本文關(guān)鍵詞：肝靶向免疫基因載體的研究　出處：《鄭州大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 肝靶向 脂質(zhì)-聚陽離子-DNA復(fù)合物 單克隆抗體 冷凍干燥

【摘要】： 本研究開發(fā)了一種新型的非病毒基因傳遞系統(tǒng)。通過利用多聚陽離子PEI壓縮質(zhì)粒DNA,然后用PEG化的脂質(zhì)體包裹PEI/pDNA壓縮體,形成脂質(zhì)復(fù)合載體LPD,并將人胰島素受體的單克隆抗體(8314-SA)與脂質(zhì)中的DSPE-PEG2000-biotin中的生物素基團(tuán)(biotin)共軛,形成以單克隆抗體(MAb)為靶向基團(tuán)的復(fù)合載體(8314-LPD),MAb提高了復(fù)合載體的靶向能力。8314-LPD是一種肝主動(dòng)靶向四元長循環(huán)基因載體制劑。其具體研究內(nèi)容及結(jié)果如(?) 首先用轉(zhuǎn)化的大腸桿菌擴(kuò)增含有報(bào)告基因的質(zhì)粒DNA (PEGFP-C1),再用QIAGEN無內(nèi)毒素大提試劑盒提取質(zhì)粒DNA,并對(duì)其濃度和質(zhì)量進(jìn)行評(píng)定。然后分別制備出PEG化的脂質(zhì)體(脂質(zhì)處方為POPC 94%, DDAB 2%, DSPE-PEG2000 3%和DSPE-PEG2000-biotin 1%)和PEI/pDNA聚陽離子壓縮體,用所得脂質(zhì)體包裹PEI/pDNA壓縮體形成了LPD復(fù)合物。單因素實(shí)驗(yàn)結(jié)合均勻設(shè)計(jì)實(shí)驗(yàn)優(yōu)化LPD處方,單因素實(shí)驗(yàn)考察結(jié)果為：質(zhì)粒濃度在40μg/ml以下,粒徑變化不大,濃度增大,復(fù)合物粒徑增大；N/P超過1.5時(shí),質(zhì)粒的包封完全,隨著N/P的增大,復(fù)合物粒徑減小,電位增大,在N/P等于15時(shí),粒徑最小,再增大N/P,粒徑基本不再發(fā)生變化；脂質(zhì)/pDNA的摩爾比在50：1時(shí),酶切電泳顯示脂質(zhì)可以完全包封PEI/pDNA壓縮體,隨著脂質(zhì)/pDNA的比例增大,LPD的粒徑減小,電位降低,在170：1時(shí)粒徑最小,脂質(zhì)/pDNA的摩爾比再增大,復(fù)合物粒徑開始變大。均勻設(shè)計(jì)實(shí)驗(yàn)優(yōu)化LPD復(fù)合物處方的結(jié)果為：在pH 7.0的HEPES緩沖液中,質(zhì)粒濃度為40μg/ml,N/P=15,脂質(zhì)/pDNA=170:1時(shí),制備的LPD平均粒徑為130 nm,平均zeta電位為1.5mV。進(jìn)一步對(duì)LPD進(jìn)行結(jié)構(gòu)修飾,在外層脂質(zhì)膜的PEG鏈上偶聯(lián)抗人胰島素受體的單克隆抗體(8314-SA),形成肝主動(dòng)靶向的單克隆抗體修飾的Lipo/PEI/pDNA復(fù)合物(8314-LPD)。透射電鏡下觀察,肝靶向8314-LPD復(fù)合物的近似球形,形態(tài)規(guī)整,無粘連；對(duì)其粒徑電位分析,平均粒徑在150nm左右,電位在4.5mv左右。以綠色熒光蛋白基因PEGFP-C1作為報(bào)告基因考察復(fù)合物對(duì)質(zhì)粒的酶切保護(hù)能力,凝膠電泳分析實(shí)驗(yàn)證明這種基因載體能有效地保護(hù)質(zhì)粒DNA免受DNA酶(?)NaseⅠ)降解；血清穩(wěn)定性實(shí)驗(yàn)表明8314-LPD復(fù)合物可以在血清中穩(wěn)定存在；長期穩(wěn)定性實(shí)驗(yàn)考察結(jié)果,4℃和25℃氮?dú)饷芊赓A存,復(fù)合物粒徑都有所增大,但變化幅度不同,說明溫度對(duì)復(fù)合物的穩(wěn)定性有明顯的作用,25℃貯存會(huì)使復(fù)合物粒徑在一個(gè)月內(nèi)發(fā)生非常大的增大。 