涎腺局部调节由防龋疫苗诱导的免疫应答的研究
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摘要
龋病仍然是影响人类健康的常见病。有资料显示,目前我国的患龋率达50%以上。提高口腔中唾液抗致龋菌变形链球菌(Streptococcus mutans,S.mutans)的特异抗体水平,是防治龋病的有效途径。免疫防龋研究在上个世纪七十年代就已开始,迄今已有较多的报道。然而,迄今为止的研究结果显示：接种防龋疫苗诱导产生的特异性抗体在口腔唾液中水平升高并不十分显著,对龋病的免疫保护效果仍不太理想。
研究表明,通过某粘膜途径接种疫苗产生的特异IgA抗原递呈细胞(IgA+ASCs)可迁徙至呼吸道、胃肠道及外分泌腺(如涎腺)的固有层。目前已经清楚,口服抗原在肠道被抗原递呈细胞捕获处理后运送到派伊尔氏结(Peyer's pat ch,PP)等粘膜相关淋巴组织(粘膜免疫诱导组织),激活抗原特异性免疫细胞,产生免疫应答。其中致敏的部分B细胞经转向重组(class switch recombination,CSR)后,分化为抗原特异IgA+ASCs,随后,经血循环迁徙至粘膜免疫效应部位(其中包括涎腺),产生并分泌IgA,发挥免疫保护效应。
趋化性细胞因子CCL28在引导IgA+ASCs经血循环选择性地向某些粘膜免疫效应部位归巢的过程中起关键作用。涎腺上皮细胞表达分泌至涎腺腺泡间组织中的CCL28可通过某种机制转运到局部毛细血管后静脉内皮细胞游离面,通过与IgA+ASCs表面的趋化性细胞因子受体CCR10特异结合来启动IgA+ASCs出血管机制,引导IgA+ASCs迁徙归巢至涎腺。此外,CCL28分子的羧基末端去富含组氨酸,可与G-、G+细菌以及某些真菌结合,改变其细胞壁包膜的通透性,具有较强的杀灭细菌、真菌的能力。可见,CCL28在口腔防御中发挥着重要作用。
变形链球菌与龋齿的发生和发展有关。其最初与附着作用有关的AgI/Ⅱ功能结构域位于分子的N-末端1/3,此区为唾液结合区段(Saliva-binding region, SBR),含有富含丙氨酸的重复区,是与牙面唾液蛋白受体结合的活性部位,并且证实它在变形链球菌与唾液包被的牙齿表面的最初结合过程中及其随后的龋病发生过程中起重要的作用。由于SBR在变形链球菌致龋过程中发挥重要的作用使其成为防治龋病的主要研究对象。
在本研究中,我们构建了一个编码基因CCL28的真核表达质粒,将该重组质粒逆行灌注到大鼠涎腺,检测其免疫效果。我们在前期的研究工作中已经成功构建了一个双启动子真核表达质粒pCN-SSIE,并证明了它作为DNA疫苗的免疫原性。以前期的研究工作为基础,我们采取将pCN-SSIE灌胃和向大鼠涎腺逆行灌注编码基因CCL28的重组质粒联合应用的方法免疫大鼠,并检测联合免疫后的免疫效果,探讨了提高唾液中IgA和CCL28水平的方法,以期为增强防龋疫苗的免疫应答提供新的思路。
材料和方法：
第一部分：本实验通过PCR方法扩增人CCL28的基因片段,连接到真核表达载体pCMV-3Tag-8上,经酶切鉴定和测序,检验重组质粒pCMV-3Tag-8-CCL28。将重组质粒转染293细胞,通过western blot方法检测转染后细胞上清中CCL28蛋白的表达。变形链球菌UA159(S. mutans UA159)在37℃、厌氧培养至生长对数期后,在新鲜的BHI培养液或293条件培养中悬浮,调节细菌浓度为1×106CFU/ml,荧光显微镜下观察S.mutans UA159的活性。依次向无菌水中加入质粒DNA和氯化锌,调节它们的浓度,分别为175μg/ml pCMV-3Tag-8-CCL28和3.6mMZn2+,制备zinc/pDNA溶液。通过大鼠腮腺导管逆行灌注zinc/pDNA溶液,分别在免疫后的第0、2、7和14天提取大鼠腮腺和唾液标本。通过RT-PCR方法和免疫荧光分别检测腮腺中CCL28和SIgA的水平,通过酶联免疫吸附实验(ELISA)检测唾液中CCL28和SIgA的表达水平。制备S. mutansUA159菌体悬浮液,接种到无菌雄性大鼠,然后将氯化锌溶液、pCMV-3Tag-8和zinc/pCMV-3Tag-8-CCL28对大鼠腮腺逆行灌注,建立动物模型。1周后,收集菌斑,用实时定量PCR方法检测变形链球菌。
第二部分：用CaCl：法将提纯的质粒pCN-SSIE转化到减毒沙门氏菌(attenuated salmonella),调节细菌浓度至1010/ml。经胃灌注pCN-SSIE防龋疫苗,3周后,在大鼠腮腺导管逆行灌注重组质粒pCMV-3Tag-8-CCL28,定期检测腮腺中CCL28的水平,以及唾液中CCL28和SIgA的表达水平；定期检查小肠黏膜组织和腮腺中CCR10的表达。
