本文選題：基因載體 + PAMAM　； 參考：《廣東藥學(xué)院》2015年碩士論文

【摘要】：基因治療是通過(guò)一定的方法、方式,將外源基因?qū)肽康募?xì)胞并使其有效表達(dá),以糾正基因的缺陷或在細(xì)胞中發(fā)揮作用,從而發(fā)揮治療疾病的作用。基因治療的關(guān)鍵就是選擇合適的載體將目的基因準(zhǔn)確的運(yùn)送到靶細(xì)胞,并控制使其有效表達(dá),發(fā)揮治療作用。病毒型載體具有較高的轉(zhuǎn)染效率,但病毒載體存在較嚴(yán)重的安全性問(wèn)題。非病毒載體主要有陽(yáng)離子聚合物、陽(yáng)離子多肽和陽(yáng)離子脂質(zhì)體等,具有靶向性,低細(xì)胞毒性,較高的安全性,并且易于進(jìn)行結(jié)構(gòu)修飾等優(yōu)點(diǎn),已成為基因治療中的熱點(diǎn),逐漸受到了越來(lái)越多的關(guān)注,對(duì)其研究已逐漸深入。PAMAM具有內(nèi)部空腔結(jié)構(gòu),其末端基團(tuán)精確可控,氨基易質(zhì)子化而帶正電,容易與DNA結(jié)合形成納米級(jí)復(fù)合物,可以保護(hù)DNA并達(dá)到轉(zhuǎn)染的目的,作為藥物載體和基因載體具有較高的穩(wěn)定性、生物兼容性良好、無(wú)免疫源性、使用劑量下低細(xì)胞毒性的優(yōu)勢(shì)。對(duì)PAMAM樹(shù)狀大分子末端基團(tuán)進(jìn)行表面修飾,減少表面的正電荷數(shù)目,以降低其細(xì)胞毒性,改善基因轉(zhuǎn)染效率。透明質(zhì)酸(Hyaluronic,簡(jiǎn)稱HA)作為天然水溶性高分子,具有多種適宜作為作為藥物載體的優(yōu)良特性,包括生物兼容性良好、非免疫原性、高生物活性及易被體內(nèi)酶作用而天然降解、靶向性。本研究以HA具有較好的腫瘤靶向性、增強(qiáng)復(fù)合物水溶性、生物兼容性,PAMAM具有較好的攜帶目的基因?qū)爰?xì)胞中的理論基礎(chǔ)為切入點(diǎn),擬構(gòu)建HA-PAMAM/p EGFP基因傳遞表達(dá)系統(tǒng),深入研究其物理化學(xué)性能、生物性能、靶向性等方面的影響;從PAMAM分子量,HA接枝率和接枝密度,聚陽(yáng)離子/基因重量比(w/w)等方面優(yōu)化該傳遞系統(tǒng),提高基因轉(zhuǎn)染效率、降低細(xì)胞毒性、提高載體靶向性、穩(wěn)定性等。本研究的主要內(nèi)容:(1)PAMAM-HA聚合物的化學(xué)合成和結(jié)構(gòu)表征:采用透明質(zhì)酸(HA)與PAMAM在含有Na Cl的四氫硼酸鈉(Na BH4)緩沖液中(p H8.5),在氰硼氫化納(Na BH3CN)催化下反應(yīng)生成PAMA-HA聚合物。不同PAMAM分子量、HA接枝量和接枝密度的PAMAM-HA樣品同步制備并作比較。PAMAM-HA樣品的化學(xué)結(jié)構(gòu)有紅外(IR)等鑒定。(2)對(duì)PAMAM-HA作為基因載體的性能考察:采用動(dòng)態(tài)光散射發(fā)測(cè)定了PAMAM-HA/DNA復(fù)合物的粒徑分布及表面電位;采用透射電鏡(TEM)觀察復(fù)合物粒子形態(tài);通過(guò)瓊脂糖凝膠阻滯實(shí)驗(yàn)考察PAMAM-HA結(jié)合并壓縮DNA的能力。(3)PAMAM-HA細(xì)胞毒性:用MTT法考察PAMAM-HA各樣品對(duì)不同腫瘤細(xì)胞的細(xì)胞毒性,并考察在PAMAM-HA/DNA的轉(zhuǎn)染比例下的毒性。(4)PAMAM-HA體外轉(zhuǎn)染研究:用Bel-7402細(xì)胞對(duì)PAMAM-HA/DNA復(fù)合物的最佳轉(zhuǎn)染效率進(jìn)行篩選并考察PAMAM-HA/DNA復(fù)合物在不同細(xì)胞之間的轉(zhuǎn)染與PEI25K相比較,同時(shí)考察血清對(duì)轉(zhuǎn)染效率的影響。(5)PAMAM-HA誘導(dǎo)細(xì)胞凋亡:用Annexin V-FITC細(xì)胞凋亡法考察PAMAM-HA各樣品在在實(shí)驗(yàn)濃度下是否可誘導(dǎo)細(xì)胞凋亡及凋亡效率與濃度和時(shí)間的關(guān)系。(6)PAMAM-HA轉(zhuǎn)染機(jī)制研究:用不同攝取途徑抑制劑考察各復(fù)合物進(jìn)入細(xì)胞的途徑,將細(xì)胞核、溶酶體和DNA分別染色標(biāo)記用激光共聚焦顯微鏡觀察考察DNA在細(xì)胞內(nèi)的分布。
[Abstract]:Gene therapy is a way of introducing foreign genes into the target cells and making them effective in order to correct the defects of the genes or to play a role in the cells. The key to gene therapy is to select the appropriate carrier to deliver the target base to the target cell accurately and control it to be effective. Viral vectors have high transfection efficiency, but viral vectors have serious safety problems. Non viral vectors mainly have cationic polymers, cationic polypeptides and cationic liposomes, which have the advantages of targeting, low cytotoxicity, high safety, and easy to carry out structural modification. More and more attention has been paid to the hot spots in gene therapy. The research has gradually deepened.PAMAM with the internal cavity structure, its terminal group is precise and controllable, the amino group is easy to protonated and positively charged, and it is easy to combine with DNA to form nano scale complex. It can protect DNA and reach the purpose of transfection as drug carrier and gene carrier. With the advantages of high stability, good biocompatibility, no immunogenic and low