體外的細(xì)胞轉(zhuǎn)染實(shí)驗(yàn)和細(xì)胞毒性實(shí)驗(yàn)分析,LPD載體的轉(zhuǎn)染效率小于PEI/pDNA壓縮體,但是經(jīng)單克隆抗體修飾后,8314-LPD基因載體能夠很好的被人肝癌細(xì)胞SMMC-7721攝取,與前兩組基因載體相比,轉(zhuǎn)染率有明顯的提高。MTT實(shí)驗(yàn)中顯示LPD和8314.-LPD復(fù)合物的毒性都明顯小于PEI/pDNA多聚陽離子壓縮體。 為了提高8314-LPD復(fù)合物的長期穩(wěn)定性,通過冷凍干燥法將其制備成凍干粉針劑。具體步驟是,首先篩選優(yōu)質(zhì)凍干保護(hù)劑,通過考察凍干粉的外觀,色澤,表面細(xì)化程度,再分散能力,發(fā)現(xiàn)海藻糖制備的凍干粉外觀不塌陷,不起泡,色澤均勻,質(zhì)地細(xì)膩；復(fù)溶后,再分散時(shí)間短；復(fù)溶后,復(fù)合物的粒徑和Zeta電位基本沒有變化,是載體的最優(yōu)保護(hù)劑。其次,對(duì)凍干粉進(jìn)行質(zhì)量評(píng)價(jià),通過對(duì)凍干粉復(fù)溶后抗酶切實(shí)驗(yàn),發(fā)現(xiàn)經(jīng)凍干復(fù)溶后的制劑仍能保持抗核酸酶能力,說明載體結(jié)構(gòu)在凍干過程中沒有受到破壞。轉(zhuǎn)染實(shí)驗(yàn)證明,經(jīng)凍干復(fù)溶的載體的轉(zhuǎn)染活性也基本沒有變化。 本研究結(jié)果表明,8314-LPD復(fù)合物制備方法簡單易行,能夠有效保護(hù)質(zhì)粒DNA不被核酸酶降解,復(fù)合物的血清中穩(wěn)定性和細(xì)胞轉(zhuǎn)染率都比較高,細(xì)胞毒性小,并可通過冷凍干燥法制備出性質(zhì)穩(wěn)定可長期保存的凍干粉針劑。這種肝主動(dòng)靶向的長循環(huán)免疫基因載體,綜合利用了陽離子多聚物和脂質(zhì)體的優(yōu)點(diǎn),克服了二者的缺點(diǎn),相信在肝癌的基因治療中,將會(huì)有很好的發(fā)展前景。
[Abstract]:This study developed a novel non viral gene delivery system. By using polycation PEI compression plasmid DNA, then liposome PEI/pDNA with PEG of a compression body, formation of lipid composite carrier LPD, and monoclonal antibody to human insulin receptor (8314-SA) and lipid in DSPE-PEG2000-biotin biotin groups (biotin) conjugate formed by monoclonal antibody (MAb) targeting composite carrier group (8314-LPD), MAb improved the composite carrier targeting ability of.8314-LPD is a liver targeting gene vector four yuan long circulating preparation. The specific research contents and results such as (?)