结果：
第一部分：我们成功构建了含CCL28的真核表达质粒pCMV-3Tag-8-CCL28。体外实验证明该质粒能在真核细胞中分泌CCL28蛋白,具有杀菌抑菌能力。将质粒通过腮腺导管逆行灌注到腮腺,结果显示它能有效地提高腮腺和唾液中CCL28的表达,提高唾液中SIgA的含量；定菌鼠唾液中的变形链球菌数目显著减少。
第二部分：联合免疫后1周,腮腺和唾液中CCL28的表达,以及唾液中SIgA的含量显著提高；小肠黏膜组织及腮腺中的CCR10的表达水平增高。
结论：
1.编码CCL28基因的真核表达质粒成功构建。体内外实验证实,构建的含CCL28基因的真核表达质粒能在真核细胞中分泌CCL28蛋白,具有抑菌杀菌的能力。用其进行涎腺导管逆行灌注转染涎腺细胞,提高了涎腺实体组织中CCL28表达水平。
2.用被pCN-SSIE转化的减毒沙门菌对大鼠灌胃进行粘膜免疫接种的同时,在腮腺导管腮腺导管逆行灌注质粒pCMV-3Tag-8-CCL28,发现无论是腮腺中的CCL28,还是唾液中特异性抗SBR SIgA分泌水平均显著提高；涎腺中CCR10的表达也增高。这说明通过在涎腺内进行基因转移,能吸引循环中更多的IgA+ASCs趋化到涎腺,分泌SIgA,因而能上调防龋疫苗诱导的免疫应答,二者在诱导机体粘膜免疫应答时具有协同作用。
Dental caries is still a common disease which affected human health. It was showed that the incidence of caries in China is higher than50%. It is agreed that promotion of specific antibody levels against cariogenic bacteria Streptococcus mutans (S. mutans) in saliva can effectively prevent dental caries. Studies on immunization against dental caries began in the seventies of the last century. So far, however, data have shown that specific antibody in saliva induced by vaccination against dental caries is not sufficient for effectively controlling the disease.
Studies have shown that specific IgA antibody secreting cells (IgA+ASCs) generated through mucosally inoculating could migrate to lamina propria of respiratory tract, gastro-intestinal tract and exocrine glands such as salivary glands. It is clear that oral antigen is captured by antigen-presenting cells in intestinal tracts and then transported to the Peyer's patch (PP, which was defined as one of mucosal immune-inducing tissues or sites) and sensitizes related antigen-specific immune cells. Some sensitized B cells by class switch recombination (CSR) differentiate into antigen-specific IgA+ASCs and subsequently migrate to the mucosal immune effector sites (including salivary glands) from bloodstream. In the effector sites, they produce and secrete IgA playing a protective role.
