[1]李婷婷,胡健艳,吴强.靶向G蛋白偶联受体91的小发夹RNA慢病毒载体的构建及功能初步检测[J].眼科新进展,2014,34(8):705-709.[doi:10.13389/j.cnki.rao.2014.0193]
　Li Ting-Ting,HU Jian-Yan,WU Qiang.Construction and identification of shRNA lentiviral vector of G protein-coupled receptor 91[J].Recent Advances in Ophthalmology,2014,34(8):705-709.[doi:10.13389/j.cnki.rao.2014.0193]
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靶向G蛋白偶联受体91的小发夹RNA慢病毒载体的构建及功能初步检测
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
34卷
期数:
2014年8期
页码:
705-709
栏目:
实验研究
出版日期:
2014-08-05

文章信息/Info

Title:
Construction and identification of shRNA lentiviral vector of G protein-coupled receptor 91
作者:
李婷婷; 胡健艳; 吴强
江苏省苏州市，苏州大学医学部
Author(s):
Li Ting-Ting; HU Jian-Yan; WU Qiang
关键词:
小发夹RNA; 慢病毒属; G蛋白偶联受体91; 糖尿病性视网膜病变; 血管内皮生长因子
Keywords:
small hairpin RNA;  lentivirus;  G protein-coupled receptor 91 ;  diabetic retinopathy ;  vascular endothelial growth factor
DOI:
10.13389/j.cnki.rao.2014.0193
文献标志码:
A
摘要:
目的　构建靶向大鼠Ｇ蛋白偶联受体９１（Ｇｐｒｏｔｅｉｎ-ｃｏｕｐｌｅｄｒｅｃｅｐｔｏｒ９１，ＧＰＲ９１）基因的小发夹ＲＮＡ（ｓｍａｌｌｈａｉｒｐｉｎＲＮＡ，ｓｈＲＮＡ）慢病毒载体，探讨ＧＰＲ９１受体对高糖诱导下血管内皮生长因子（ｖａｓｃｕｌａｒｅｎｄｏｔｈｌｉａｌｇｒｏｗｔｈｆａｃｔｏｒ，ＶＥＧＦ）释放的调节作用。方法　设计合成４对针对大鼠ＧＰＲ９１（ＮＭ＿００１００１５１８）的特异性单链寡核苷酸链，两端分别引入ＡｇｅＩ和ＥｃｏＲＩ酶切位点，退火后得到小片段带黏性末端的双链ＤＮＡ，克隆入慢病毒载体ｐＧＣＳＩＬ-ＧＦＰ，行聚合酶链反应（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ，ＰＣＲ）和ＤＮＡ测序鉴定重组体。将各慢病毒ｓｈＲＮＡ干扰载体和辅助包装载体共转染２９３Ｔ细胞，收集病毒颗粒并行浓缩滴度检测。将各慢病毒载体转染ＲＧＣ-５细胞后，Ｗｅｓｔｅｒｎｂｌｏｔ法筛选有效的慢病毒ｓｈＲＮＡ干扰载体。使用４５ｍｍｏｌ?Ｌ－１的高糖刺激ＲＧＣ-５细胞２４ｈ，用ＥＬＩＳＡ法观察干扰ＧＰＲ９１后ＶＥＧＦ的表达情况。结果　ＰＣＲ及ＤＮＡ测序结果均显示慢病毒载体ｐＧＣＳＩＬ-ＧＦＰ-ｓｈＧＰＲ９１构建正确；包装病毒颗粒后，ｐＧＣＳＩＬ-ＧＦＰ-ｓｈＧＰＲ９１-１、２、３、４组病毒浓缩液的滴度依次为１．５×１０９ ＴＵ?ｍＬ－１、１．５×１０９ＴＵ?ｍＬ－１、３．０×１０９ＴＵ?ｍＬ－１、３．０×１０９ＴＵ?ｍＬ－１。将慢病毒颗粒感染ＲＧＣ-５细胞后，Ｗｅｓｔｅｒｎｂｌｏｔ检测显示ＮＣ组ＧＰＲ９１蛋白（０．６０±０．０８）空白组（０．６２±０．０７）的表达无明显差异（Ｆ＝４９．０３，Ｐ＞０．０５）。而与空白组相比，４个慢病毒载体组（０．４８±０．０５、０．３４±０．０６、０．３０±０．０４和０．１１±０．０６）均能不同程度沉默ＧＰＲ９１的表达，差异有统计学意义（Ｆ＝４９．０３，Ｐ＜００１），其中ｐＧＣＳＩＬ-ＧＦＰ-ｓｈＧＰＲ９１-３干扰效率最高。ＥＬＩＳＡ结果显示空白组、高糖组、高糖＋ＮＣ组、高糖＋ｐＧＣＳＩＬ-ＧＦＰ-ｓｈＧ-ＰＲ９１-３组ＶＥＧＦ蛋白表达分别为（２５．６３±４．５２）ｐｇ?ｍＬ－１、（７２．７４±８．２４）ｐｇ?ｍＬ－１、（７１．６８±８．３１）ｐｇ?ｍＬ－１和（４６．７７±６２１）ｐｇ?ｍＬ－１，表明ＧＰＲ９１病毒干扰载体可显著降低高糖引起的ＶＥＧＦ分泌，差异有统计学意义（Ｆ＝３０．８５２，Ｐ＜０．０１）。结论　本实验成功构建了靶向大鼠ＧＰＲ９１的ｓｈＲＮＡ慢病毒载体并进行病毒颗粒包装。所构建的慢病毒载体能够降低高糖作用下的ＶＥＧＦ表达，为进一步研究ＧＰＲ９１基因在糖尿病视网膜病变中的作用机制和动物基因治疗奠定基础。
Abstract:
