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

发布时间：2018-11-16 08:34

【摘要】：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,P0.05)。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,P0.01),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,P0.01)。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 small hairpin RNA (small hairpin RNA,shRNA lentivirus vector targeting G-protein coupled receptor 91 (G protein-coupled receptor 91) gene in rats and to explore the effect of GPR91 receptor on vascular endothelial growth factor (vascular endothlial growth factor, induced by high glucose. VEGF). Methods four pairs of specific single-stranded oligonucleotide chains targeting rat GPR91 (NM_001001518) were designed and synthesized. Age I and EcoR I restriction sites were introduced at both ends, respectively. After annealing, small fragments of double-stranded DNA, with adhesive ends were cloned into lentivirus vector pGCSIL-GFP,. The recombinant was identified by polymerase chain reaction (polymerase chain reaction,PCR) and DNA sequencing. The lentivirus shRNA interference vector and the auxiliary packaging vector were co-transfected into 293T cells and the virus particles were collected and the titer concentration was detected. The lentivirus shRNA interference vectors were screened by, Western blot after transfection of lentivirus vectors into RGC-5 cells. RGC-5 cells were stimulated with 45 mmol L-1 of high glucose for 24 h. The expression of VEGF was observed by ELISA method after GPR91 interference. Results the results of PCR and DNA sequencing showed that the construction of lentivirus vector pGCSIL-GFP-shGPR91 was correct. After packaging virus particles, the titer of pGCSIL-GFP-shGPR91-1,2,3,4 group virus concentrate was 1.5 脳 10 ~ 9 TU mL-1,1.5 脳 10 ~ 9 TU mL-1,3.0 脳 10 ~ 9 TU mL-1.. The expression of GPR91 protein in NC group (0.60 卤0.08) was not significantly different from that in blank group (0.62 卤0.07) after lentivirus particles were infected with RGC-5 cells (P 0.05). Compared with the blank group, the four lentivirus vector groups (0.48 卤0.05) (0.48 卤0.05) (0.30 卤0.04) and 0.11 卤0.06 (0.11 卤0.06) were able to silence the expression of GPR91 in different degrees, and the difference was statistically significant (F _ (49.03) P _ (0.01). PGCSIL-GFP-shGPR91-3 interference efficiency was the highest. ELISA results showed that the expression of VEGF protein in blank group, high glucose NC group and high glucose pGCSIL-GFP-shGPR91-3 group was (25.63 卤4.52) pg mL-1, respectively. (72.74 卤8.24) pg mL-1, (71.68 卤8.31) pg mL-1 and (46.77 卤6.21) pg mL-1, showed that GPR91 virus interference vector could significantly reduce VEGF secretion induced by high glucose. P0.01) Conclusion the shRNA lentivirus vector targeting rat GPR91 was successfully constructed and packaged. The constructed lentivirus vector can reduce the expression of VEGF in diabetic retinopathy and lay a foundation for further study on the role of GPR91 gene in diabetic retinopathy and animal gene therapy.
【作者单位】：苏州大学医学部;上海交通大学附属第六人民医院眼科;
【基金】：国家自然科学基金(编号:81070738) 上海市自然科学基金(编号:11JC1407702)~~
【分类号】：R587.2;R774.1

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