本文關(guān)鍵詞：用于防齲DNA疫苗粘膜給藥的陽(yáng)離子脂質(zhì)體載體的研究　出處：《華中科技大學(xué)》2009年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 非病毒載體 陽(yáng)離子脂質(zhì)體 DNA脂質(zhì)復(fù)合物 轉(zhuǎn)染

【摘要】：基因治療成功的關(guān)鍵是需要高效、安全和靶向的載體系統(tǒng),將DNA轉(zhuǎn)移至靶細(xì)胞核內(nèi)進(jìn)行表達(dá)。陽(yáng)離子脂質(zhì)體是用于基因治療中最有前景的非病毒載體系統(tǒng)之一。陽(yáng)離子脂質(zhì)體/DNA復(fù)合物,也叫脂質(zhì)復(fù)合物,是自組裝納米體系,能將DNA輸送至多種類(lèi)型的細(xì)胞中。在本研究中,我們制備了DOTAP/DOPE脂質(zhì)體,對(duì)其與DNA的相互作用進(jìn)行了理化表征,并研究了它們的體外轉(zhuǎn)染效率。完成的工作包括以下幾個(gè)方面: (1)薄膜超聲法制備DOTAP/DOPE陽(yáng)離子脂質(zhì)體,考察確定了此工藝中水化時(shí)間與超聲時(shí)間,并對(duì)制得的脂質(zhì)體的粒徑分散性、zeta電位分布、形貌以及初步物理穩(wěn)定性進(jìn)行表征。結(jié)果表明此制備工藝有較好的重復(fù)性。最后制得的陽(yáng)離子脂質(zhì)體平均粒徑為128.6±4.6 nm,PDI為0.168±0.016,zeta電位為68.7±4.6 mV。在4℃或37℃貯存1個(gè)月后,其膠體穩(wěn)定性較好。 (2)制備了陽(yáng)離子脂質(zhì)體/DNA復(fù)合物,并考察了復(fù)合物的理化性質(zhì)。粒徑和電位測(cè)試、瓊脂糖凝膠電泳的結(jié)果表明在N/P比為1.5以上時(shí),DNA與陽(yáng)離子脂質(zhì)體形成了穩(wěn)定且?guī)д姷膹?fù)合物。核酸酶酶切實(shí)驗(yàn)的結(jié)果表明復(fù)合物能保護(hù)DNA抵御核酸酶的降解。我們還對(duì)在生理離子強(qiáng)度下制備的復(fù)合物進(jìn)行粒徑和電位考察,結(jié)果表明在N/P比為3以上時(shí),復(fù)合物帶正電,且復(fù)合物顆粒有成簇聚集現(xiàn)象。此外,我們對(duì)動(dòng)物實(shí)驗(yàn)要求的脂質(zhì)復(fù)合物的制備進(jìn)行摸索。 (3)通過(guò)對(duì)CHO和NIH-3T3細(xì)胞的MTT細(xì)胞毒性和轉(zhuǎn)染實(shí)驗(yàn)表明,復(fù)合物的細(xì)胞毒性較小;陽(yáng)離子脂質(zhì)體可以將DNA轉(zhuǎn)移到兩種細(xì)胞內(nèi)得到有效的表達(dá),轉(zhuǎn)染的最佳N/P比為1.5到2。在CHO細(xì)胞中的轉(zhuǎn)染效率比NIH-3T3細(xì)胞中高。 綜上所述,DOTAP/DOPE陽(yáng)離子脂質(zhì)體與DNA通過(guò)靜電作用自組裝形成的復(fù)合物,能有效介導(dǎo)基因的體外轉(zhuǎn)染,為后續(xù)的研究打下良好的基礎(chǔ)。
[Abstract]:Gene therapy is the key to success needs to be efficient, safe and carrier system to the target, will be transferred to the DNA nuclei of the target cells. The cationic liposome is used as one of the most promising nonviral systems in gene therapy. The cationic liposome /DNA complexes, also called lipid complexes, self-assembly nanometer system and DNA can be transported to various types of cells. In this study, we prepared DOTAP/DOPE liposome was prepared, and its interaction on the DNA of physicochemical characterization, and study the transfection efficiency in vitro. Their work includes the following aspects:
(1) preparation of DOTAP/DOPE cationic liposome film ultrasonic method, investigate and determine the process of hydration time and ultrasonic time, and the liposomes prepared by particle size dispersion, zeta potential distribution, morphology and physical stability was investigated. The preliminary results show that the preparation process has good repeatability. Finally prepared the cationic liposomes with an average particle size of 128.6 + 4.6 nm, PDI was 0.168 + 0.016, zeta potential is 1 months 68.7 + 4.6 mV. storage at 4 degrees or 37 degrees Celsius, the colloidal stability is good.
(2) cationic liposome /DNA complexes were prepared, and the effects of physicochemical properties of the complexes. The particle size and zeta potential test, agarose gel electrophoresis results showed that the N/P ratio above 1.5, DNA and cationic liposomes formed stable and positively charged complexes nuclease digestion assay. The results show that the degradation compound can protect DNA against nuclease. We also prepared complexes at physiological ionic strength of the particle size and zeta potential investigation results show that in the N/P ratio above 3, the positively charged complexes, and composite particles aggregated to form. In addition, we on the lipid complex animal experiment requirements of the preparation were explored.
(3) by CHO and NIH-3T3 MTT cell cytotoxicity and transfection experiments showed that the cytotoxicity of small complexes; cationic liposome DNA can be transferred to the two kinds of effective expression in cells transfected with N/P, the best ratio of 1.5 to 2., the transfection efficiency in CHO cells than in NIH-3T3 cells.
In conclusion, DOTAP/DOPE cationic liposomes and DNA complex formed by electrostatic interaction can effectively mediate gene transfection in vitro, and lay a good foundation for subsequent research.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R392.1

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本文編號(hào)：1366977