本文選題：聚乙烯亞胺（Polyethyleneimine + PEI）　； 參考：《吉林大學(xué)》2014年博士論文

【摘要】：近年來(lái)，惡性腫瘤已經(jīng)成為威脅人類生命健康的主要?dú)⑹�，我�?guó)每年因惡性腫瘤而死亡的人數(shù)達(dá)到220萬(wàn)，占我國(guó)居民死亡原因的第一位。因此，惡性腫瘤的治療已經(jīng)成為亟待解決的世界性難題，具有極其重要的戰(zhàn)略意義�，F(xiàn)如今，臨床常用的腫瘤治療手段包括手術(shù)、化療和放療等，手術(shù)只能使一部分腫瘤患者受益，而化療和放療對(duì)正常組織器官毒副作用大，嚴(yán)重影響患者的生活質(zhì)量。此外，長(zhǎng)期使用化學(xué)藥物治療腫瘤，會(huì)產(chǎn)生多藥耐藥現(xiàn)象（Multidrug resistance，，MDR），降低了化療藥物的治療效果。而與傳統(tǒng)治療手段相比，基因治療有望從根本上治療疾病，已成為目前國(guó)際學(xué)術(shù)界研究的焦點(diǎn)，它被認(rèn)為是醫(yī)學(xué)和藥學(xué)領(lǐng)域的一次革命，是當(dāng)今生物醫(yī)學(xué)發(fā)展中最重要的里程碑之一，同時(shí)也必將對(duì)傳統(tǒng)制藥業(yè)產(chǎn)生深遠(yuǎn)影響和沖擊，有望成為未來(lái)在腫瘤臨床治療中的“新寵”。 基因治療主要有三個(gè)方面：基因藥物、基因診斷和基因傳輸。隨著人類基因組計(jì)劃（human genome project, HGP）的成功實(shí)施，基因診斷和基因藥物的制備不再是基因治療的障礙；但如何安全、有效的將藥物基因運(yùn)載到靶細(xì)胞并起到治療效果，成為獲得基因治療良好效果的瓶頸之一。常見的基因遞送方式可以分為物理方法、化學(xué)方法和生物方法�；瘜W(xué)方法和生物方法都是利用載體承載基因藥物，載體的地位極其重要，也成了基因治療發(fā)展中的關(guān)鍵性問(wèn)題。生物學(xué)上根據(jù)來(lái)源將基因載體分為質(zhì)粒載體和病毒載體；而化學(xué)研究人員習(xí)慣將質(zhì)粒看做DNA的一種，而將載體分為病毒類載體（ViralVector）和非病毒類載體（Non-viral Vector）。目前在臨床使用中應(yīng)用最多的仍然是病毒類載體，部分轉(zhuǎn)染效率可高達(dá)90%以上，但病毒類載體存在嚴(yán)重的免疫源性等自身難以克服的缺陷。相對(duì)而言，非病毒類載體由于其特有的優(yōu)勢(shì)而越來(lái)越受到關(guān)注。聚乙烯亞胺(Polyethyleneimine, PEI)分子已經(jīng)成為了非病毒類基因載體領(lǐng)域里發(fā)展最快、最具有研究?jī)r(jià)值和應(yīng)用潛質(zhì)的一類基因傳輸載體。 對(duì)于轉(zhuǎn)染效率而言，理想的PEI分子量范圍約為11.9×103~70.0×103，其中重均分子量為25K的超支化PEI（PEI25K）轉(zhuǎn)染效率最高，因而成為衡量非病毒基因載體各項(xiàng)性能的“黃金標(biāo)準(zhǔn)”。PEI獨(dú)特的“質(zhì)子海綿效應(yīng)”有利于實(shí)現(xiàn)細(xì)胞內(nèi)的內(nèi)涵體逃逸。然而，PEI25K作為非病毒基因載體材料仍然有很多缺陷，如相對(duì)較低的基因轉(zhuǎn)染效率、細(xì)胞毒性大、聚合物結(jié)構(gòu)的不可降解性等。因此，對(duì)其進(jìn)行結(jié)構(gòu)改造就顯得尤為必要。 本論文立足于腫瘤基因治療這一當(dāng)前研究熱點(diǎn)，設(shè)計(jì)了兩類新型PEI衍生物材料，我們首先將不同數(shù)量的帶有疏水基團(tuán)的N-乙酰-亮氨酸，以EDC/NHS催化，使其與PEI25K的伯氨基進(jìn)行偶聯(lián)，來(lái)研究N-乙酰-亮氨酸引入的數(shù)量對(duì)PEI25K作為基因傳遞材料能力的影響。然后在前期工作基礎(chǔ)上，進(jìn)行了N-異丙基丙烯酰胺改性PEI25K基因載體的制備及性能研究，通過(guò)一系列的化學(xué)及細(xì)胞實(shí)驗(yàn)對(duì)衍生物的材料結(jié)構(gòu)、細(xì)胞毒性、核酸結(jié)合能力、基因轉(zhuǎn)染效率進(jìn)行研究，并采用改性后的PEI衍生物（PEN）將外源的p53基因遞送到p53基因缺失的前列腺癌PC-3細(xì)胞系與p53野生型細(xì)胞系Hela中進(jìn)行對(duì)比研究。 第一部分：N-乙酰-亮氨酸(N-Ac-Leu)改性PEI25k基因載體的構(gòu)建與評(píng)價(jià) 1.本章采用EDC/NHS催化反應(yīng)合成了三種N-Ac-Leu修飾的PEI衍生物，分別為N-Ac-Leu-PEI (60:1)、 N-Ac-Leu-PEI (80:1)、N-Ac-Leu-PEI (100:1)并利用核磁共振氫譜（1H NMR）對(duì)聚合物的結(jié)構(gòu)進(jìn)行表征。 2.通過(guò)凝膠電泳可以表明，三種N-Ac-Leu-PEI聚合物均能很好的與pEGFP質(zhì)粒結(jié)合，但結(jié)合能力低于未修飾的PEI25K。通過(guò)表面電位測(cè)定可以得知三種N-Ac-Leu-PEI的表面電位在+30mV，恰好可以說(shuō)明N-Ac-Leu-PEI與質(zhì)粒結(jié)合能力的降低。粒徑測(cè)定結(jié)果表明N-Ac-Leu-PEI聚合物的粒徑在180~220nm范圍內(nèi)。 3. N-Ac-Leu-PEI基因載體材料的溶血實(shí)驗(yàn)證明載體材料溶血效應(yīng)相對(duì)于PEI25K明顯降低，具有很好的血液相容性；蛋白吸附實(shí)驗(yàn)中，N-Ac-Leu-PEI蛋白吸附值相對(duì)于PEI25K明顯降低，且隨時(shí)間的增加蛋白吸附值緩慢降低。由此證明N-Ac-Leu基團(tuán)的引入可改善PEI的生物相容性，可望用于活體實(shí)驗(yàn)中。 4. MTT實(shí)驗(yàn)結(jié)果表明，N-Ac-Leu-PEI在HeLa細(xì)胞中的細(xì)胞毒性較PEI25K有明顯降低。 5.三種N-Ac-Leu-PEI聚合物均能有效的介導(dǎo)基因轉(zhuǎn)染，且轉(zhuǎn)染能力高于PEI25K。N-Ac-Leu-PEI (100:1)在質(zhì)量比為3.0的條件下轉(zhuǎn)染效率最高。 第二部分：PEI衍生物基因載體的設(shè)計(jì)制備以及性能研究 1.首先在PEI骨架上偶聯(lián)上N-異丙基丙烯酰胺，然后通過(guò)細(xì)化投料比，得到五種PEI衍生物（PEN）。使用凝膠阻滯實(shí)驗(yàn)和細(xì)胞毒性試驗(yàn)初步篩選出一種載體。之后通過(guò)核磁共振氫譜對(duì)所選材料的結(jié)構(gòu)進(jìn)行了表征。 2.使用納米粒度電位儀檢測(cè)了載體、質(zhì)粒以及載體質(zhì)粒復(fù)合物的粒徑大小以及表面電位。 3.通過(guò)MTT實(shí)驗(yàn)檢測(cè)了載體的細(xì)胞毒性，結(jié)果顯示，修飾后的載體毒性低于PEI。 4. DNase I酶降解實(shí)驗(yàn)發(fā)現(xiàn)，在載體質(zhì)粒質(zhì)量比不小于0.8時(shí)，載體就能夠?qū)崿F(xiàn)對(duì)DNA的完全保護(hù)。