载幽门螺杆菌Lpp20基因壳聚糖纳米粒的制备及其黏膜免疫小鼠的实验研究
发布时间：2018-08-07 18:49
【摘要】： 目的:构建包裹幽门螺杆菌脂蛋白Lpp20基因的壳聚糖纳米粒,通过黏膜途径免疫BALB/c小鼠,观察其所产生的体液免疫和细胞免疫应答水平,为壳聚糖纳米粒作为H. pylori DNA疫苗载体的应用提供理论和实验依据。 方法:采用复凝聚法制备载幽门螺杆菌脂蛋白Lpp20基因的壳聚糖(CS)纳米粒,用凝胶阻滞实验分析壳聚糖和DNA的结合能力;用透射电镜观察粒子的形态和大小;用纳米粒度仪测定粒径分布和Zeta电位;用紫外分光光度法检测纳米粒包封率;释放实验评价CS / DNA NPs的稳定性;用DNaseI消化实验观察CS /DNA NPs抗核酸酶降解的能力;用MTT法评价CS /DNA NPs细胞毒性。将裸质粒pcDNA3.1(+)/Lpp20和载基因壳聚糖纳米粒通过黏膜免疫(滴鼻和口服)雌性BALB/c小鼠,测定免疫小鼠血清特异性IgG和肠黏膜sIgA水平,ELISA法检测免疫小鼠脾淋巴细胞培养上清中IFN-γ、IL-4水平,MTT比色法检测免疫小鼠脾淋巴细胞增殖反应,免疫组化法检测Lpp20蛋白在小鼠鼻黏膜组织中的表达情况。 结果: (1)凝胶阻滞分析结果表明质粒DNA与壳聚糖之间通过静电作用而完全结合,包封率大于90%。 (2)制备的载基因壳聚糖纳米粒形态规则、大多成球形,粒径分布150-300nm,Zeta电位约为13.4mV。 (3)壳聚糖对质粒DNA有保护作用,能有效抵抗核酸酶降解。稳定性试验表明,4℃保存60d,壳聚糖纳米粒仍能较稳定地包裹Lpp20。MTT法证明载基因壳聚糖纳米粒在高浓度才出现低细胞毒性。 (4)裸质粒pcDNA3.1(+)/Lpp20与CS/DNA NPs通过黏膜免疫均能诱导小鼠产生有效的免疫反应。CS/DNA NPs滴鼻和口服免疫组诱导的抗体明显升高,与裸质粒pcDNA3.1(+)/Lpp20组相比有明显差异(P0.05),同时CS/DNA NPs滴鼻和口服免疫组小鼠脾淋巴细胞经特异性抗原刺激后,培养上清中IFN-γ和IL-4含量明显升高,与裸质粒pcDNA3.1(+)/Lpp20组、壳聚糖和PBS对照组之间差异具有显著性(P0.01),且壳聚糖纳米粒滴鼻免疫组高于口服组,差异也具有显著性(P0.05);CS/DNA NPs滴鼻和口服免疫组小鼠脾淋巴细胞经特异性抗原刺激后,刺激指数(分别为1.466±0.29和1.169±0.17)明显高于裸质粒pcDNA3.1(+)/Lpp20组(0.788±0.11)、壳聚糖组(0.473±0.09)和PBS组(0.442±0.12)(P0.01),且纳米粒滴鼻免疫组刺激指数高于口服组(P0.05)。 结论: (1)成功构建了包裹幽门螺杆菌脂蛋白Lpp20基因壳聚糖纳米粒; (2)壳聚糖纳米粒能增强pcDNA3.1(+)/Lpp20核酸疫苗的黏膜免疫(滴鼻和口服免疫)效果; (3)载Lpp20基因壳聚糖纳米粒滴鼻免疫比口服免疫能诱导更强的细胞和体液免疫应答。
[Abstract]:Objective: to construct chitosan nanoparticles coated with Helicobacter pylori lipoprotein Lpp20 gene and immunize BALB/c mice by mucosal pathway, and observe the humoral and cellular immune responses. To provide theoretical and experimental basis for the application of chitosan nanoparticles as H. pylori DNA vaccine carrier. Methods: chitosan (CS) nanoparticles carrying Helicobacter pylori lipoprotein Lpp20 gene were prepared by complex coacervation method. The binding ability of chitosan to DNA was analyzed by gel block assay, and the morphology and size of the particles were observed by transmission electron microscope (TEM). The particle size distribution and Zeta potential were measured by nano-particle size analyzer, the encapsulation efficiency of nanoparticles was detected by ultraviolet spectrophotometry, the stability of CS / DNA NPs was evaluated by release experiment, the ability of anti-nuclease degradation of CS / NPs was observed by DNaseI digestion experiment. The cytotoxicity of CS / NPs was evaluated by MTT assay. Female BALB/c mice were immunized with naked plasmid pcDNA3.1 () -Lpp20 and gene loaded chitosan nanoparticles through mucosal administration (nasal and oral). The levels of serum specific IgG and intestinal mucosal sIgA in immunized mice were determined by Elisa. The level of IFN- 纬 -IL-4 in the supernatant of splenic lymphocyte culture in immunized mice was determined by Elisa and the proliferation of spleen lymphocytes in immunized mice was detected by colorimetric assay. The expression of Lpp20 protein in mouse nasal mucosa was