壳聚糖CpG-ODN纳米粒的制备及其免疫增强活性研究
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摘要
随着人类对基因的认识和研究,功能基因不断涌现。大量研究事实表明含CpG基序的DNA或人工合成的CpG-ODN是一种强有力的免疫刺激剂,可激活多种免疫细胞,诱导分泌以IL-12为主的Th1型细胞因子,诱导Th1型免疫应答,在抗肿瘤、抗感染、抑止过敏反应、作为免疫佐剂等方面得到广泛应用和研究。CpG核酸要发挥激活功能,必须作用于细胞内的Toll样受体-9(TLR-9)。然而,核酸不仅在体内过程中容易被酶解,而且由于本身带负电荷,不易结合到带负电的细胞表面,因此CpG核酸是否能被细胞有效摄取很大程度上影响其免疫激活功能,基因传递成为其发挥作用的关键环节。壳聚糖(chitosan,CS)具有良好的生物相容性、生物可降解性以及无毒性,近几年被用作基因载体材料已成为研究热点。研究显示,CS纳米粒能有效传递基因,结合效应细胞,提高基因的转染及表达效率。然而迄今未见用CS作为材料制备纳米粒运载CpG-ODN用于正常小鼠体内研究其免疫增强活性的报道,本论文将构建CS-CpG-ODN基因纳米粒,初步探究其理化性质及其它相关特性,继而免疫接种于正常小鼠体内研究免疫活性,期望获得更强烈的免疫刺激效应,以证实CS基因纳米粒可促进CpG-ODN发挥免疫刺激作用。本论文完成的研究工作主要包括以下三个方面:
1.用复凝法制备CS-CpG-ODN基因纳米粒,紫外分光光度法和水合茚三酮法分别测试CpG-ODN的包封率和载药量,并通过单因素分析考察制备过程中几种因素对纳米粒包封率的影响。实验结果表明,CS-CpG-ODN基因纳米粒平均包封率为90.67%,平均载药量为38.78%,均符合规定。在一定范围内,各因素对包封率影响较小。
2.在透射电子显微镜下观察纳米粒形态,初步统计粒径分布范围,激光粒度分析仪测定纳米粒粒径、表面电位等。采用3%琼脂糖凝胶电泳法进行阻滞试验,验证纳米粒复合物是否形成及其带电电性,开展酶保护试验以考察纳米粒抗酶特性。以L-02细胞和HepG2细胞作为模型细胞,研究CS纳米粒的细胞转染特性。结果显示,纳米粒得以形成,在透射电镜下观察呈近似球形,大小较均一,粒径主要分布在75-100nm之间,粒度分析仪测试粒径均值为93.7nm,纳米粒带正电荷,表面电位均值为20.2mV。纳米粒载体能有效抵抗高于生理条件下浓度核酶的裂解作用,保护CpG核酸不被酶解,转染实验结果显示,该纳米粒能被摄入至L-02细胞和HepG2细胞内。
3.小鼠随机分成4组,包括对照组、CS组、CpG-ODN组和CS-CpG-ODN组,采用后大腿肌肉注射方式免疫接种小鼠,两周后同法加强免疫一次。末次免疫两周后,处死小鼠,取脾脏供细胞免疫分析用,收集抗凝全血检测T细胞亚群,收集细胞上清和血清以测定免疫球蛋白及细胞因子。MTT法进行脾淋巴细胞增殖试验,流式细胞术分析外周血T淋巴细胞亚群CD4+、CD8+各自百分比以及CD4+/CD8+比值,ELISA法测定细胞上清IFN-γ、IL-4分泌水平,检测血清中IL-2、IL-12水平以及IgG水平。研究结果表明,小鼠经免疫接种后,CS组和CpG-ODN组显示出增强的T淋巴细胞增殖效应,表明T细胞免疫应答被激发;还表明外周血中CD4+T细胞百分比和CD4+/CD8+比值升高,细胞上清IFN-γ分泌增多,血清中IL-2、IL-12及IgG水平升高。于CS-CpG-ODN组,这些效应和变化表现更为明显,此外,IL-4分泌显著减少。
本论文采用复凝法成功构建了CS-CpG-ODN基因纳米粒,具有较小的粒径,获得了满意的包封率和载药量,具备较强保护CpG核酸效能,该带正电的纳米粒能够被细胞摄取;首次将CS-CpG-ODN基因纳米粒载体运用于正常小鼠体内研究免疫活性。IL-2、IL-12和IFN-γ代表Th1型细胞因子,IL-4代表Th2型细胞因子,CpG核酸经CS纳米粒包载后,加强了它的免疫刺激作用尤其是诱导Th1型免疫应答的能力。通过调节CD4+CD8+T细胞比例,重建T细胞免疫,也可能提高免疫功能。因此,CS纳米粒能有效递送并促进CpG-ODN发挥作用,研究结果对CpG-ODN的基因治疗提供重要的参考价值。
Many functional genes emerge prominently one after another along with the never-ending recognition and study on genes. Bacterial DNA and synthetic oligdeoxynucleotides (ODN) with motifs consisting of a central unmethylated CpG dinucleotides are immunostimulatory and can activate many kinds of immunological cells, and trigger Th1-polarized immune response. Owing to their strong immunostimulatory activity, a number of CpG-ODN are at various stages of preclinical and clinical evaluation as anti-tumor, antiviral, anti-anaphylaxis agents and as adjuvant in immunotherapy. To elicit immunostimulatory activity CpG-ODN need to be internalized by cells and act on Toll-like receptor-9. However, nucleic acid inclines to degradation in face of nuclease in vivo, besides, electronegative nucleic acid appears to be hard to bind to cell surface loading negative charge, so delivery of CpG-ODN is the critical step for CpG-ODN activity. Chitosan(CS), a kind of biodegradable, biocompatible and innoxious cationic polymer, has been popularly investigated as gene carrier. It is known that chitosan gene nanoparticles can be good for delivering genes and bind to effector cells, and enhance transfection efficiency of genes. Little is known as to the delivery of CpG-ODN using chitosan nanoparticles so far, in the current study, we therefore prepared CS-CpG-ODN nanoparticles, initially investigated their physico-chemical properties and other characteristics, then mice were immunized to research the immunocompetence in vivo, we hypothesized that the‘protected and controlled’gene delivery of CpG-ODN would enhance the immunostimulating effect of CpG-ODN in mice. The main contents are as follows in this paper:
1. CS-CpG-ODN nanoparcles were prepared by complex coacervation. The nanoparcles entrapment efficiency and loading capacity were detected by UV and ninhydrin assay respectively. Meanwhile, some factors of effecting entrapment efficiency were investigated in preparation process. The results obtained indicated that the nanoparticles showed 90.67% of mean entrapment efficiency and 38.78% of mean loading capacity respectively. To some extent, there was less effect of each factor to entrapment efficiency.
2. Size distribution and surface morphology of the particles were identified by transmission electron microscopy (TEM) measurements. The mean diameter, polydispersity, as well as mean zeta potential of nanoparticles were determined by a particle size / zeta potential analyzer. Gel retardation assay of 3% agarose was used to test the nature of surface charge and formed complexes, enzyme protection assay was carried out to investigate the inhibition of degradation by degrading enzymes. L-02 cells and HepG2 cells were used as model cells to study the transfection activity of nanoparcles. As seen in the electron microscope figure, The morphology of nanoparticles exhibited spherical shape, and the size was uniform with a size distribution range of 75-100nm on the whole. The mean diameter was 93.7 nm, the mean zeta potential was +20.2mV. The complexes could effectively bind CpG-ODN and protect it from nuclease degradation, the result of cellular transfection showed that these gene nanoparticles could be taken in by L-02 cells and HepG2 cells.
3. All mice were randomized into four groups: control group, CS group, CpG-ODN group and CS-CpG-ODN group,injected into posterior thigh muscles, then were given another shot after 2 weeks using the same formulation. Mice were killed two weeks later after secondary immunization, spleens were collected for cellular immunity analysis, blood serum was harvested and assayed for IL-2, IL-12 and IgG, cellular supernatant was extracted and assayed for IL-4 and IFN-γ, heparinized blood was gained for T lymphocyte subsets analysis. After that, Splenic lymphocytes proliferation was detected by MTT method, levels of IL-4, IFN-γ, IL-2, IL-12 and IgG were measured by ELISA. T lymphocyte subsets CD4+CD8+ from peripheral blood were analyzed by flow cytometry. After in vivo immunization, CS group and CpG-ODN group showed enhanced proliferative effects of T lymphocytes, increased percentages of CD4+ T cells and CD4+/CD8+ ratios, high levels of IFN-γin cellular supernatant and IL-2, IL-12, IgG in serum. These cases in CS-CpG-ODN group appeared more remarkable, in addition, evidently reduced level of IL-4 was observed in cellular supernatant.
