Study On Enhancing SIgA In Saliva Through Transfecting Salivary Glands With The Gene Encoding CCL25

Objective:The frequently-occurring oral diseases such as caries and periodontal disease are causes of tooth defects, tooth losing, and very harmful to the physical and mental health of human being. These diseases are concerned with bacterial infections which depend on the bacterial colonization on oral tissue. Either cariogenic bacteria or periodontal pathogens, must firstly colonize on the surface of tooth and periodontal tissue to form the plaque, then can play a role in the pathogeneses of the diseases. Therefore, to avoid the bacterial adhesion and colonization on the oral tissue is the effective way to prevent such oral infectious diseases.Secretory immunoglobulin A (sIgA) mainly exists in the exocrine fluid acting as a player of mucosal immunologic function. SIgA in the saliva could make the pathogenic microorganisms agglutinated, lose movement. It also could interact with the pathogenic microorganisms, block the specific combining site on the microbial surface, and consequently prevent the microbial from attaching on the oral surface and acts as the important natural immune barrier to withstand infection. Thereby, increasing sIgAin saliva could reduce the adhesion of bacterias on oral surface so as to effectively prevent oral infections.CCL25, the CC family of mucosa-associated chemokine, could guide the memory/effector T cells and part of the IgA antibody-secreting cells (IgA+ASC) to migrate from bloodstream to interstitial tissues. Recent studies showed that both of CCL25and its cognate receptor, CCR9, are expressed in the small intestine. Up to more than90%of T lymphocytes in the small intestine express CCR9. In addition to the induction of T lymphocytes, CCL25/CCR9also guides the homing of the B lymphocytes, and CCR9is find to be expressed in IgA+ASCs in murine and human's small intestinal.The expression and roles of CCL25in the small intestine has been confirmed, but it was not found in the salivary glands. Salivary glands are the effector sites of so-called common mucosal immune system. For the fact that sIgA in the saliva is synthesized by plasma cells in the salivary glands, so in this study, we aimed to guide more IgA+ASCs homing to the salivary glands to improve the level of sIgA throuth transfecting the salivary glands with the gene encoding CCL25. This strategy might provide basic data for the prevention of oral tissue infection.Methods:1. Total RNA was extracted from human small intestine. Amplify, purify and introduct the CCL25gene fragment into eukaryotic expression plasmid pCMV-3Tag-9by using the restriction sites Sac â…  and Hind â…¢, restriction electrophoresis verify CCL25connected correctly and test the sequence of CCL25.2. The experiment had5groups:the blank control group (B), transfec reagent control group (M), pCMV-3Tag-9/CCL25group (S), pCMV-3Tag-9/GFP group (G), pCMV-3Tag-9no-load group (N). Analysed the effect of transfection in human embryonic kidney293T cells by Real-time PCR and Western blot.3. The experiment had5groups:group A (the control group), group B (retroductal perfusion of plasmid pCMV-3Tag-9group), group C (injection in the glands of plasmid pCMV-3Tag-9group), Group D(retroductal perfusion of plasmid pCMV-3Tag-9/CCL25group), Group E (injection in the glands of plasmid pCMV-3Tag-9/CCL25group). The saliva of Wistar rats was taken at6time points (1day,2days,3days, lweek,2weeks and lmonth). The submandibular glands were randomly taken at the third day after experiment. Tested by Immunohistochemistry and ELISA.Results:1. The electrophoresis results revealed that the recombinant of the plasmid pCMV-3Tag-9/CCL25was correct by showing high bright bands around500bp and5000bp. The cDNA sequence of CCL25was correct by sequencing test.2. The293T cells were in good condition after they were transfected by the eukaryotic expression plasmid pCMV-3Tag-9/CCL25. GFP signals could be detected24h,48h and72h after transfection in more than70%cells. All Real-time PCR amplification curve show "s" shape which showed the normal amplification. Compared with other groups, the S group had a significant high level expression of CCL25at24h,48h after transfection. Western blot results showed that only S group had obvious strip in about20KD (fusion protein about20KD), and expression of72h was much higher than48h.3. The immunohistochemical results of Group D showed uniformly distributed brown granules and strong positive staining in the glands. Group E had local intensive positive staining. The sIgA level of Group D and Group E was significantly higher than other groups (P<0.01); and the sIgA level of group D incresead by time (within1month); the sIgA level of Group E decreased by time(within1month).Conclusion:1. Transfer of the plasmid harboring the gene encoding CCL25to salivary glands, can enhance the level of sIgA in saliva and avoid the potential safety problems caused by viral vectors. The method provides a safe protection for oral health.2. It is the first study on promoting the oral health through transferring chemokine CCL25gene fragment to the salivary glands so far.