相比較而言， PEN對(duì)DNA的保護(hù)能力較PEI略有下降。 5.通過(guò)綠色熒光蛋白質(zhì)粒轉(zhuǎn)染實(shí)驗(yàn)和PGL-3質(zhì)粒轉(zhuǎn)染實(shí)驗(yàn)，分別定性定量考察了材料 作為基因載體的傳遞性能。實(shí)驗(yàn)數(shù)據(jù)表明，在PEI上引入N-異丙基丙烯酰胺，降低了聚合物的表面電荷，并引入了一定量的疏水基團(tuán)。但是疏水基團(tuán)的過(guò)多引入反而會(huì)降低載體的基因轉(zhuǎn)染效率。在定性轉(zhuǎn)染試驗(yàn)中，改變載體的加入量發(fā)現(xiàn)，在載體質(zhì)粒質(zhì)量比為2時(shí)，轉(zhuǎn)染效果最好。在定量轉(zhuǎn)染實(shí)驗(yàn)中，PEI展現(xiàn)了不錯(cuò)的轉(zhuǎn)染效率，PEN雖然不及PEI，但和商業(yè)轉(zhuǎn)染試劑相比，轉(zhuǎn)染效率相差無(wú)幾。 6.在前列腺癌細(xì)胞系PC-3中，通過(guò)RT-PCR和Western blot分別從mRNA水平和蛋白水平證明p53基因的表達(dá)。之后通過(guò)檢測(cè)相關(guān)蛋白表達(dá)的變化證明表達(dá)出的p53蛋白的確具有正常的功能。之后通過(guò)檢測(cè)caspase家族蛋白活性和線粒體膜電位變化輔助說(shuō)明上述問(wèn)題。 7.使用流式細(xì)胞儀檢測(cè)周期阻滯和細(xì)胞凋亡情況，在檢測(cè)周期阻滯時(shí)發(fā)現(xiàn)Hela細(xì)胞系不存在周期阻滯現(xiàn)象，通過(guò)DAPI染色，證明細(xì)胞存在凋亡現(xiàn)象。 8.綜合各方面的實(shí)驗(yàn)結(jié)果，證明經(jīng)過(guò)修飾，雖然在一定程度上降低了PEI的基因轉(zhuǎn)染效率，但所得到的衍生物的毒性低，而且其基因轉(zhuǎn)染效率可以和商業(yè)轉(zhuǎn)染試劑相媲美。而且在遞送p53基因致使癌癥細(xì)胞凋亡的試驗(yàn)中，展現(xiàn)了較好的轉(zhuǎn)染性能，后續(xù)的凋亡相關(guān)實(shí)驗(yàn)也證明外源的p53基因的確可以代替或者補(bǔ)償內(nèi)源性缺失或者無(wú)效的p53基因。
[Abstract]:In recent years, malignant tumors have become the main killer of human life and health. The number of deaths from malignant tumors in China is 2 million 200 thousand, which accounts for the first cause of death in our country. Therefore, the treatment of malignant tumors has become a worldwide problem to be solved urgently. It is of great strategic significance. Now, it is commonly used in clinical practice. The treatment of cancer includes surgery, chemotherapy and radiotherapy, and the operation can only benefit a part of the tumor patients. Chemotherapy and radiotherapy have large side effects on normal tissues and organs, which seriously affect the quality of life of the patients. In addition, the long-term use of chemical drugs in the treatment of tumors can produce multidrug resistance (Multidrug resistance, MDR) and reduce the chemotherapy. Compared with the traditional treatment, gene therapy is expected to cure disease fundamentally. It has become the focus of international academic research. It is considered as a revolution in the field of medicine and pharmacy. It is one of the most important milestones in the development of biomedicine, and it will also have a far-reaching effect on the traditional pharmaceutical industry. Impact and impact is expected to become the "new favorite" in clinical treatment of cancer in the future.
Gene therapy has three main aspects: gene medicine, gene diagnosis and gene transmission. With the successful implementation of the human genome project (human genome project, HGP), gene diagnosis and the preparation of gene drugs are no longer a barrier to gene therapy; however, how to safely, effectively carry the drug genes to the target cells and play a therapeutic effect, One of the bottlenecks in the good effect of gene therapy is that the common way of gene delivery can be divided into physical, chemical and biological methods. Both chemical and biological methods use carriers to carry genetic drugs. The status of carriers is extremely important, and it is also a key problem in the development of gene therapy. Biological sources will be based on the source. Gene vectors are divided into plasmid vectors and viral vectors, and chemical researchers are used to treat plasmids as one of DNA and divide the vectors into viral vectors (ViralVector) and non viral vectors (Non-viral Vector). The most used in clinical use are viral vectors, and the partial transfection efficiency can be as high as 90%. Polyethyleneimine (PEI) has become more and more popular in the field of non viral gene carriers, which have become the fastest, most valuable and applied potential in the field of non viral gene carriers. Gene transmission carrier.