Objective To construct a lentiviral vector of small hairpin RNA ( shRNA) of rat G protein-coupled receptor 91 gene ( GPR91 ) , and explore the regulative effects of GPR91 0n the vascular endothelial growth factor ( VEGF) secretion induced by high glucose. Methods Four special single strand oligonucleotide were selected according to rat GPR91 mRNA sequence , and double strand DNA containing the target sequence was chemically synthesized . annealed , and inserted into the lentivirus expression vector pGCSIL-GFP by double digestion with Age I and EcoR I. After being identified by polymerase chain reaction and sequencing , these plasnuds were cotransfected int0 293T cells to package lentiviral particles. The titer of virus was tested. The lentiviral vector particles were transducted into RGC-5 cells. Western blot was used to assess the gene silencing efficacy of these recombinants. High glucose (45 mmol . L -l ) -mediated VEGF expression was determined by ELISA in RGC-5 cells transducted with or without lentiviral vector. Results PCR and sequencing confirmed that lentiviral vectors had the correct structure and could express high titer of virus :1. 5 x 109 TU . mL.l .1. 5 x 109 TU . ITiL’1 ,3. 0 x 109 TU . mL.l .3. 0 x lOv TU . mL -’. After being transducted into RGC-5 cells, the expression of GPR91 had no obvious statistical difference between control group (0. 62 + 0. 07 ) and blank vector control group ( 0. 60 + 0. 08 ) (F = 49. 03 .P >0. 05 ) . GPR91 expression was knocked down sigruficantly by all of these lentiviral vectors (0. 48 + 0. 05 .0. 34 + 0. 06 .0. 30 + 0. 04 .0. 11 + 0. 06) at protein levels compared to the control group , the difference was statistically significant ( F = 49. 03 .P < 0. 01 ) . and the pGCSIL-GFP-shGPR91-3 had the most efficient interference ( F = 49. 03 .P < 0. 01) . ELISA showed the VEGF secretion of control group ,high glucose group , high glucose +NC group and high glucose + pGCSIL-GFP-shGPR91-3 were ( 25. 63 +4. 52) pg . mL-l , (72. 74 +8. 24) pg . mL ’l . (71. 68 + 8. 31) pg . mL -l and (46. 77 +6. 21 ) pg . mL ’l , respectively,the high glucose-induced VEGF expression was significant down-regulated after silencing GPR91 gene , the difference was statistically significant ( F = 30. 852 ,P <0. 01). Conclrision The lentivirus shRNA vector of GPR91 is constructed successfully. The lentivirus shRNA vector of GPR91 can decrease high glucose-induced VEGF expression in RGC-5 cells , which provides basement for assessing diabetic retinopathy mechanism and arumal gene treatment via GPR91 gene.

备注/Memo

备注/Memo:
国家自然科学基金（编号：８１０７０７３８），上海市自然科学基金（编号：１１ＪＣ１４０７７０２）
更新日期/Last Update: 2014-07-30