本文選題：DNA + siRNA��； 參考：《華東師范大學(xué)》2016年碩士論文

【摘要】：基因和蛋白質(zhì)治療是一種新興的疾病治療手段,它相比于傳統(tǒng)的治療方法,具有靶向性強(qiáng),副作用小,治療效果明顯等優(yōu)勢(shì),因此成為當(dāng)今生物醫(yī)學(xué)領(lǐng)域的研究熱點(diǎn)�；蚝偷鞍踪|(zhì)自身很難進(jìn)入細(xì)胞,在實(shí)際應(yīng)用過(guò)程中需要載體來(lái)遞送。陽(yáng)離子高分子是當(dāng)前研究最多的一類基因和蛋白質(zhì)載體。然而,這些高分子載體普遍存在轉(zhuǎn)染效率不高,毒副作用大等問(wèn)題,因而需要對(duì)其進(jìn)行各種功能化修飾來(lái)改善這些載體的性能。常用的高分子載體改性方法包括脂肪鏈修飾,糖基化修飾.,氨基酸修飾,小肽和蛋白質(zhì)修飾,納米材料修飾等。但這些修飾方法無(wú)法從根本上解決陽(yáng)離子高分子轉(zhuǎn)染過(guò)程中帶來(lái)的細(xì)胞毒性等問(wèn)題。因此,開(kāi)發(fā)高效、低毒的基因和蛋白質(zhì)載體一直是該研究領(lǐng)域亟待解決的關(guān)鍵問(wèn)題。氟化修飾是一種改善陽(yáng)離子高分子基因轉(zhuǎn)染效率的新策略。這種方法修飾的高分子材料可以在極低的氮磷比條件下實(shí)現(xiàn)高效基因轉(zhuǎn)染,低氮磷比也會(huì)降低復(fù)合物表面的電荷密度,從而緩解陽(yáng)離子高分子對(duì)細(xì)胞的毒副作用。但是,氟化修飾的高分子材料要獲得高轉(zhuǎn)染效率往往需要在高分子表面接枝高比例的含氟脂肪鏈,這樣會(huì)帶來(lái)一系列的問(wèn)題。比如,高表面接枝率會(huì)造成高分子表面產(chǎn)生嚴(yán)重的空間位阻,影響高分子材料與核酸分子結(jié)合,也限制了在高分子材料上進(jìn)行其它的功能化修飾；另外,接枝大量的含氟脂肪鏈也可能會(huì)產(chǎn)生嚴(yán)重的細(xì)胞毒性。本論文針對(duì)這些問(wèn)題,擬采用在高分子表面修飾含雙氟鏈的化合物來(lái)使得高分子可以在低接枝率條件下實(shí)現(xiàn)高轉(zhuǎn)染效率。即在不同的高分子材料表面修飾2-氯-4,6-雙[3-(全氟己基)丙氧基]-1,3,5-三嗪(CBT),探索其作為DNA, siRNA和蛋白質(zhì)載體的可行性。論文的主要研究結(jié)果介紹如下：在不同代數(shù)(Generation, G)的聚酰胺-胺樹(shù)形高分子(PAMAM)和分子量為1800 Da的支化聚乙烯亞胺(bPEI1.8K)上修飾不同比例的CBT分子,并通過(guò)茚三酮和氟元素分析等方法表征這些材料表面CBT的平均接枝數(shù)量。所得材料分別定義為GI-CBT1.9, G2-CBT1.5,G5-CBTi.3和bPEI1.8K-CBTi.3 (CBT后的數(shù)字下標(biāo)為CBT的平均接枝數(shù))。研究結(jié)果表明所有CBT修飾的高分子材料均可以緊密結(jié)合DNA。僅僅修飾1-2條CBT后,所有的高分子材料轉(zhuǎn)染綠色熒光蛋白和熒光素酶的效率均大幅提高。其中,GI-CBT1.9的轉(zhuǎn)染效率提高最為顯著,在HEK293細(xì)胞中的轉(zhuǎn)染效率達(dá)到了70%以上,與商業(yè)化轉(zhuǎn)染試劑Lipofectamine2000近乎相當(dāng),顯著優(yōu)于SuperFect和PolyFect。這些CBT修飾的高分子材料除了高轉(zhuǎn)染效率外,在轉(zhuǎn)染條件下幾乎未對(duì)細(xì)胞產(chǎn)生細(xì)胞毒性。除了具有較高的DNA轉(zhuǎn)染能力,CBT修飾的高分子材料還能夠有效遞送siRNA。比如,G1-CBT1.9, G2-CBT1.5, G5-CBT1.3和bPEI1.8K-CBTi.3在穩(wěn)定表達(dá)熒光素酶的HeLa和MDA-MB-231細(xì)胞中均能有效干擾熒光素酶基因的表達(dá)水平,而且在RNA干擾過(guò)程中未檢測(cè)到脫靶現(xiàn)象。其中,GI-CBT1.9的RNA干擾效率與Lipofectamine 2000相當(dāng),而且基因沉默過(guò)程中細(xì)胞毒性更低。GI-CBT1.9不僅在體外具有較高的RNA干擾水平,在小鼠體內(nèi)也展示了一定的基因沉默能力,顯著優(yōu)于未進(jìn)行CBT修飾的G1樹(shù)形高分子材料。這一結(jié)果表明GI-CBT1.9具有作為潛在的基因治療載體的可能。雖然氟化修飾的高分子材料作為DNA和siRNA的遞送載體已有相關(guān)報(bào)道,但是其作為蛋白質(zhì)載體的可行性還未進(jìn)行探索。研究結(jié)果表明GI-CBT1.9, G2-CBT1.5和bPEI1.8K-CBTi.3均可將修飾綠色熒光素的牛血清蛋白(BSA-FITC)遞送進(jìn)HeLa細(xì)胞。三種載體中,GI-CBT1.9對(duì)BSA-FITC遞送效率最高,這與前面DNA和siRNA的轉(zhuǎn)染結(jié)果一致,表明GI-CBT1.9遞送這幾種生物分子采用了類似的機(jī)理。除了BSA-FITC之外,GI-CBT1.9還可以將β-半乳糖苷酶(β-Galactosidase,β-Gal)遞送進(jìn)入細(xì)胞質(zhì)中。綜上所述,本論文提供了一種新的氟化修飾方法用于改善陽(yáng)離子高分子對(duì)基因和蛋白質(zhì)的遞送性能。相對(duì)于傳統(tǒng)的含氟脂肪鏈修飾,雙氟鏈修飾法可以在極低的氟鏈接枝率(1-2條CBT分子)條件下實(shí)現(xiàn)高效、低毒的基因和蛋白質(zhì)轉(zhuǎn)染。這種方法簡(jiǎn)單易行,具有較高的商業(yè)化應(yīng)用前景。該研究進(jìn)一步完善了氟化修飾高分子的基因遞送系統(tǒng),并且拓展了氟化高分子在蛋白質(zhì)遞送中的應(yīng)用。