For the transfection efficiency, the ideal PEI molecular weight range is about 11.9 x 103~70.0 x 103, and the hyperbranched PEI (PEI25K) transfection efficiency is the highest with the weight average molecular weight 25K. Thus, the "gold standard".PEI unique "proton sponge effect" is beneficial to the realization of intracellular endosomal escape. However, as a non viral gene carrier, PEI25K still has many defects, such as relatively low gene transfection efficiency, large cytotoxicity, and non degradable polymer structure. Therefore, it is particularly necessary to reconstruct its structure.
This paper, based on the current research hotspot of tumor gene therapy, designs two new types of PEI derivatives. First, we catalyze N- acetyl leucine with different numbers of hydrophobic groups, catalyze it with EDC/NHS, and study the number of N- acetylleucine introduced to PEI25K as a gene transfer. On the basis of previous work, the preparation and performance of N- isopropylacrylamide modified PEI25K gene carrier was studied on the basis of previous work. Through a series of chemical and cell experiments, the material structure, cytotoxicity, nucleic acid binding capacity and gene transfection efficiency of the derivatives were studied, and the modified PEI derivatives (P) were used. EN) the exogenous p53 gene was delivered to the p53 gene deficient prostate cancer PC-3 cell line and p53 wild-type cell line Hela.
Part one: Construction and evaluation of N- acetylleucine (N-Ac-Leu) modified PEI25k gene vector.
1. in this chapter, three kinds of N-Ac-Leu modified PEI derivatives were synthesized by EDC/NHS catalytic reaction, which were N-Ac-Leu-PEI (60:1), N-Ac-Leu-PEI (80:1), N-Ac-Leu-PEI (100:1) and the structure of the polymer was characterized by NMR (1H NMR).
2. the gel electrophoresis shows that the three N-Ac-Leu-PEI polymers can be well combined with pEGFP plasmids, but the binding ability is lower than the unmodified PEI25K. by the surface potential measurement. The surface potential of the three N-Ac-Leu-PEI can be found in +30mV, which shows the decrease of the binding capacity of N-Ac-Leu-PEI and plasmids. The results of particle size determination show that The particle size of N-Ac-Leu-PEI polymer is in the range of 180~220nm.
The hemolysis test of the 3. N-Ac-Leu-PEI gene carrier material showed that the hemolysis effect of the carrier material was significantly lower than that of the PEI25K and had a good blood compatibility. In the protein adsorption experiment, the adsorption value of N-Ac-Leu-PEI protein was significantly lower than that of the PEI25K, and the adsorption value of the protein decreased slowly with the increase of time. Thus, the N-Ac-Leu group was proved to be introduced. It can improve the biocompatibility of PEI and is expected to be used in vivo experiments.