cytotoxicity at the dosage, surface modification of the terminal group of PAMAM tree macromolecules to reduce the number of positive charges on the surface in order to reduce its cytotoxicity and improve gene transfection efficiency. Hyaluronic (HA) as a natural water-soluble high score It has a variety of excellent properties as a drug carrier, including good biocompatibility, non immunogenicity, high bioactivity and easy to be degraded naturally by enzymes in the body. The HA has good tumor targeting, enhanced water solubility and biocompatibility, and PAMAM has good carrier gene guidance. The theoretical basis of cell entry is the breakthrough point. The HA-PAMAM/p EGFP gene transfer and expression system will be constructed to further study its physical and chemical properties, biological performance and targeting. The transfer system is optimized from PAMAM molecular weight, HA grafting rate and grafting density, polycation / gene weight ratio (w /w) and so on, and gene transfection efficiency is improved. The main contents of this study are: (1) the chemical synthesis and structural characterization of PAMAM-HA polymers: the use of hyaluronic acid (HA) and PAMAM in the buffer solution (P H8.5) of four sodium borate (Na BH4) containing Na Cl (P H8.5), and the formation of a PAMA-HA polymer under the catalysis of cyanoborohydride (Na BH3CN). The molecular weight, the HA grafting quantity and the graft density of PAMAM-HA samples were synchronized and compared with the.PAMAM-HA samples. The chemical structure of the.PAMAM-HA samples was identified as infrared (IR). (2) the performance of PAMAM-HA as a gene carrier: the particle size distribution and surface potential of the PAMAM-HA/DNA complex were measured by dynamic light scattering, and the composite was observed by transmission electron microscopy (TEM). Particle morphology; the ability of PAMAM-HA binding and compressing DNA by agarose gel block test. (3) PAMAM-HA cytotoxicity: MTT method was used to investigate the cytotoxicity of PAMAM-HA samples to different tumor cells and the toxicity of PAMAM-HA/DNA in the transfection ratio. (4) PAMAM-HA in vitro transfection study: Bel-7402 cells to PAMAM-HA/DNA The optimal transfection efficiency of the complex was screened and the transfection of PAMAM-HA/DNA complex between different cells was compared with PEI25K, and the effect of serum on transfection efficiency was investigated. (5) PAMAM-HA induced apoptosis: Annexin V-FITC cell apoptosis method was used to investigate whether various PAMAM-HA products could induce apoptosis at the experimental concentration. The relationship between apoptosis efficiency and concentration and time. (6) study on the mechanism of PAMAM-HA transfection: investigate the pathway of each compound into cells by different uptake inhibitors, and observe the distribution of DNA in cell by laser confocal microscopy, respectively, with the staining of nuclei, lysosomes and DNA.
【學(xué)位授予單位】：廣東藥學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R450

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 趙亮;蘇暢;馬洪林;;陽(yáng)離子聚合物基因載體的理論研究[J];中國(guó)組織工程研究與臨床康復(fù);2009年42期

，

本文編號(hào)：2110068