口服Ag85A DNA疫苗对小鼠肠上皮内淋巴细胞亚群和NK细胞毒活性的影响

发布时间：2018-06-23 15:09

本文选题：Ag85A + DNA疫苗　；参考：《中国医科大学》2007年硕士论文

【摘要】： 前言在我实验室完成国家自然基金资助项目《肠上皮淋巴细胞在消化道溃疡中生物活性的变化》和辽宁省科学技术基金项目((Ag85A基因疫苗治疗膀胱癌的研究》的基础上，已经建立了与iIEL研究相关的一整套实验技术，如iIEL及其亚群的分离技术，细胞因子检测技术，细胞毒及NK活性检测技术等。本实验为国家自然科学基金《自制口服DNA疫苗的黏膜表达部位与诱导肠iIEL效应机制的研究》中的一部分，拟采用构建的可在真核细胞内表达Ag85A基因的质粒，制成可供口服的DNA疫苗，经口服途径投与小鼠，以观察DNA疫苗对IEL的生物学活性的影响为重点，进行口服DNA疫苗的黏膜局部免疫应答效应的研究，阐明口服DNA疫苗诱导肠道局部黏膜免疫反应的机制。本实验将对口服DNA疫苗诱导肠道IEL的功能变化及黏膜局部免疫应答效应有较深入的了解，为调节肠道局部免疫功能提供新思路，并且将为口服DNA疫苗的临床应用进一步提供理论和实验依据。实验材料和方法按照Pure Yield Plasmid Maxiprep System质粒抽提试剂盒的说明从大肠杆菌中提取Ag85A质粒，然后用BglⅡ限制性内切酶消化质粒，经琼脂糖凝胶电泳鉴定Ag85ADNA的存在。C57BL／6小鼠30只，雄性，6—8周龄，体重18—22克，随机分成3组①生理盐水组10只，经口灌饲100μl生理盐水；②空质粒组10只，经口灌饲100μl lg／L以1：1-1：0.5的比例在室温状态下与脂质体乳化融合的空质粒；③Ag85A疫苗组10只，经口灌饲100μl lg／L Ag85A疫苗，共免疫三次，每次间隔14天，于末次免疫后的第10天颈脱位法处死各组小鼠，收集iIEL和脾细胞。用PE-cy5-CD3，FITC-CD4和PE-CD8荧光抗体标记，经流式细胞仪分析。同时用乳酸脱氢酶法测定脾NK细胞毒活性。实验结果以均数±标准差((?)±s)表示，用t检验进行显著性分析。实验结果末次免疫后10天，Ag85A疫苗组和生理盐水组CD4~-CD8~+iIEL相比差异显著降低(P＜0.05)，空质粒组和生理盐水组CD4~+CD8~-iIEL百分比相比无显著差异；Ag85A疫苗组，空质粒组和生理盐水组CD4~-CD8~+iIEL百分比无显著差异。Ag85A疫苗组和生理盐水组脾NK细胞毒活性见有意义的变化(P＜0.05)。结果显示口服Ag85A DNA疫苗后小鼠CD4~-CD8~+ iIEL百分比的数量显著降低，而脾NK细胞毒活性则增加。讨论本实验中小鼠在口服Ag85A DNA疫苗后出现了iIEL免疫功能下调和脾NK细胞毒活性提高，推测其机制可能如下，DNA疫苗的接种方法及途径将影响其所含目的基因的表达、抗原的提呈及其诱导的免疫应答的类型。Tanglle等曾用肌肉注射和皮内基因枪轰击方法将Ag85A DNA疫苗投于BALB／c小鼠和C57BL小鼠。结果发现，基因枪轰击方法可诱导BALB／c小鼠的抗体水平升高和CTL应答增强，但Th1型细胞因子IL-2和IFN-7分泌水平较低；而C57BL小鼠则对基因枪轰击方法反应较弱，对经肌肉注射接种反应较强，IL-2和IFN-γ分泌水平较BALB／c小鼠明显升高。另外经口免疫耐受与粘膜相关淋巴组织关系密切，一般来说低剂量口服抗原通过主动抑制起作用，高剂量抗原诱导克隆缺失和克隆无能。口服抗原作用于GALT中的调节性CD4~+T细胞，诱导其分泌抑制性细胞因子，如Th2或Th3类细胞因子来诱导免疫抑制。Tsuji等给予BALB／C小鼠口服β-乳球蛋白后发现，，小肠PP淋巴结中出现抗原特异性CD4~+CD25~+调节性细胞克隆，这些克隆具有无能的特性，且能主动抑制β-乳球蛋白特异性抗体的产生。激活的CD8~+γdT细胞除本身可以分泌TGF-β、IL-10等抑制因子外，还可以促使CD4~+aβT效应细胞向Th2／Th3表型转化，并使得TGF-β、IL-4、IL-10等细胞因子的分泌增加，进而发挥免疫抑制作用。CD8~+T细胞也表达抑制性表型，与肠上皮细胞共同培养后，CD8~+Tr细胞可进行寡克隆扩增，而这些细胞在炎症性肠道疾病患者中是缺失的，这表明CD8~+T细胞也参与了免疫耐受。结论 1、口服Ag85ADNA疫苗后小鼠CD4~-CD8~+iIEL百分比明显降低，CD4~+CD8~-ilEL百分比未见明显变化。 2、口服Ag85ADNA疫苗后小鼠脾NK细胞毒活性明显提高。
[Abstract]:Foreword