Chemotactic chemokine CCL28is crucial in guiding homing of IgA+ASCs in blood circulation selectively to mucosal immune effector sites. CCL28produced by salivary gland epithelial cells can be secreted into the salivary gland acinar lumina as well as transported to the free surface of vein endothelial cells through presently unknown mechanism where it interacts with chemokine receptor CCR10on IgA+ASCs and then initiates the cells homing to the salivary glands. Moreover, histidine-rich CCL28carboxy-terminal exerts the strong ability of killing G-/G+bacteria and some fungi through changing the permeability of the cell wall envelope. Therefore, it is clear that CCL28plays an important role in the oral defense.
S. mutans is associated with the initiation and progression of dental caries formation. The functional domain of AgⅠ/Ⅱ of the microbe responsible for initial adherence is localized on the N-terminal one-third of the molecule, which contains an alanine-rich repeat region and is termed saliva-binding region (SBR). Studies have suggested its importance in the initial adherence of S. mutans to saliva-coated tooth surfaces and subsequent development of dental caries. Because its importance in the pathogenesis of the caries, SBR have been became a significant candidate for anti-caries vaccine.
In this study, we have constructed a eukaryotic expression plasmid harboring the gene encoding CCL28and investigated its immune enhancing effects through retrograde perfusion of the recombinant plasmid to the rat salivary glands. At the same time, we immunized the animals with our previously constructed dual-promoter expression plasmid pCN-SSIE which acts as the DNA vaccine. And then, we detected the immune effects induced by the combined applications of intragastric administration of the plasmid pCN-SSIE and retrograde perfusion of the plasmid encoding CCL28to the rat salivary glands. We explore the way to increase the levels of both IgA and CCL28in saliva in order to provide new ideas to enhance the immune responses of anti-caries vaccines.
Material and Method:
Part I:Human CCL28gene fragment obtained by PCR amplification was inserted into the eukaryotic expression vector pCMV-3Tag-8. The recombinant plasmid pCMV-3Tag-8-CCL28was testeded by restriction enzyme digestion and gene sequencing. The successfully constructed plasmid pCMV-3Tag-8-CCL28was transfected into293cells and then the expression of CCL28protein in the supernatant after transfection was detected by western blot. Streptococcus mutans UA159(S. mutans UA159) was cultured at37℃in the anaerobic condition. Microbial cells in logarithmic growth phase were resuspended at1×106cells/ml in fresh BHI medium or293conditions and the activity of S. mutans UA159was observed under the fluorescence microscopy. The zinc/pDNA solution was prepared, which contained175μg/ml pCMV-3Tag-8-CCL28and3.6mM Zn2+in sterile water. Rat parotid glands and saliva were extracted at0,2,7and14days after retrograde perfusion zinc/pDNA solution to parotid ducts. After immunization, the levels of CCL28and SIgA in parotid glands were detected by RT-PCR and immunofluorescence; the expressions of CCL28and SIgA in saliva were tested by enzyme-linked immunosorbent assay (ELISA). Bacterial suspensions of S. mutans UA159were prepared and then were inoculated into the sterile male rats. Using zinc chloride solution, pCMV-3Tag-8and zinc/pCMV-3Tag-8-CCL28respectively immunized the gnotobiotic rats by retrograde perfusion to the parotid glands. One week later, the dental plaque was collected and investigated Streptococcus mutans by real-time quantitative PCR.
Part Ⅱ:The plasmid pCN-SSIE was purified and transformed into attenuated salmonella by CaCl2method. Rats were immunized by the attenuated salmonella transferred with pCN-SSIE at a concentration of bacteria to1010/ml. After3weeks, the zinc/pCMV-3Tag-8-CCL28solutions were administered into parotid glands through retrograde perfusion. The levels of CCL28in parotid glands and the expressions of CCL28and SIgA in saliva were detected. The levels of CCR10in small intestinal mucosa and salivary glands were detected.