4. MTT test results showed that the cytotoxicity of N-Ac-Leu-PEI in HeLa cells was significantly lower than that in PEI25K.
5. the three N-Ac-Leu-PEI polymers all can effectively mediate gene transfection, and the transfection efficiency is higher than that of PEI25K.N-Ac-Leu-PEI (100:1) with the highest transfection efficiency under the condition of mass ratio of 3.
The second part: design, preparation and properties of PEI derivative gene vector.
1. first, N- isopropyl acrylamide was coupled on the PEI skeleton, and then five PEI derivatives (PEN) were obtained by refining the feeding ratio. A carrier was screened by gel block experiment and cytotoxicity test. The structure of the selected materials was characterized by NMR.
2. the particle size and surface potential of vectors, plasmids and plasmid plasmids were detected by nanoparticle potentiometer.
3. the cytotoxicity of the carrier was detected by MTT test. The results showed that the toxicity of the modified vector was lower than PEI..
The 4. DNase I enzyme degradation experiment showed that the carrier could fully protect the DNA when the plasmid mass was less than 0.8. Compared with the PEI, the protection ability of DNA was slightly lower than that of PEI.
5. through green fluorescent protein plasmid transfection and PGL-3 plasmid transfection experiments, the materials were qualitatively and quantitatively investigated.
As the transmission performance of the gene carrier, the experimental data show that the introduction of N- isopropyl acrylamide on PEI reduces the surface charge of the polymer and introduces a certain amount of hydrophobic groups. But the excessive hydrophobic group will reduce the gene transfection efficiency of the carrier. When the plasmid mass ratio was 2, the transfection efficiency was the best. In the quantitative transfection experiment, PEI showed a good transfection efficiency, while PEN was not as good as PEI, but the transfection efficiency was similar to that of commercial transfection reagents.
6. in the prostate cancer cell line PC-3, the expression of the p53 gene was demonstrated by RT-PCR and Western blot respectively from the mRNA level and protein level. Then the expression of the expressed protein was proved to have normal function by detecting the changes in the expression of the associated protein. Then, the changes in the activity of caspase family protein and the changes of mitochondrial membrane potential were detected. The above question.
7. the flow cytometry was used to detect the cell cycle arrest and cell apoptosis. The Hela cell line was found to have no periodic block during the detection period, and the cell apoptosis was demonstrated by DAPI staining.
8. the experimental results showed that, although the transfection efficiency of PEI was reduced to a certain extent, the toxicity of the derived derivatives was low, and the gene transfection efficiency was comparable to that of commercial transfection reagents. Moreover, the transfection efficiency of the gene transfection was better than that of the commercial transfection reagent. The subsequent apoptosis related experiments also showed that the exogenous p53 gene could replace or compensate for endogenous deletion or ineffective p53 gene.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R318.08;R73-36