In this experiment , we have established a complete set of experimental techniques related to iIEL research , such as the separation technology of iIEL and its subpopulation , the technique of cytokine detection , cytotoxicity and NK activity detection .

experimental materials and methods

Ag85A plasmid was extracted from E . coli according to the instructions of the extraction kit of the plasmid of the plasmid extracted from Escherichia coli . The presence of Ag85ADNA was identified by agarose gel electrophoresis . Thirty male C57BL / 6 mice , 6 - 8 weeks old and 18 - 22 g of body weight were randomly divided into 3 groups ( 1 normal saline group ) , and 100 渭l of physiological saline was fed through oral gavage ;
( 2 ) 10渭l lg / L of empty plasmid group and 1 : 1 - 1 : 0.5 at room temperature ;
( 3 ) Ag85A vaccine group was immunized with 100 渭l lg / L Ag85A vaccine by oral gavage for three times . Each group of mice was sacrificed at the 10th day after the last immunization . IEL and spleen cells were collected . The cytotoxicity of spleen NK cells was determined by flow cytometry . The results of the experiment were expressed by mean 卤 SD ( 卤 s ) , and the significance analysis was performed with t test .

experimental results

Compared with the normal saline group , the percentage of CD4 ~ + CD8 ~ - iIEL was significantly lower in the Ag85A vaccine group and the normal saline group ( P < 0.05 ) 10 days after the last immunization , and the percentage of CD4 ~ + CD8 ~ - iIEL in the empty plasmid group and the saline group was not significantly different .
There was no significant difference in the percentage of CD4 ~ - CD8 ~ + iIEL in the Ag85A vaccine group , the empty plasmid group and the physiological saline group .

discuss

In this experiment , the immune function of iIEL and the cytotoxic activity of spleen NK appeared after oral administration of Ag85A DNA vaccine . It was speculated that the mechanism might be as follows : The inoculation method and pathway of DNA vaccine could affect the expression of the target gene , the antigen presentation and the type of immune response induced . The results showed that the gene gun bombardment method could induce the increase of antibody level and CTL response in BALB / c mice , but the level of Th1 type cytokines IL - 2 and IFN - 7 secretion was lower .
Compared with BALB / c mice , the level of IL - 2 and IFN - 纬 secretion was higher in C57BL mice than in BALB / c mice .

In addition , CD8 ~ + T cells can secrete TGF - 尾 , IL - 10 and so on . The CD8 ~ + T cells can secrete TGF - 尾 , IL - 10 and so on .

Conclusion

1 . After oral administration of Ag85ADNA vaccine , the percentage of CD4 ~ - CD8 ~ + iEL was obviously decreased , and the percentage of CD4 ~ + CD8 ~ - ilEL was not changed .

2 . NK cytotoxicity of mice after oral administration of Ag85ADNA vaccine was significantly improved .
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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