本文選題：miRNA + 載體��； 參考：《復(fù)旦大學(xué)》2013年碩士論文

【摘要】：第一部分 miR-200b/c/429過(guò)表達(dá)和敲降載體構(gòu)建及慢病毒包轉(zhuǎn) 目的：通過(guò)文獻(xiàn)檢索和生物信息學(xué)分析發(fā)現(xiàn)可能調(diào)控棕色脂肪發(fā)育過(guò)程中關(guān)鍵的轉(zhuǎn)錄因子PRDM16的miRNA,為了驗(yàn)證niRNA通過(guò)PRDM16對(duì)于棕色脂肪發(fā)育分化的調(diào)控,首先構(gòu)建miRNA的過(guò)表達(dá)質(zhì)粒和敲降miRNA表達(dá)的質(zhì)粒。并且通過(guò)慢病毒包轉(zhuǎn)驗(yàn)證慢病毒載體的構(gòu)建。 方法：1.克隆約500bp的包含miR的前體序列的片段,通過(guò)酶切、連接、轉(zhuǎn)化和測(cè)序等獲得重組的miR-200b/c/429過(guò)表達(dá)質(zhì)粒。2.根據(jù)miR-200b/c/429的成熟序列設(shè)計(jì)并合成anti-miRNA序列,然后通過(guò)克隆的構(gòu)建anti-miR-200b/c/429的重組質(zhì)粒。3.通過(guò)三質(zhì)粒體系包裝miRNA的過(guò)表達(dá)的慢病毒和敲降的miRNA的慢病毒。 結(jié)果：經(jīng)過(guò)PCR擴(kuò)增后瓊脂糖凝膠電泳驗(yàn)證目的片段大小約為500bp,經(jīng)過(guò)酶切和連接后構(gòu)建出重組質(zhì)粒。陽(yáng)性克隆測(cè)序結(jié)果顯示目的片段正確克隆入載體中。三質(zhì)粒體系構(gòu)建的轉(zhuǎn)染包裝細(xì)胞系后48小時(shí),細(xì)胞顯示綠色熒光。 結(jié)論：本研究成功構(gòu)建了niR-200b/c/429過(guò)表達(dá)載體和anti-miR-200b/c/429載體,并且慢病毒包裝成功。 第二部分 mir-200b/c/429的表達(dá)及其對(duì)靶基因的調(diào)控 目的：觀察miR-200b/c/429在高脂誘導(dǎo)老鼠的皮下白色脂肪和3T3-L1細(xì)胞系誘導(dǎo)成熟分化過(guò)程中的表達(dá)變化。構(gòu)建包含PRDM16基因3'UTR的雙熒光報(bào)告載體,通過(guò)雙熒光素實(shí)驗(yàn)驗(yàn)證]miR-200b/c/429對(duì)于PRDM16基因表達(dá)的調(diào)控。通過(guò)通過(guò)Q-PCR驗(yàn)證慢病毒干擾敲降3T3-L1中的miR-200b/c/429后,其中的miR-200b/c/429的表達(dá),并觀察1miR-200b/c/429表達(dá)量改變后,在3T3-L1其對(duì)PRDM16表達(dá)的影響。 方法：1、用Q-PCR的方法觀察miR-200b/c/429在高脂肪誘導(dǎo)的C57小鼠的皮下白色脂肪中的表達(dá),以及1niR-200b/c/429在3T3-L1前脂肪細(xì)胞系中誘導(dǎo)分化成熟過(guò)程中的表達(dá)的變化。4、通過(guò)構(gòu)建PRDM16的基因的雙熒光報(bào)告載體,并與miR-200b/c/429過(guò)表達(dá)質(zhì)粒共轉(zhuǎn)染到293FT細(xì)胞系中,觀察熒光表達(dá)的變化。5、通過(guò)慢病毒包轉(zhuǎn)后侵染3T3-L1細(xì)胞系,經(jīng)過(guò)8天的誘導(dǎo)分化,觀察PRDM16基因的表達(dá)的變化。 結(jié)果：1.成功構(gòu)建了PRDM16基因的3'UTR,并且通過(guò)與miR-200b/c/429的共轉(zhuǎn)染,觀察到miR-200b/c下調(diào)了熒光素的表達(dá),證明miR-200b/c可以調(diào)控PRDM16基因的3'UTR。2.在高脂肪誘導(dǎo)的C57BL/6小鼠的皮下脂肪中,miR-200b/c的表達(dá)量顯著地下調(diào),在3T3-L1誘導(dǎo)分化成熟過(guò)程中]miR-200b/c/429的表達(dá)量顯著性上調(diào)。3.通過(guò)慢病毒侵染anti-miR-200b/c/429干擾miR-200b/c/429的表達(dá)后,經(jīng)過(guò)誘導(dǎo)分化發(fā)現(xiàn)PRDM16的表達(dá)量上調(diào)。 結(jié)論：1.雙熒光素酶實(shí)驗(yàn)證明miR-200b/c下調(diào)PRDM16, PRDM16的表達(dá)受到miR-200b/c/429的調(diào)控。2. miR-200b/c/429在3T3-L1的誘導(dǎo)分化過(guò)程中表達(dá)量上調(diào),在高脂肪誘導(dǎo)的老鼠的皮下白色脂肪中miR-200b/c表達(dá)量下調(diào)。
[Abstract]:Part one Construction of miR-200b/c/429 overexpression and knock down Vector and Lentivirus Envelope Transformation Objective: to find out the possible regulation of PRDM16, a key transcription factor in brown fat development, by literature retrieval and bioinformatics analysis, in order to verify the regulation of niRNA on brown adipose development and differentiation by PRDM16. Firstly, the overexpression plasmid of miRNA and the plasmid of knockdown miRNA expression were constructed. The construction of lentivirus vector was verified by lentivirus envelope transfer. Method 1: 1. The fragment containing the precursor sequence of miR was cloned from about 500bp, and the recombinant miR-200b/c/429 overexpression plasmid. 2 was obtained by enzyme digestion, ligation, transformation and sequencing. According to the mature sequence of miR-200b/c/429, the anti-miRNA sequence was designed and synthesized, and then the recombinant plasmid. 