detected by immunohistochemical method. Results: (1) the gel block analysis showed that the plasmid DNA was completely bound to chitosan by electrostatic interaction, and the encapsulation efficiency was greater than 90. (2) the morphology of chitosan nanoparticles containing genes was regular. Most of them were spherical, and the Zeta potential was about 13.4 MV. (3) chitosan had protective effect on plasmid DNA and could effectively resist the degradation of nuclease. The stability test showed that chitosan nanoparticles could still be stably encapsulated by Lpp20.MTT method after preservation at 4 鈩，
本文编号：2171023
[Abstract]:Objective: to construct chitosan nanoparticles coated with Helicobacter pylori lipoprotein Lpp20 gene and immunize BALB/c mice by mucosal pathway, and observe the humoral and cellular immune responses. To provide theoretical and experimental basis for the application of chitosan nanoparticles as H. pylori DNA vaccine carrier. Methods: chitosan (CS) nanoparticles carrying Helicobacter pylori lipoprotein Lpp20 gene were prepared by complex coacervation method. The binding ability of chitosan to DNA was analyzed by gel block assay, and the morphology and size of the particles were observed by transmission electron microscope (TEM). The particle size distribution and Zeta potential were measured by nano-particle size analyzer, the encapsulation efficiency of nanoparticles was detected by ultraviolet spectrophotometry, the stability of CS / DNA NPs was evaluated by release experiment, the ability of anti-nuclease degradation of CS / NPs was observed by DNaseI digestion experiment. The cytotoxicity of CS / NPs was evaluated by MTT assay. Female BALB/c mice were immunized with naked plasmid pcDNA3.1 () -Lpp20 and gene loaded chitosan nanoparticles through mucosal administration (nasal and oral). The levels of serum specific IgG and intestinal mucosal sIgA in immunized mice were determined by Elisa. The level of IFN- 纬 -IL-4 in the supernatant of splenic lymphocyte culture in immunized mice was determined by Elisa and the proliferation of spleen lymphocytes in immunized mice was detected by colorimetric assay. The expression of Lpp20 protein in mouse nasal mucosa was detected by immunohistochemical method. Results: (1) the gel block analysis showed that the plasmid DNA was completely bound to chitosan by electrostatic interaction, and the encapsulation efficiency was greater than 90. (2) the morphology of chitosan nanoparticles containing genes was regular. Most of them were spherical, and the Zeta potential was about 13.4 MV. (3) chitosan had protective effect on plasmid DNA and could effectively resist the degradation of nuclease. The stability test showed that chitosan nanoparticles could still be stably encapsulated by Lpp20.MTT method after preservation at 4 鈩，
本文编号：2171023
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