[Abstract]:Gene and protein therapy is a new means of treatment for disease. Compared with the traditional therapy, it has the advantages of strong targeting, small side effect and obvious therapeutic effect. Therefore, it has become a hot topic in the field of biomedicine. Gene and protein are difficult to enter cells. In practical application, the carrier is needed to deliver. Ionic polymers are the most widely studied genes and protein carriers. However, these polymer carriers generally have problems such as low transfection efficiency and toxic and side effects. Therefore, various functional modifications are needed to improve the performance of these carriers. The commonly used polymer modified methods include fatty chain modification and glycosylation. Modification, amino acid modification, small peptide and protein modification, nanomaterial modification, etc. but these modification methods can not fundamentally solve the cytotoxicity of cationic polymers in the process of transfection. Therefore, the development of high efficient, low toxic genes and protein carriers has been the key problem to be solved urgently in this field. Fluorination modification. It is a new strategy to improve the efficiency of cationic polymer gene transfection. This modified polymer can achieve efficient gene transfection at very low nitrogen and phosphorus ratio. The low N and P ratio will also reduce the charge density on the surface of the compound, thus relieving the side effects of the cationic polymer on the cells. However, the high grade of fluorinated modification is high. In order to obtain high transfection efficiency, a high ratio of fluorine fat chain is often needed to be grafted on the surface of the polymer, which will lead to a series of problems. For example, the high surface grafting rate will cause serious space hindrance on the surface of the polymer, the binding of polymer to nucleic acid molecules, and other restrictions on the polymer materials. In addition, the grafting of large amounts of fluorine fat chains may also produce severe cytotoxicity. In this paper, we propose to use compounds containing double fluorine chains on the surface of polymer to make high efficiency of high transfection efficiency under low grafting rate. That is, the surface modification of 2- chlorine -4 on the surface of different polymer materials 6- double [3- (perfluoro hexyl) promethoxy]-1,3,5- three azine (CBT) as a DNA, siRNA and protein carrier is explored. The main results of this paper are as follows: modified polyamide amine tree (PAMAM) and branched polyethylenimide (bPEI1.8K) of different Algebras (Generation, G) and molecular weight of 1800 Da (bPEI1.8K) The average grafting number of CBT on the surface of these materials was characterized by the method of BT and fluorine analysis. The obtained materials were defined as GI-CBT1.9, G2-CBT1.5, G5-CBTi.3 and bPEI1.8K-CBTi.3 (the number of digital subscript after CBT was the average grafting number of