APB-DOCK8转基因番茄疫苗防龋的实验研究  

Experimental study of APB-DOCK8 transgenic tomato vaccine for caries prevention

作　　者：龙茜 廖成成 肖琳琳 刘建国 管晓燕 LONG Qian;LIAO Cheng-cheng;XIAO Lin-lin;LIU Jian-guo;GUAN Xiao-yan(Department of Orthodontics Ⅱ,Affiliated Stomatological Hospital of Zunyi Medical University,Zunyi 563000;Oral Disease Research Key Laboratory of Guizhou Tertiary Institution,School of Stomatology,Zunyi Medical University,Zunyi 563006,Guizhou Province,China)

机构地区：[1]遵义医科大学附属口腔医院正畸科二组,贵州遵义563000 [2]贵州省普通高等学校口腔疾病研究特色重点实验室暨遵义市口腔疾病研究重点实验室,贵州遵义563006

出　　处：《上海口腔医学》2023年第6期561-567,共7页Shanghai Journal of Stomatology

基　　金：国家自然科学基金(81260164);贵州省科技计划项目[SY字(2012)3086号];遵义市科技计划项目[遵市科合HZ字(2022)423号]。

摘　　要：目的:观察转基因番茄防龋疫苗经灌胃免疫SD大鼠后的防龋效果,初步探索其免疫机制。方法:采用SD大鼠建立实验性龋齿模型,培育并鉴定转基因防龋番茄,将表达目的蛋白的转基因番茄防龋疫苗分次灌胃免疫SD大鼠,Elisa法检测SD大鼠唾液及血液样品中特异性抗PAcA的SIgA和IgG含量,取上下颌骨进行Keyes龋齿计分,取脾脏进行RNA-seq测序分析。采用SPSS 18.0软件包对数据进行统计学分析。结果:转基因番茄防龋疫苗中目的蛋白浓度为36.28μg/mL,免疫SD大鼠后,D组(8 mL/kg)在第6周产生的特异性SIgA和IgG抗体水平最高,且与其他组存在显著差异(P<0.05),龋齿计分也较其他组差异显著(P<0.05)。提取该组SD大鼠脾脏mRNA经RNA-seq测序,得到40个mRNA表达差异显著基因(P-adjust<0.05&|Fold Change|≥1.5)。26个基因显著上调,包括IGFBP6、COL15A1等,GO富集到体液免疫反应、B细胞活化、免疫球蛋白受体结合等;KEGG富集到56条信号通路,包括PI3K-AKT、NF-κB等信号通路,且F<0.001。14个基因显著下调,但下调基因GO和KEGG富集分析无统计学意义(F>0.1)。结论:转基因番茄防龋疫苗可能通过上调SD大鼠IGFBP6基因,介导PI3K-AKT信号通路的激活而减少龋齿发生。PURPOSE:To observe the anti-caries effect of transgenic tomato anti-caries vaccine after immunization with SD rats by gavage and to explore its immunity mechanism initially.METHODS:SD rats were used to establish an experimental caries model.The transgenic anti-caries tomatoes expressing the target protein were cultivated and identified.The SIgA and IgG contents of specific anti-PAcA in saliva and blood samples of SD rats were detected by ELISA.Then,the SD rats were sacrificed,the maxillary and mandibular bones were taken for Keyes dental caries score,and spleens were taken for the analysis of RNA-seq.Statistical analysis was performed with SPSS 18.0 software package.RESULTS:The target protein concentration in the transgenic tomato anti-caries vaccine was 36.28μg/mL.After vaccine immunization of SD rats,group D(8 mL/kg)produced the highest levels of specific SIgA and IgG antibodies at week 6 and was significantly different from the other groups(P<0.05),and caries counting score was also significantly different than the other groups(P<0.05).The spleen mRNA of SD rats in group D was extracted and sequenced by RNA-seq,and 40 genes with significant differences in mRNA expression were obtained(P-adjust<0.05&|Fold Change|≥1.5).26 genes were significantly upregulated,including IGFBP6 and COL15A1.The upregulated gene GO enrichment was enriched to humoral immune response,B-cell activation,and immunoglobulin receptor binding;KEGG enrichment was enriched to 56 signaling pathways,including PI3K-AKT and NF-κB,and F<0.001.Fourteen genes were significantly downregulated,but the analysis of downregulated gene GO and KEGG enrichment was not statistically significant(F>0.1).CONCLUSIONS:Transgenic tomato anti-caries vaccine may reduce caries occurrence by upregulating the activation of PI3K-AKT signaling pathway mediated by IGFBP6 in SD rats.

关 键 词：转基因番茄防龋疫苗 龋病 IGFBP6 PI3K-AKT 

分 类 号：R780.1[医药卫生—口腔医学]