First transformed Escherichia coli amplification of plasmid DNA containing reporter gene (PEGFP-C1), then QIAGEN endotoxin free Maxi kit extraction of plasmid DNA, and to evaluate its quality and concentration. Then prepared liposomes of PEG (lipid formulation for POPC 94%, DDAB 2%, DSPE-PEG2000 3% and DSPE-PEG2000-biotin 1%) PEI/pDNA and polycation compression body, the formation of LPD complexes with the liposome PEI/pDNA compression. The single factor experiment combined with the optimization of prescription LPD uniform design experiments, single factor experiments results are as follows: the plasmid concentration at 40 g below /ml, the particle size changes little, the increase of the concentration of composite particle size increased more than N/P; 1.5, plasmid encapsulation completely, with the increase of N/P composite particle size decreases, the potential increase in N/P is equal to 15, the smallest particle size, increasing the particle size of N/P, basically no change; the molar ratio of lipid /pDNA In 50:1, enzyme digestion electrophoresis showed that the lipid can be completely encapsulated with PEI/pDNA compression, lipid /pDNA ratio increased, the grain size of LPD decreases, the 170:1 potential decreased, the smallest particle size and the molar ratio of lipid /pDNA and then increase the complex size began to become larger. The optimization of LPD compound prescription uniform design experiment results: in HEPES 7 pH buffer, plasmid concentration was 40 g/ml, N/P=15, lipid /pDNA=170:1, LPD prepared by the average particle size is 130 nm, the average zeta potential of 1.5mV. LPD for further structure modification in the outer lipid membrane PEG chain receptor monoclonal antibody conjugated anti human insulin (8314-SA), Lipo/PEI/pDNA complexes modified by monoclonal antibody targeting of the liver (8314-LPD). Transmission electron microscope, the liver targeting of 8314-LPD complex spherical, regular shape, no adhesion; the particle size of potential analysis, the average particle size At about 150nm, the potential is about 4.5mv. The green fluorescent protein gene PEGFP-C1 as a reporter gene study complex on plasmid protection ability analysis experiments prove that this gene vector can effectively protect plasmid DNA from DNA enzyme gel electrophoresis (?) Nase 1) degradation; experimental serum stability indicated that 8314-LPD complex can exist stably in the serum; long-term stability experiment results, 4 degrees and 25 degrees of nitrogen sealed storage, complex size has increased, but the extent of different description of temperature on stability of composites with obvious effect, 25 C storage will make the complex size occurred within a month of very large increases.
Analysis of cell transfection experiments in vitro cytotoxicity and transfection efficiency of LPD vector, less than PEI/pDNA compression, but the monoclonal antibody modified 8314-LPD gene vector can well be human hepatocellular carcinoma cell SMMC-7721 uptake, compared with the previous two group gene vector, transfection rate showed that LPD and 8314.-LPD complexes increased obviously.MTT experiment the toxicity of less than PEI/pDNA polyoxocations compression body.
In order to improve the long-term stability of the 8314-LPD complex, by freeze drying method to prepare freeze-dried powder. The specific steps, the first selection of quality cryoprotector, through the investigation of freeze-dried powder of appearance, color, surface refinement, dispersing ability, found that the lyophilized preparation of trehalose appearance does not collapse, no bubble, uniform color, delicate texture; after rehydration, dispersing time is short; after reconstitution, composite particle size and Zeta potential did not change, is the best protection agent carrier. Secondly, to evaluate the quality of freeze-dried powder, the freeze-dried powder dissolved after anti enzyme digestion experiment, found by lyophilization preparation after rehydration can still maintain the nuclease resistance, indicating vector structure in freeze drying process have not been damaged. Transfection experiments showed that the freeze-dried carrier dissolved after the transfection activity is basically no change.
The results of this study show that the 8314-LPD composite preparation method is simple and feasible and can effectively protect the plasmid DNA by nuclease degradation, serum complexes stability and cell transfection rate are high, low cytotoxicity, and can be prepared by freeze drying properties of stable long term preservation of freeze-dried powder of long cycle. The liver immune gene carrier to the active target, comprehensive utilization of the advantages of cationic polymer and liposome, overcomes two shortcomings, I believe in the gene therapy of liver cancer, there will be good prospects for development.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R346

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 季寶芳;肝癌靶向的阿霉素—脂質(zhì)—聚陽離子-DNA復(fù)合物的制備及體內(nèi)評(píng)價(jià)[D];鄭州大學(xué);2012年

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本文編號(hào)：1361012