CBT). The results showed that all the CBT modified polymer materials could be tightly combined. The efficiency of all polymer materials transfected to green fluorescent protein and luciferase was greatly improved after only 1-2 CBT modified by DNA.. The transfection efficiency of GI-CBT1.9 was the most significant. The transfection efficiency in HEK293 cells was more than 70%, which was almost equal to that of commercial transfection reagent Lipofectamine2000, which was significantly better than SuperFect and Po. LyFect. in addition to high transfection efficiency, these CBT modified polymer materials have no cytotoxicity to the cells under the transfection condition. In addition to high DNA transfection capacity, CBT modified polymer materials can also deliver siRNA., such as G1-CBT1.9, G2-CBT1.5, G5-CBT1.3 and bPEI1.8K-CBTi.3, in the stable expression of luciferase He. Both La and MDA-MB-231 cells could effectively interfere with the expression level of luciferase gene, and no miss target was detected during RNA interference. The RNA interference efficiency of GI-CBT1.9 was equal to that of Lipofectamine 2000, and the lower cytotoxicity of.GI-CBT1.9 in the process of gene silencing was not only with a high level of RNA interference in vitro. The mice also showed a certain gene silencing ability, which was significantly better than the G1 tree without CBT modification. This result indicates that GI-CBT1.9 has the potential as a potential vector for gene therapy. Although fluorinated polymer materials have been reported as delivery carriers of DNA and siRNA, it is used as a protein carrier. The results show that GI-CBT1.9, G2-CBT1.5 and bPEI1.8K-CBTi.3 can deliver the bovine serum protein (BSA-FITC) modified with green fluorescein (BSA-FITC) into HeLa cells. Among the three carriers, GI-CBT1.9 has the highest delivery efficiency to BSA-FITC, which is in accordance with the transfection results of the previous DNA and siRNA, indicating that GI-CBT1.9 delivery is a few kinds of birth. In addition to BSA-FITC, GI-CBT1.9 can also deliver beta galactosidase (beta -Galactosidase, beta -Gal) into the cytoplasm. To sum up, this paper provides a new fluorinated modification method to improve the delivery performance of cationic polymers to genes and proteins. Adipose chain modification and double fluorine chain modification can be used to achieve high efficiency, low toxic gene and protein transfection at a very low fluorine linked branching rate (1-2 CBT molecule). This method is simple and has a high commercial application prospect. This study further perfected the gene delivery system of fluorinated high fraction and expanded fluorinated polymers. Application in protein delivery.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q789

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