表达高糖环境组(lentivirus overexpressed K93T QDPR exposed to High glucose,K93T-QDPR-HG)9敲低对照组(LV-SHCON)10敲低对照高糖组(LV-SHCON-HG)11 QDPR基因敲低组(LV-SHQDPR)12 QDPR基因敲低对照组(LV-SHQDPR-HG)。每组细胞重复3皿,测定高糖环境下QDPR及K93T突变对其酶活性的影响;对各组用western-blot法检测高糖环境下QDPR表达改变及K93T突变对TGF-β1、CTGF和VEGF蛋白含量的影响。结果1 QDPR蛋白在大鼠肝、脑、肾皮质、肾髓质中高表达,胰腺中等量表达。QDPR蛋白在大鼠和人的肾脏,肾小管,尤其是近曲小管上皮细胞高表达。2通过慢病毒转染成功构建了QDPR基因过表达、敲低及K93T突变肾小管上皮细胞模型。3正常糖环境下K93T-QDPR组QDPR酶活性低于LV-QDPR组(P0.05),高糖环境下LV-QDPR-HG组酶活性低于LV-QDPR组(P0.05)。4高糖环境下NHG组TGF-β1蛋白含量高于NC组(P0.01)。正常糖环境下空载病毒LV-OCON组TGF-β1表达水平无明显改变;而高糖环境对TGF-β1含量影响不明显。过表达QDPR基因LV-QDPR组与LV-OCON组相比TGF-β1蛋白含量增高(P0.05);而高糖环境下LV-QDPR-HG与LV-QDPR组相比TGF-β1表达量显著增高(P0.01),与LV-OCON-HG组相比显著增高(P0.001)。正常糖环境下敲低对照组LV-SHCON与NC组相比TGF-β1表达量降低;而高糖环境下TGF-β1表达量无明显变化。敲低QDPR基因组LV-SHQDPR在正常和高糖环境下对TGF-β1表达量无明显影响。正常糖环境下K93T突变组与LV-QDPR组相比TGF-β1含量降低,在高糖环境下能使TGF-β1表达量增高,但低于LV-QDPR-HG组(P0.05)。5高糖环境下NHG组CTGF蛋白含量高于NC组(P0.05)。正常糖环境下空载病毒LV-OCON组与NC组相比CTGF表达含量无明显改变;而高糖环境下CTGF无明显改变。正常糖环境下过表达QDPR基因LV-QDPR组与LVOCON组相比CTGF表达含量无明显改变;而高糖环境下CTGF表达量与LVQDPR组相比增高(P0.01),与LV-OCON-HG组相比显著增高(P0.01)。正常糖环境下敲低对照组LV-SHCON与NC组相比CTGF表达量有降低的趋势;而高糖环境下LV-SHQDPR组CTGF表达量无明显改变。正常糖环境下K93T突变与LV-QDPR组相比CTGF表达含量增高(P0.05);而高糖环境能使CTGF表达量增高,但低于LV-QDPR-HG组(P0.05)。6高糖环境下NHG组VEGF蛋白含量低于NC组(P0.05)。空载病毒LV-OCON组与NC组相比VEGF水平增高;高糖环境下与NHG相比VEGF水平增高(P0.05)。正常糖环境下过表达QDPR基因LV-QDPR组与LV-OCON组相比VEGF蛋白含量无明显改变;高糖环境下VEGF表达量与LV-QDPR组相比降低(P0.05),也显著低于LVOCON-HG组(P0.05)。正常糖环境下敲低对照组LV-SHCON组与NC组相比VEGF表达量增高(P0.05);而高糖环境下VEGF表达量显著降低(P0.01)。敲低QDPR基因组LV-SHQDPR与LV-SHCON组相比VEGF表达量降低(P0.01);而高糖环境能使VEGF有减少的趋势。在正常糖环境下K93T突变组与LV-OCON、LV-QDPR组相比VEGF表达量变化不大;而在高糖环境下能使VEGF表达量降低,但高于LV-QDPR-HG组(P0.05)。结论1 QDPR基因能影响TGF-β1、CTGF及VEGF蛋白含量,提示过表达QDPR基因在一定程度上促进DN的发生发展。2敲低QDPR基因及K93T突变导致QDPR酶活性降低,在一定程度上延缓DN的进展。
[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缁勭浉姣擳GF-尾1琛ㄨ揪閲忔樉钁楀�為�，

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