体外合成JSRV-env mRNA和FGF21 mRNA及二者生物功能初步研究

发布时间：2018-06-02 20:10

本文选题：体外合成mRNA + 加亚嘉西科逆转录病毒　；参考：《天津医科大学》2017年硕士论文

【摘要】：目的:本研究采用体外合成mRNA技术,设计并体外转录合成稳定表达JSRV-env mRNA和FGF21 mRNA;利用体外合成JSRV-env mRNA免疫小鼠产生特异性抗体,揭示体外合成mRNA在感染性疾病中的治疗潜力,探索肺腺癌的发病因素,为进一步预防我国呼吸道传染病奠定体外实验基础;检测体外合成FGF21mRNA对胰岛素抵抗模型肝细胞的葡萄糖代谢的影响,为进一步研究FGF21改善胰岛素抵抗降血糖的机制奠定基础,为T2DM的治疗探讨新方法。方法:优化PT7TS载体序列,插入目的序列经电泳测序验证后,体外转录合成JSRV-env mRNA,将体外合成mRNA与鱼精蛋白结合后皮下注射免疫BALB/c小鼠;利用慢病毒包装系统制作JSRV假病毒,取免疫小鼠血清进行假病毒为基础的抗体中和试验,检测小鼠体内针对JSRV-env mRNA的特异性抗体。体外合成GFP荧光标记的FGF21 mRNA;将肝细胞细胞置于含34.4μmol/L重组胰岛素和1μmol/L地塞米松10%FBS RPMI-1640培养基中培养72 h,诱导建立胰岛素抵抗(IR))肝细胞模型;建立IR模型对照组、IR胰岛素组、IR FGF21组、IR胰岛素+FGF21组;利用葡萄糖氧化酶(GOD-POD)法试剂盒测定四组细胞葡萄糖吸收量,实时荧光定量PCR检测细胞GLUT1 mRNA表达的影响,Western blot检测细胞GLUT1的蛋白表达水平。结果:成功体外合成具有稳定性的JSRV-env mRNA和FGF21 mRNA;重组了具有感染能力JSRV假病毒,假病毒体外中和试验检测出JSRV-env mRNA免疫小鼠体内产生针对JSRV的特异性抗体。IR状态下肝细胞转染FGF21后,葡萄糖吸收量与IR对照组相比上升(P0.05),并与胰岛素产生协同作用,细胞的GLUT1 mRNA和蛋白表达量显著增加(P0.05)。结论:成功体外合成JSRV-env mRNA和FGF21 mRNA。JSRV-env mRNA能够表达产生针对JSRV-env的中和抗体。FGF21 mRNA可以改善胰岛素抵抗模型细胞对葡萄糖的摄取。
[Abstract]:Objective: in this study, we designed and transcribed JSRV-env mRNA and FGF21 mRNAs stably by in vitro synthesis of mRNA, and synthesized JSRV-env mRNA in vitro to immunize mice to produce specific antibodies, so as to reveal the therapeutic potential of mRNA synthesis in infectious diseases in vitro. To explore the pathogenesis of lung adenocarcinoma, to lay a foundation for further prevention of respiratory infectious diseases in China in vitro, to detect the effect of synthesis of FGF21mRNA on glucose metabolism in hepatocytes of insulin resistance model. To further study the mechanism of FGF21 to improve insulin resistance and hypoglycemia, and to explore a new method for the treatment of T2DM. Methods: the PT7TS vector sequence was optimized and inserted into the target sequence after electrophoresis sequencing, then JSRV-env mRNAs were transcribed and synthesized in vitro, then the mRNA synthesized by in vitro binding with protamine was injected subcutaneously into BALB/c mice, and JSRV pseudoviruses were prepared by lentivirus packaging system. The serum of immunized mice was neutralized by pseudovirus based antibody neutralization test to detect the specific antibody against JSRV-env mRNA in mice. GFP fluorescent labeled FGF21 mRNAs were synthesized in vitro, and hepatocytes were cultured on 34.4 渭 mol/L recombinant insulin medium and 1 渭 mol/L dexamethasone 10s RPMI-1640 medium for 72 hours. We established IR model control group, IR insulin group, IR FGF21 group, IR insulin FGF21 group, the glucose oxidase assay kit was used to determine the glucose absorption of the four groups. The effect of real-time fluorescence quantitative PCR on the expression of GLUT1 mRNA in cells; Western blot was used to detect the protein expression of GLUT1. Results: stable JSRV-env mRNA and FGF21 mRNAs were successfully synthesized in vitro, and JSRV pseudovirus was recombined. In vitro neutralization test showed that JSRV-env mRNA immunized mice with specific antibody against JSRV. Ir induced hepatocyte transfection into FGF21. Compared with IR control group, glucose absorption increased P0.05 and synergistic effect with insulin. The expression of GLUT1 mRNA and protein increased significantly. Conclusion: the successful synthesis of JSRV-env mRNA and FGF21 mRNA.JSRV-env mRNA in vitro can produce neutralizing antibody against JSRV-env. FGF21 mRNA can improve glucose uptake of insulin resistant model cells.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R450

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