鼠尾静脉注射装载脑抗原的纳米粒子治疗手术脑损伤的实验研究

Experimental study on treatment of surgical brain injury by rat tail vein injection of biotargeting nanoparticles loaded with brain antigens

目的:研究装载髓鞘碱性蛋白(MBP)的纳米粒子的物理、化学特性,探讨鼠尾静脉注射装载MBP的纳米粒子诱导免疫耐受治疗手术脑损伤(SBI)继发性炎症的疗效。方法:①采用乳化溶剂挥发法合成含有支链普鲁兰多糖的聚乙烯醇(PVA)修饰聚-(β氨基酯)(PBAE)/聚乳酸-羟基乙酸共聚物(PLGA)芯的纳米粒子;②30只雄性SD大鼠按随机数字表法分为假手术组、对照组及实验组,每组10只。假手术组只开骨窗,保持硬脑膜的完整;对照组和实验组建立SBI模型,对照组经鼠尾静脉注射装载生理盐水(NS)的纳米粒子,实验组经鼠尾静脉注射装载同种异体MBP的纳米粒子。分别于SBI后1、7、14、21 d通过ELISA检测外周血血清中IL-2、IL-4水平,流式细胞术检测外周血CD4/^(+)CD8^(+)T细胞比值,进行改良神经功能缺陷评分(MNSS)。SBI后第21天以脑组织切片免疫组织化学染色法检测神经小胶质细胞的表面标记物离子钙接头分子(Iba-1)表达水平。结果:①成功制备装载MBP或NS的纳米粒子,平均直径分别为266.4 nm和287.5 nm,平均Zeta电位值分别为14.2 mV和9.7 mV,电子显微镜观察具有典型的“核-壳结构”;②术后第7、14、21天,实验组MNSS低于对照组,对照组高于假手术组(P<0.05)。术后第7、14天,假手术组和实验组IL-2浓度低于对照组(P<0.05),假手术组和对照组IL-4浓度低于实验组(P<0.05)。术后第7、14、21天,假手术组和实验组CD4/^(+)CD8^(+)T细胞低于对照组(P<0.05)。术后21 d大鼠脑组织切片Iba-1表达阳性的细胞显微镜下呈棕黄色,实验组Iba-1表达量较对照组明显降低。结论:①本实验设计的新型纳米粒子对MBP具有高效和稳定的装载性;②鼠尾静脉注射装载MBP的纳米粒子能诱导机体产生免疫耐受,降低SBI术后继发性炎症反应及脑神经功能缺陷,可激活小胶质细胞从而保护脑神经细胞。

Objective:To study the physical and chemical characteristics of myelin basic protein(MBP)-loaded nanoparticles,and to explore the efficacy of rat tail vein injection of MBP-loaded nanoparticles to induce immune tolerance for the treatment of secondary inflammatory reaction caused by surgical brain injury.Methods:①By the emulsion solvent evaporation method,using polyvinyl alcohol(PVA)containing branched pullulan modified poly(β-amino ester)(PBAE)/poly(lactic-co-glycolic acid)(PLAG),synthesize a novel nanoparticle and its core consist of PVA/PBAE/PLGA;②30 male SD rats were divided into sham operation group,control group and experimental group with 10 rats in each group according to the random number table.Sham operation group only opened the bone window to keep the dura mater intact;control group and experimental group received SBI surgery,control group was injected with nanoparticles loaded with normal saline(NS)through the tail vein of rat,and experimental group was intravenous injected with the same species allogeneic MBP nanoparticles.Respectively 1,7,14,21 days after SBI,ELISA was used to detect IL-2 and IL-4 levels in peripheral blood serum;flow cytometry was used to test peripheral blood CD4/^(+)CD8^(+)T cell ratio,modified neurological severity score(MNSS)was also carried out.21 days after SBI,expression level of the surface marker ionized calcium binding adaptor molecule-1(Iba-1)of neuromicroglia was detected by immunohistochemical staining of brain tissue sections.Results:①Nanoparticles loaded with MBP or NS were successfully prepared,with average diameter of 266.4 nm and 287.5 nm,average Zeta potential values of 14.2 mV and 9.7 mV,indicating good stability and transmission electron microscope observation of typical"core-shell"structure;②7,14 and 21 days after surgery,MNSS in experimental group was lower than that in control group,and control group was higher than sham operation group(P<0.05).7,14 days after surgery,IL-2 concentration in sham operation group and experimental group was lower than that in control group(P<0.05),IL-4 concentration in sham operation group and control group was lower than that in experimental group(P<0.05).7,14 and 21 days after surgery,ratio of CD4/^(+)CD8^(+)T cells in sham operation group and experimental group were lower than control group(P<0.05).Immunohistochemistry of paraffin sections of brain tissue of rats at 21 days after operation showed that Iba-1 positive cells were brownish yellow under the microscope.Expression of Iba-1 in experimental group was significantly lower than that in control group.Conclusion:①The novel nanoparticles designed in this experiment have high and stable loading capacity for MBP;②Rat intravenous injected with MBP-loaded nanoparticles can induce immune tolerance and reduce secondary inflammation damage and cerebral nerve function after SBI defects can activate microglia to protect brain nerve cells.