本文選題：體外合成mRNA + 加亞嘉西科逆轉(zhuǎn)錄病毒��； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:本研究采用體外合成mRNA技術(shù),設(shè)計(jì)并體外轉(zhuǎn)錄合成穩(wěn)定表達(dá)JSRV-env mRNA和FGF21 mRNA;利用體外合成JSRV-env mRNA免疫小鼠產(chǎn)生特異性抗體,揭示體外合成mRNA在感染性疾病中的治療潛力,探索肺腺癌的發(fā)病因素,為進(jìn)一步預(yù)防我國(guó)呼吸道傳染病奠定體外實(shí)驗(yàn)基礎(chǔ);檢測(cè)體外合成FGF21mRNA對(duì)胰島素抵抗模型肝細(xì)胞的葡萄糖代謝的影響,為進(jìn)一步研究FGF21改善胰島素抵抗降血糖的機(jī)制奠定基礎(chǔ),為T2DM的治療探討新方法。方法:優(yōu)化PT7TS載體序列,插入目的序列經(jīng)電泳測(cè)序驗(yàn)證后,體外轉(zhuǎn)錄合成JSRV-env mRNA,將體外合成mRNA與魚精蛋白結(jié)合后皮下注射免疫BALB/c小鼠;利用慢病毒包裝系統(tǒng)制作JSRV假病毒,取免疫小鼠血清進(jìn)行假病毒為基礎(chǔ)的抗體中和試驗(yàn),檢測(cè)小鼠體內(nèi)針對(duì)JSRV-env mRNA的特異性抗體。體外合成GFP熒光標(biāo)記的FGF21 mRNA;將肝細(xì)胞細(xì)胞置于含34.4μmol/L重組胰島素和1μmol/L地塞米松10%FBS RPMI-1640培養(yǎng)基中培養(yǎng)72 h,誘導(dǎo)建立胰島素抵抗(IR))肝細(xì)胞模型;建立IR模型對(duì)照組、IR胰島素組、IR FGF21組、IR胰島素+FGF21組;利用葡萄糖氧化酶(GOD-POD)法試劑盒測(cè)定四組細(xì)胞葡萄糖吸收量,實(shí)時(shí)熒光定量PCR檢測(cè)細(xì)胞GLUT1 mRNA表達(dá)的影響,Western blot檢測(cè)細(xì)胞GLUT1的蛋白表達(dá)水平。結(jié)果:成功體外合成具有穩(wěn)定性的JSRV-env mRNA和FGF21 mRNA;重組了具有感染能力JSRV假病毒,假病毒體外中和試驗(yàn)檢測(cè)出JSRV-env mRNA免疫小鼠體內(nèi)產(chǎn)生針對(duì)JSRV的特異性抗體。IR狀態(tài)下肝細(xì)胞轉(zhuǎn)染FGF21后,葡萄糖吸收量與IR對(duì)照組相比上升(P0.05),并與胰島素產(chǎn)生協(xié)同作用,細(xì)胞的GLUT1 mRNA和蛋白表達(dá)量顯著增加(P0.05)。結(jié)論:成功體外合成JSRV-env mRNA和FGF21 mRNA。JSRV-env mRNA能夠表達(dá)產(chǎn)生針對(duì)JSRV-env的中和抗體。FGF21 mRNA可以改善胰島素抵抗模型細(xì)胞對(duì)葡萄糖的攝取。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R450

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