Results:
Part Ⅰ:We have successfully constructed eukaryotic expression plasmid pCMV-3Tag-8-CCL28containing CCL28gene. Results of experiments in vitro showed that this plasmid secreted CCL28protein in eukaryotic cells and elicited bactericidal activity. The administration of plasmid through retrograde perfusion to the parotid gland could effectively increase the expressions of CCL28in the parotid gland and in saliva, and enhanced the levels of SIgA in saliva; the amount of S. mutans in gnotobiotic rats dramatically decreased.
Part II:After1week, the expressions of CCL28in the parotid gland and in saliva and SIgA in saliva were significantly increased via the strategy of combined immunization; the expressions of CCR10in in small intestinal mucosa and salivary glands increased.
Conclusions:
1. The construction of the eukaryotic expression plasmid harboring the gene encoding CCL28was successful. Experiments in vitro and in vivo confirmed that CCL28protein possessed strong antibacterial and bactericidal functions. Salivary duct retrograde perfusion with the plasmid constructed in the present study made the transfection of the salivary gland with the gene encoding CCL28a success.
2. Rats were treated by retrograde perfusion of plasmid pCMV-3Tag-8-CCL28in the parotid ducts and immunized throuth oral delivery of attenuated Salmonella transformed with pCN-SSIE. The levels of both CCL28in the parotid glands and specific anti-SBR SIgA in saliva were significantly increased. The expressions of CCR10in salivary glands were also enhanced. These results showed that much more IgA+ASCs migrated to the salivary glands and secreted SIgA. This strategy has a synergistic effect on inducing mucosal immune responses against cario-pathagens.
研究表明,通过某粘膜途径接种疫苗产生的特异IgA抗原递呈细胞(IgA+ASCs)可迁徙至呼吸道、胃肠道及外分泌腺(如涎腺)的固有层。目前已经清楚,口服抗原在肠道被抗原递呈细胞捕获处理后运送到派伊尔氏结(Peyer's pat ch,PP)等粘膜相关淋巴组织(粘膜免疫诱导组织),激活抗原特异性免疫细胞,产生免疫应答。其中致敏的部分B细胞经转向重组(class switch recombination,CSR)后,分化为抗原特异IgA+ASCs,随后,经血循环迁徙至粘膜免疫效应部位(其中包括涎腺),产生并分泌IgA,发挥免疫保护效应。
趋化性细胞因子CCL28在引导IgA+ASCs经血循环选择性地向某些粘膜免疫效应部位归巢的过程中起关键作用。涎腺上皮细胞表达分泌至涎腺腺泡间组织中的CCL28可通过某种机制转运到局部毛细血管后静脉内皮细胞游离面,通过与IgA+ASCs表面的趋化性细胞因子受体CCR10特异结合来启动IgA+ASCs出血管机制,引导IgA+ASCs迁徙归巢至涎腺。此外,CCL28分子的羧基末端去富含组氨酸,可与G-、G+细菌以及某些真菌结合,改变其细胞壁包膜的通透性,具有较强的杀灭细菌、真菌的能力。可见,CCL28在口腔防御中发挥着重要作用。