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 姜濤,姜輝,宋希雙,李憲承,李泉林;前列腺癌中P53的表達(dá)及其臨床意義[J];中華男科學(xué)雜志;2005年06期

2 李鳴;許昌泰;;前列腺癌生物標(biāo)志物研究現(xiàn)狀[J];武警醫(yī)學(xué)院學(xué)報(bào);2010年08期

3 于曉謨;;前列腺癌Bcl-2、p53和PCNA表達(dá)的研究[J];現(xiàn)代中西醫(yī)結(jié)合雜志;2010年36期

4 莫林鍵;莫曾南;;前列腺慢性炎癥與前列腺癌[J];醫(yī)學(xué)新知雜志;2007年03期

5 Mack Roach Ⅲ;孔琳;;前列腺癌:近期研究結(jié)果和新方向[J];中國(guó)癌癥雜志;2007年03期

6 孫穎浩;我國(guó)前列腺癌的研究現(xiàn)狀[J];中華泌尿外科雜志;2004年02期

7 童大躍;伍新堯;;雄激素非依賴性前列腺癌分子機(jī)理研究進(jìn)展[J];中華泌尿外科雜志;2006年S1期

8 許清泉;;前列腺癌局部治療后PSA水平低的患者有必要行骨掃描嗎?[J];中華泌尿外科雜志;2006年11期

9 肖云翔;;前列腺癌根治術(shù)后性功能障礙：發(fā)生率、治療、治療局限性和苦惱[J];中華泌尿外科雜志;2006年12期

本文編號(hào)：1835418