【摘要】： 目的:構(gòu)建包裹幽門螺桿菌脂蛋白Lpp20基因的殼聚糖納米粒,通過(guò)黏膜途徑免疫BALB/c小鼠,觀察其所產(chǎn)生的體液免疫和細(xì)胞免疫應(yīng)答水平,為殼聚糖納米粒作為H. pylori DNA疫苗載體的應(yīng)用提供理論和實(shí)驗(yàn)依據(jù)。 方法:采用復(fù)凝聚法制備載幽門螺桿菌脂蛋白Lpp20基因的殼聚糖(CS)納米粒,用凝膠阻滯實(shí)驗(yàn)分析殼聚糖和DNA的結(jié)合能力;用透射電鏡觀察粒子的形態(tài)和大小;用納米粒度儀測(cè)定粒徑分布和Zeta電位;用紫外分光光度法檢測(cè)納米粒包封率;釋放實(shí)驗(yàn)評(píng)價(jià)CS / DNA NPs的穩(wěn)定性;用DNaseI消化實(shí)驗(yàn)觀察CS /DNA NPs抗核酸酶降解的能力;用MTT法評(píng)價(jià)CS /DNA NPs細(xì)胞毒性。將裸質(zhì)粒pcDNA3.1(+)/Lpp20和載基因殼聚糖納米粒通過(guò)黏膜免疫(滴鼻和口服)雌性BALB/c小鼠,測(cè)定免疫小鼠血清特異性IgG和腸黏膜sIgA水平,ELISA法檢測(cè)免疫小鼠脾淋巴細(xì)胞培養(yǎng)上清中IFN-γ、IL-4水平,MTT比色法檢測(cè)免疫小鼠脾淋巴細(xì)胞增殖反應(yīng),免疫組化法檢測(cè)Lpp20蛋白在小鼠鼻黏膜組織中的表達(dá)情況。 結(jié)果: (1)凝膠阻滯分析結(jié)果表明質(zhì)粒DNA與殼聚糖之間通過(guò)靜電作用而完全結(jié)合,包封率大于90%。 (2)制備的載基因殼聚糖納米粒形態(tài)規(guī)則、大多成球形,粒徑分布150-300nm,Zeta電位約為13.4mV。 (3)殼聚糖對(duì)質(zhì)粒DNA有保護(hù)作用,能有效抵抗核酸酶降解。穩(wěn)定性試驗(yàn)表明,4℃保存60d,殼聚糖納米粒仍能較穩(wěn)定地包裹Lpp20。MTT法證明載基因殼聚糖納米粒在高濃度才出現(xiàn)低細(xì)胞毒性。 (4)裸質(zhì)粒pcDNA3.1(+)/Lpp20與CS/DNA NPs通過(guò)黏膜免疫均能誘導(dǎo)小鼠產(chǎn)生有效的免疫反應(yīng)。CS/DNA NPs滴鼻和口服免疫組誘導(dǎo)的抗體明顯升高,與裸質(zhì)粒pcDNA3.1(+)/Lpp20組相比有明顯差異(P0.05),同時(shí)CS/DNA NPs滴鼻和口服免疫組小鼠脾淋巴細(xì)胞經(jīng)特異性抗原刺激后,培養(yǎng)上清中IFN-γ和IL-4含量明顯升高,與裸質(zhì)粒pcDNA3.1(+)/Lpp20組、殼聚糖和PBS對(duì)照組之間差異具有顯著性(P0.01),且殼聚糖納米粒滴鼻免疫組高于口服組,差異也具有顯著性(P0.05);CS/DNA NPs滴鼻和口服免疫組小鼠脾淋巴細(xì)胞經(jīng)特異性抗原刺激后,刺激指數(shù)(分別為1.466±0.29和1.169±0.17)明顯高于裸質(zhì)粒pcDNA3.1(+)/Lpp20組(0.788±0.11)、殼聚糖組(0.473±0.09)和PBS組(0.442±0.12)(P0.01),且納米粒滴鼻免疫組刺激指數(shù)高于口服組(P0.05)。 結(jié)論: (1)成功構(gòu)建了包裹幽門螺桿菌脂蛋白Lpp20基因殼聚糖納米粒; (2)殼聚糖納米粒能增強(qiáng)pcDNA3.1(+)/Lpp20核酸疫苗的黏膜免疫(滴鼻和口服免疫)效果; (3)載Lpp20基因殼聚糖納米粒滴鼻免疫比口服免疫能誘導(dǎo)更強(qiáng)的細(xì)胞和體液免疫應(yīng)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- 緯 -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，

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