变形链球菌与龋齿的发生和发展有关。其最初与附着作用有关的AgI/Ⅱ功能结构域位于分子的N-末端1/3,此区为唾液结合区段(Saliva-binding region, SBR),含有富含丙氨酸的重复区,是与牙面唾液蛋白受体结合的活性部位,并且证实它在变形链球菌与唾液包被的牙齿表面的最初结合过程中及其随后的龋病发生过程中起重要的作用。由于SBR在变形链球菌致龋过程中发挥重要的作用使其成为防治龋病的主要研究对象。
在本研究中,我们构建了一个编码基因CCL28的真核表达质粒,将该重组质粒逆行灌注到大鼠涎腺,检测其免疫效果。我们在前期的研究工作中已经成功构建了一个双启动子真核表达质粒pCN-SSIE,并证明了它作为DNA疫苗的免疫原性。以前期的研究工作为基础,我们采取将pCN-SSIE灌胃和向大鼠涎腺逆行灌注编码基因CCL28的重组质粒联合应用的方法免疫大鼠,并检测联合免疫后的免疫效果,探讨了提高唾液中IgA和CCL28水平的方法,以期为增强防龋疫苗的免疫应答提供新的思路。
材料和方法：
第一部分：本实验通过PCR方法扩增人CCL28的基因片段,连接到真核表达载体pCMV-3Tag-8上,经酶切鉴定和测序,检验重组质粒pCMV-3Tag-8-CCL28。将重组质粒转染293细胞,通过western blot方法检测转染后细胞上清中CCL28蛋白的表达。变形链球菌UA159(S. mutans UA159)在37℃、厌氧培养至生长对数期后,在新鲜的BHI培养液或293条件培养中悬浮,调节细菌浓度为1×106CFU/ml,荧光显微镜下观察S.mutans UA159的活性。依次向无菌水中加入质粒DNA和氯化锌,调节它们的浓度,分别为175μg/ml pCMV-3Tag-8-CCL28和3.6mMZn2+,制备zinc/pDNA溶液。通过大鼠腮腺导管逆行灌注zinc/pDNA溶液,分别在免疫后的第0、2、7和14天提取大鼠腮腺和唾液标本。通过RT-PCR方法和免疫荧光分别检测腮腺中CCL28和SIgA的水平,通过酶联免疫吸附实验(ELISA)检测唾液中CCL28和SIgA的表达水平。制备S. mutansUA159菌体悬浮液,接种到无菌雄性大鼠,然后将氯化锌溶液、pCMV-3Tag-8和zinc/pCMV-3Tag-8-CCL28对大鼠腮腺逆行灌注,建立动物模型。1周后,收集菌斑,用实时定量PCR方法检测变形链球菌。
第二部分：用CaCl：法将提纯的质粒pCN-SSIE转化到减毒沙门氏菌(attenuated salmonella),调节细菌浓度至1010/ml。经胃灌注pCN-SSIE防龋疫苗,3周后,在大鼠腮腺导管逆行灌注重组质粒pCMV-3Tag-8-CCL28,定期检测腮腺中CCL28的水平,以及唾液中CCL28和SIgA的表达水平；定期检查小肠黏膜组织和腮腺中CCR10的表达。
结果：
第一部分：我们成功构建了含CCL28的真核表达质粒pCMV-3Tag-8-CCL28。体外实验证明该质粒能在真核细胞中分泌CCL28蛋白,具有杀菌抑菌能力。将质粒通过腮腺导管逆行灌注到腮腺,结果显示它能有效地提高腮腺和唾液中CCL28的表达,提高唾液中SIgA的含量；定菌鼠唾液中的变形链球菌数目显著减少。
第二部分：联合免疫后1周,腮腺和唾液中CCL28的表达,以及唾液中SIgA的含量显著提高；小肠黏膜组织及腮腺中的CCR10的表达水平增高。
结论：
1.编码CCL28基因的真核表达质粒成功构建。体内外实验证实,构建的含CCL28基因的真核表达质粒能在真核细胞中分泌CCL28蛋白,具有抑菌杀菌的能力。用其进行涎腺导管逆行灌注转染涎腺细胞,提高了涎腺实体组织中CCL28表达水平。
2.用被pCN-SSIE转化的减毒沙门菌对大鼠灌胃进行粘膜免疫接种的同时,在腮腺导管腮腺导管逆行灌注质粒pCMV-3Tag-8-CCL28,发现无论是腮腺中的CCL28,还是唾液中特异性抗SBR SIgA分泌水平均显著提高；涎腺中CCR10的表达也增高。这说明通过在涎腺内进行基因转移,能吸引循环中更多的IgA+ASCs趋化到涎腺,分泌SIgA,因而能上调防龋疫苗诱导的免疫应答,二者在诱导机体粘膜免疫应答时具有协同作用。
Dental caries is still a common disease which affected human health. It was showed that the incidence of caries in China is higher than50%. It is agreed that promotion of specific antibody levels against cariogenic bacteria Streptococcus mutans (S. mutans) in saliva can effectively prevent dental caries. Studies on immunization against dental caries began in the seventies of the last century. So far, however, data have shown that specific antibody in saliva induced by vaccination against dental caries is not sufficient for effectively controlling the disease.
