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

Construction and identification of shRNA lentiviral vector of G protein-coupled receptor 91

目的构建靶向大鼠G蛋白偶联受体91(G protein-coupled receptor 91,GPR91)基因的小发夹RNA(small hairpin RNA,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(vascular endothlial growth factor,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Age I和EcoR I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(polymerase chain reaction,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,Western blot法筛选有效的慢病毒shRNA干扰载体。使用45 mmol·L-1的高糖刺激RGC-5细胞24 h,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、1.5×109 TU·mL-1、3.0×109 TU·mL-1、3.0×109 TU·mL-1。将慢病毒颗粒感染RGC-5细胞后,Western blot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-shGPR91-3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。

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 on 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 plasmids were cotransfected into 293 T 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- 1）-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 × 109 TU·mL- 1,1. 5 × 109 TU·mL- 1,3. 0 × 109 TU·mL- 1,3. 0 × 109 TU·mL- 1. 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 ＆gt; 0. 05）. GPR91 expression was knocked down significantly 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 ＆lt; 0.01）, and the pGCSIL-GFP-shGPR91-3 had the most efficient interference（ F = 49. 03,P ＆lt; 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- 1,（ 72. 74 ±8. 24） pg·mL- 1,（ 71. 68 ± 8. 31） pg·mL- 1 and（ 46. 77 ± 6. 21） pg·mL- 1, respectively,the high glucose-induced VEGF expression was significant down-regulated after silencing GPR91 gene,the difference was statistically significant（ F = 30. 852,P ＆lt; 0. 01）. Conclusion 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 animal gene treatment via GPR91 gene.