Delivery of Human Antibacterial Peptide Gene Into Mouse Submandibular Gland

Objectives: Background: Human beta-defensin-2 (hBD-2) is one of the host-derived bactericidal and cationic peptides. Aggregatibacter actinomycetemcomitans (Aa) is gram-negative bacteria and relates to development of the aggressive periodontitis. Objective: To evaluate the antimicrobial effects of hBD-2 produced by epithelial cells and salivary glands after gene transfer.
Methods: Methods: Two recombinants, rAAV-hbd-2 and rAAV-gfp (green fluorescent protein gene), were constructed and transfected into HeLa cells and submandibular glands (SMGs) of 8 weeks-old male BALB/c mice via retro-ductal delivery. After 72h of transfection, Aa was added into culture medium and detected Aa invaded into HeLa cells. After sacrifice of mice, SMGs were harvested and analyzed for hBD-2 expression by immunohistochemistry and for hbd-2 mRNA accumulation in SMGs tissue by real-time PCR.
Results: Results: Numbers of Aa in HeLa cells was significantly decreased in rAAV-hbd-2 group compare to those in rAAV-gfp group 8 weeks after transfection (p < 0.05). Immunohistochemistry revealed that hBD-2 were produced in SMGs of the rAAV-hbd-2 treated group. In addition, accumulation of hbd-2 mRNA was significantly increased in that group (p < 0.05). rAAV-hbd-2 group showed a tendency to decreases at 15 weeks-old in total number of oral bacteria, however there were no significant differences between two groups at 16 weeks-old.
Conclusions: Conclusion: These results suggest the expression of hBD-2 in saliva may be beneficial for the control of oral infection. Further analyses of microflora are in progress.