Studies have shown that specific IgA antibody secreting cells (IgA+ASCs) generated through mucosally inoculating could migrate to lamina propria of respiratory tract, gastro-intestinal tract and exocrine glands such as salivary glands. It is clear that oral antigen is captured by antigen-presenting cells in intestinal tracts and then transported to the Peyer's patch (PP, which was defined as one of mucosal immune-inducing tissues or sites) and sensitizes related antigen-specific immune cells. Some sensitized B cells by class switch recombination (CSR) differentiate into antigen-specific IgA+ASCs and subsequently migrate to the mucosal immune effector sites (including salivary glands) from bloodstream. In the effector sites, they produce and secrete IgA playing a protective role.
Chemotactic chemokine CCL28is crucial in guiding homing of IgA+ASCs in blood circulation selectively to mucosal immune effector sites. CCL28produced by salivary gland epithelial cells can be secreted into the salivary gland acinar lumina as well as transported to the free surface of vein endothelial cells through presently unknown mechanism where it interacts with chemokine receptor CCR10on IgA+ASCs and then initiates the cells homing to the salivary glands. Moreover, histidine-rich CCL28carboxy-terminal exerts the strong ability of killing G-/G+bacteria and some fungi through changing the permeability of the cell wall envelope. Therefore, it is clear that CCL28plays an important role in the oral defense.
S. mutans is associated with the initiation and progression of dental caries formation. The functional domain of AgⅠ/Ⅱ of the microbe responsible for initial adherence is localized on the N-terminal one-third of the molecule, which contains an alanine-rich repeat region and is termed saliva-binding region (SBR). Studies have suggested its importance in the initial adherence of S. mutans to saliva-coated tooth surfaces and subsequent development of dental caries. Because its importance in the pathogenesis of the caries, SBR have been became a significant candidate for anti-caries vaccine.
In this study, we have constructed a eukaryotic expression plasmid harboring the gene encoding CCL28and investigated its immune enhancing effects through retrograde perfusion of the recombinant plasmid to the rat salivary glands. At the same time, we immunized the animals with our previously constructed dual-promoter expression plasmid pCN-SSIE which acts as the DNA vaccine. And then, we detected the immune effects induced by the combined applications of intragastric administration of the plasmid pCN-SSIE and retrograde perfusion of the plasmid encoding CCL28to the rat salivary glands. We explore the way to increase the levels of both IgA and CCL28in saliva in order to provide new ideas to enhance the immune responses of anti-caries vaccines.
Material and Method:
Part I:Human CCL28gene fragment obtained by PCR amplification was inserted into the eukaryotic expression vector pCMV-3Tag-8. The recombinant plasmid pCMV-3Tag-8-CCL28was testeded by restriction enzyme digestion and gene sequencing. The successfully constructed plasmid pCMV-3Tag-8-CCL28was transfected into293cells and then the expression of CCL28protein in the supernatant after transfection was detected by western blot. Streptococcus mutans UA159(S. mutans UA159) was cultured at37℃in the anaerobic condition. Microbial cells in logarithmic growth phase were resuspended at1×106cells/ml in fresh BHI medium or293conditions and the activity of S. mutans UA159was observed under the fluorescence microscopy. The zinc/pDNA solution was prepared, which contained175μg/ml pCMV-3Tag-8-CCL28and3.6mM Zn2+in sterile water. Rat parotid glands and saliva were extracted at0,2,7and14days after retrograde perfusion zinc/pDNA solution to parotid ducts. After immunization, the levels of CCL28and SIgA in parotid glands were detected by RT-PCR and immunofluorescence; the expressions of CCL28and SIgA in saliva were tested by enzyme-linked immunosorbent assay (ELISA). Bacterial suspensions of S. mutans UA159were prepared and then were inoculated into the sterile male rats. Using zinc chloride solution, pCMV-3Tag-8and zinc/pCMV-3Tag-8-CCL28respectively immunized the gnotobiotic rats by retrograde perfusion to the parotid glands. One week later, the dental plaque was collected and investigated Streptococcus mutans by real-time quantitative PCR.