The delivery system of CS-CpG-ODN nanoparcles was successfully made in this paper, and favourable entrapment efficiency, loading capacity and effect of enzyme protection were obtained. Furthermore, the nanoparcles result in effective cell uptake. Increased amounts of IL-2, IL-12 and IFN-γTh1 cytokines compared to decreased amount of IL-4 Th1 cytokine are believed to support the strongly biased towards Th1 response.The immunoenhancing potency could be related to availably re-establishing immunologic balance of T cells by adjusting the percentages of CD4+CD8+ T cells. The results in our work demonstrate for the first time the delivery system of CS-CpG-ODN nanoparcles may be associated with the strengthened immunostimulating effect of CpG-ODN, and then have an important base on CpG-ODN used for gene therapy.
1.用复凝法制备CS-CpG-ODN基因纳米粒,紫外分光光度法和水合茚三酮法分别测试CpG-ODN的包封率和载药量,并通过单因素分析考察制备过程中几种因素对纳米粒包封率的影响。实验结果表明,CS-CpG-ODN基因纳米粒平均包封率为90.67%,平均载药量为38.78%,均符合规定。在一定范围内,各因素对包封率影响较小。
2.在透射电子显微镜下观察纳米粒形态,初步统计粒径分布范围,激光粒度分析仪测定纳米粒粒径、表面电位等。采用3%琼脂糖凝胶电泳法进行阻滞试验,验证纳米粒复合物是否形成及其带电电性,开展酶保护试验以考察纳米粒抗酶特性。以L-02细胞和HepG2细胞作为模型细胞,研究CS纳米粒的细胞转染特性。结果显示,纳米粒得以形成,在透射电镜下观察呈近似球形,大小较均一,粒径主要分布在75-100nm之间,粒度分析仪测试粒径均值为93.7nm,纳米粒带正电荷,表面电位均值为20.2mV。纳米粒载体能有效抵抗高于生理条件下浓度核酶的裂解作用,保护CpG核酸不被酶解,转染实验结果显示,该纳米粒能被摄入至L-02细胞和HepG2细胞内。
3.小鼠随机分成4组,包括对照组、CS组、CpG-ODN组和CS-CpG-ODN组,采用后大腿肌肉注射方式免疫接种小鼠,两周后同法加强免疫一次。末次免疫两周后,处死小鼠,取脾脏供细胞免疫分析用,收集抗凝全血检测T细胞亚群,收集细胞上清和血清以测定免疫球蛋白及细胞因子。MTT法进行脾淋巴细胞增殖试验,流式细胞术分析外周血T淋巴细胞亚群CD4+、CD8+各自百分比以及CD4+/CD8+比值,ELISA法测定细胞上清IFN-γ、IL-4分泌水平,检测血清中IL-2、IL-12水平以及IgG水平。研究结果表明,小鼠经免疫接种后,CS组和CpG-ODN组显示出增强的T淋巴细胞增殖效应,表明T细胞免疫应答被激发;还表明外周血中CD4+T细胞百分比和CD4+/CD8+比值升高,细胞上清IFN-γ分泌增多,血清中IL-2、IL-12及IgG水平升高。于CS-CpG-ODN组,这些效应和变化表现更为明显,此外,IL-4分泌显著减少。
本论文采用复凝法成功构建了CS-CpG-ODN基因纳米粒,具有较小的粒径,获得了满意的包封率和载药量,具备较强保护CpG核酸效能,该带正电的纳米粒能够被细胞摄取;首次将CS-CpG-ODN基因纳米粒载体运用于正常小鼠体内研究免疫活性。IL-2、IL-12和IFN-γ代表Th1型细胞因子,IL-4代表Th2型细胞因子,CpG核酸经CS纳米粒包载后,加强了它的免疫刺激作用尤其是诱导Th1型免疫应答的能力。通过调节CD4+CD8+T细胞比例,重建T细胞免疫,也可能提高免疫功能。因此,CS纳米粒能有效递送并促进CpG-ODN发挥作用,研究结果对CpG-ODN的基因治疗提供重要的参考价值。
Many functional genes emerge prominently one after another along with the never-ending recognition and study on genes. Bacterial DNA and synthetic oligdeoxynucleotides (ODN) with motifs consisting of a central unmethylated CpG dinucleotides are immunostimulatory and can activate many kinds of immunological cells, and trigger Th1-polarized immune response. Owing to their strong immunostimulatory activity, a number of CpG-ODN are at various stages of preclinical and clinical evaluation as anti-tumor, antiviral, anti-anaphylaxis agents and as adjuvant in immunotherapy. To elicit immunostimulatory activity CpG-ODN need to be internalized by cells and act on Toll-like receptor-9. However, nucleic acid inclines to degradation in face of nuclease in vivo, besides, electronegative nucleic acid appears to be hard to bind to cell surface loading negative charge, so delivery of CpG-ODN is the critical step for CpG-ODN activity. Chitosan(CS), a kind of biodegradable, biocompatible and innoxious cationic polymer, has been popularly investigated as gene carrier. It is known that chitosan gene nanoparticles can be good for delivering genes and bind to effector cells, and enhance transfection efficiency of genes. Little is known as to the delivery of CpG-ODN using chitosan nanoparticles so far, in the current study, we therefore prepared CS-CpG-ODN nanoparticles, initially investigated their physico-chemical properties and other characteristics, then mice were immunized to research the immunocompetence in vivo, we hypothesized that the‘protected and controlled’gene delivery of CpG-ODN would enhance the immunostimulating effect of CpG-ODN in mice. The main contents are as follows in this paper:
1. CS-CpG-ODN nanoparcles were prepared by complex coacervation. The nanoparcles entrapment efficiency and loading capacity were detected by UV and ninhydrin assay respectively. Meanwhile, some factors of effecting entrapment efficiency were investigated in preparation process. The results obtained indicated that the nanoparticles showed 90.67% of mean entrapment efficiency and 38.78% of mean loading capacity respectively. To some extent, there was less effect of each factor to entrapment efficiency.
2. Size distribution and surface morphology of the particles were identified by transmission electron microscopy (TEM) measurements. The mean diameter, polydispersity, as well as mean zeta potential of nanoparticles were determined by a particle size / zeta potential analyzer. Gel retardation assay of 3% agarose was used to test the nature of surface charge and formed complexes, enzyme protection assay was carried out to investigate the inhibition of degradation by degrading enzymes. L-02 cells and HepG2 cells were used as model cells to study the transfection activity of nanoparcles. As seen in the electron microscope figure, The morphology of nanoparticles exhibited spherical shape, and the size was uniform with a size distribution range of 75-100nm on the whole. The mean diameter was 93.7 nm, the mean zeta potential was +20.2mV. The complexes could effectively bind CpG-ODN and protect it from nuclease degradation, the result of cellular transfection showed that these gene nanoparticles could be taken in by L-02 cells and HepG2 cells.
3. All mice were randomized into four groups: control group, CS group, CpG-ODN group and CS-CpG-ODN group,injected into posterior thigh muscles, then were given another shot after 2 weeks using the same formulation. Mice were killed two weeks later after secondary immunization, spleens were collected for cellular immunity analysis, blood serum was harvested and assayed for IL-2, IL-12 and IgG, cellular supernatant was extracted and assayed for IL-4 and IFN-γ, heparinized blood was gained for T lymphocyte subsets analysis. After that, Splenic lymphocytes proliferation was detected by MTT method, levels of IL-4, IFN-γ, IL-2, IL-12 and IgG were measured by ELISA. T lymphocyte subsets CD4+CD8+ from peripheral blood were analyzed by flow cytometry. After in vivo immunization, CS group and CpG-ODN group showed enhanced proliferative effects of T lymphocytes, increased percentages of CD4+ T cells and CD4+/CD8+ ratios, high levels of IFN-γin cellular supernatant and IL-2, IL-12, IgG in serum. These cases in CS-CpG-ODN group appeared more remarkable, in addition, evidently reduced level of IL-4 was observed in cellular supernatant.
The delivery system of CS-CpG-ODN nanoparcles was successfully made in this paper, and favourable entrapment efficiency, loading capacity and effect of enzyme protection were obtained. Furthermore, the nanoparcles result in effective cell uptake. Increased amounts of IL-2, IL-12 and IFN-γTh1 cytokines compared to decreased amount of IL-4 Th1 cytokine are believed to support the strongly biased towards Th1 response.The immunoenhancing potency could be related to availably re-establishing immunologic balance of T cells by adjusting the percentages of CD4+CD8+ T cells. The results in our work demonstrate for the first time the delivery system of CS-CpG-ODN nanoparcles may be associated with the strengthened immunostimulating effect of CpG-ODN, and then have an important base on CpG-ODN used for gene therapy.
引文
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