3. 3 of anti-miR-200b/c/429 was cloned and constructed. Three plasmids were used to package lentivirus and lentivirus of miRNA and miRNA. Results: the size of the target fragment was about 500 BP by agarose gel electrophoresis after PCR amplification. The recombinant plasmid was constructed after digestion and ligation. The results of positive cloning and sequencing showed that the target fragment was correctly cloned into the vector. The three plasmids showed green fluorescence at 48 hours after transfection. Conclusion: niR-200b/c/429 overexpression vector and anti-miR-200b/c/429 vector were successfully constructed and lentivirus packaging was successful. Part two Expression of mir-200b/c/429 and its regulation on target gene Aim: to observe the expression of miR-200b/c/429 in hyperlipidemic mice induced by subcutaneous white fat and 3T3-L1 cell line induced maturation. The double fluorescent report vector containing PRDM16 gene 3'UTR was constructed and the regulation of PRDM16 gene expression by miR-200b/c/429 was verified by double fluorescein experiment. The expression of miR-200b/c/429 in lentivirus interference knockdown 3T3-L1 was detected by Q-PCR, and the effect of 3T3-L1 on PRDM16 expression was observed after the change of 1miR-200b/c/429 expression. Methods Q-PCR was used to observe the expression of miR-200b/c/429 in subcutaneous white fat of C57 mice induced by high fat. And the change of 1niR-200b/c/429 expression during differentiation and maturation in 3T3-L1 preadipoid cell line. The double fluorescent report vector of PRDM16 gene was constructed and co-transfected with miR-200b/c/429 overexpression plasmid into 293FT cell line. The changes of fluorescence expression were observed. The changes of PRDM16 gene expression were observed after infection of 3T3-L1 cell line with lentivirus envelope for 8 days. The result is 1: 1. The PRDM16 gene was successfully constructed, and by co-transfection with miR-200b/c/429, it was observed that miR-200b/c down-regulated the expression of fluorescein, which proved that miR-200b/c could regulate 3UTR.2of PRDM16 gene. The expression of miR-200b / c was significantly down-regulated in the subcutaneous fat of C57BL/6 mice induced by high fat, and the expression of miR-200b/c/429 was significantly up-regulated during the differentiation and maturation induced by 3T3-L1. After lentivirus infecting anti-miR-200b/c/429 to interfere with the expression of miR-200b/c/429, it was found that the expression of PRDM16 was up-regulated after induced differentiation. Conclusion 1. Double luciferase assay showed that miR-200b/c down-regulated PRDM16, and the expression of PRDM16 was regulated by miR-200b/c/429. 2.The expression of miR-200b/c/429 was up-regulated during the differentiation induced by 3T3-L1, and the expression of miR-200b/c was down-regulated in the subcutaneous white fat of high-fat induced mice.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R3416
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本文編號(hào)：1926634