Part Ⅱ:The plasmid pCN-SSIE was purified and transformed into attenuated salmonella by CaCl2method. Rats were immunized by the attenuated salmonella transferred with pCN-SSIE at a concentration of bacteria to1010/ml. After3weeks, the zinc/pCMV-3Tag-8-CCL28solutions were administered into parotid glands through retrograde perfusion. The levels of CCL28in parotid glands and the expressions of CCL28and SIgA in saliva were detected. The levels of CCR10in small intestinal mucosa and salivary glands were detected.
Results:
Part Ⅰ:We have successfully constructed eukaryotic expression plasmid pCMV-3Tag-8-CCL28containing CCL28gene. Results of experiments in vitro showed that this plasmid secreted CCL28protein in eukaryotic cells and elicited bactericidal activity. The administration of plasmid through retrograde perfusion to the parotid gland could effectively increase the expressions of CCL28in the parotid gland and in saliva, and enhanced the levels of SIgA in saliva; the amount of S. mutans in gnotobiotic rats dramatically decreased.
Part II:After1week, the expressions of CCL28in the parotid gland and in saliva and SIgA in saliva were significantly increased via the strategy of combined immunization; the expressions of CCR10in in small intestinal mucosa and salivary glands increased.
Conclusions:
1. The construction of the eukaryotic expression plasmid harboring the gene encoding CCL28was successful. Experiments in vitro and in vivo confirmed that CCL28protein possessed strong antibacterial and bactericidal functions. Salivary duct retrograde perfusion with the plasmid constructed in the present study made the transfection of the salivary gland with the gene encoding CCL28a success.
2. Rats were treated by retrograde perfusion of plasmid pCMV-3Tag-8-CCL28in the parotid ducts and immunized throuth oral delivery of attenuated Salmonella transformed with pCN-SSIE. The levels of both CCL28in the parotid glands and specific anti-SBR SIgA in saliva were significantly increased. The expressions of CCR10in salivary glands were also enhanced. These results showed that much more IgA+ASCs migrated to the salivary glands and secreted SIgA. This strategy has a synergistic effect on inducing mucosal immune responses against cario-pathagens.
引文
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15. Douglas C, Russell R. The adsorption of human salivary components to strans of the bacterium Streptococcus mutans [J]. Archs Oral Biol,1984,29(1):751-757.
16.樊明文.口腔微生物的附着[J].国外医学口腔医学分册,1986：13-73.
17.樊明文,张平,边专.免疫防龋的研究新进展[J].中华口腔医学杂志,1999,34(2)：69-73.
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19.贾荣,樊明文,边专等GTF-PAc融合防龋DNA疫苗研究(Ⅲ) pGLUA-P免疫定菌鼠防龋研究[J].口腔医学纵横,2002,18(1)：1-3.
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22. Jinghua Sun, Xuechao Yang, Qing-An Xu, Zhuan Bian. Zhi Chen, Mingwen Fan. Protective efficacy of two new anti-caries DNA vaccines. Vaccine 2009,27(52):7459-7466.
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8. Iwaki M, Okahashi N, Takahashi J, et al. Oral immunization with recombinant Streptococcus lactis earring the Streptococcus mutans surface ptotein antigen [J]. Infect Immun,1990, 58(9):2929-2934.
9. Motegi Y, Kita H, Kato M, et al. Role of secretory IgA,secretory componentand eosinophils in mucosal inflammation [J]. In Arch Allergy Immonol.2000,122(1):25-27.
10. Mazaanec MB, Kaetzel CS, Neudrud JG. Intracellμlar neutralization of virus by immunoglobin A antibodies [J]. ProcNatl Acad Sci USA,1992,89(5):6901-6905.
11. Kaetzl CS, Robinson JK, Chintalacharavn KR. The polymeric immunoglobμlin receptor mediates transport of immune complexes across epithelial cell:a local denfence function for IgA [J]. Proc Natl Acad Sci USA,1991,88(19):8796-8880.
12. Williams RC and Gibbons RJ. Inhibition of bacterialadherence by secretory immunoglobulin A:a mechanismof antigen disposal [J]. Science,1972.177(50):697-699.
13. Russell MW, M Kilian and ME Lamm. Biological activities of IgA in mucosal immunology. Academic Press,1999,11(2):225-240.
14.魏临.龋病的病因和预防[M].中华口腔医学杂志.1979：14-119.
15. Douglas C, Russell R. The adsorption of human salivary components to strans of the bacterium Streptococcus mutans [J]. Archs Oral Biol,1984,29(1):751-757.
16.樊明文.口腔微生物的附着[J].国外医学口腔医学分册,1986：13-73.
17.樊明文,张平,边专.免疫防龋的研究新进展[J].中华口腔医学杂志,1999,34(2)：69-73.
18.樊明文,边专,彭志翔等.编码PAc结构基因的DNA疫苗免疫动物的实验研究[J].中华口腔医学杂志,2002,37(1)：4-7.
19.贾荣,樊明文,边专等GTF-PAc融合防龋DNA疫苗研究(Ⅲ) pGLUA-P免疫定菌鼠防龋研究[J].口腔医学纵横,2002,18(1)：1-3.
20.杨德琴,刘天佳,曹福娴等.变形链球菌表面蛋白和葡糖基转移酶基因疫苗免疫防龋实验研究：唾液和血清抗体水平测定[J].华西口腔医学杂志,2003,21(5)：696-399.
21. Chang Liu, Mingwen Fan, Zhuan Bian, Zhi Chen, Yuhong Li. Effects of targeted fusion anti-caries DNA vaccine pGJA-P/VAX in rats with caries. Vaccine,2008,26(51):6685-6689.
22. Jinghua Sun, Xuechao Yang, Qing-An Xu, Zhuan Bian. Zhi Chen, Mingwen Fan. Protective efficacy of two new anti-caries DNA vaccines. Vaccine 2009,27(52):7459-7466.
23. Russell MW.Hajishengallis G,Childer NK, et al. Secretory immunity in defense against cariogenic mutans streptococci [J]. Caries Res,1999,33(1):4-15.
24. Czerkinsky C, Svennerholm AM, Quiding M, Jonsson R, Holmgren J. Antibody-producing blood and salivary glands after oral cholera vaccination of humans[J]. Infect Immun,1991, 59(3):996-1001.
25. Brandtzaeg P, IN Farstad, and G Haraldsen. Regional specialization in the mucosal immune system:primed cells do not always home along the same track [J]. Immunol, Today,1999, 20(6):20-267.
26.张延龄,张晖.疫苗学[M].北京,科学出版社,2004：286-306.
27. Yoshie O, Imai T, Nomiyama H. Chemokines in immunity [J]. Adv Immunol,2001, 78(1):57-110.
28. Youn BS, Mantel C, Broxmeyer HE, et al. Chemokines, chemokine receptors and hematopoiesis [J]. Immunol Rev,2000,177(1):150-174.
29. Ma Q, Jones D, Borghesani PR, et al. Impaired Blymphopoiesis, myelopoiesis and derailed cerebellar neuron migration in CXCR24 and SDF deficient mice [J]. PNAS,1998.95(16):9 448-9 453.
30. Hedrick JA, Zlotnik A. Chemokines and chemokine receptorsin T cell development [J]. Chem Immunol,1999,72:57-68.
31. Bowman EP, Campbell JJ, Soler D, et al. Developmental switches in chemokine response profiles during B cell differentiation and maturation [J]. J Exp Me,2000,191(8):130-1317.
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