AQP1 Gene Therapy Of Sj(?)gren's Syndrome Mediated By Recombinant Adenovirus Vector

Sj?gren's Syndrome (SS) is a autoimmune disease which mainly effect exocrine glands, such as salivary and lacrimal glands are involved in. The patients often showed dry mouth and eyes, ardor of oral mucosa, and abnormal taste. The severe symptoms include difficulty of speaking, chewing and swallowing, followed by oral ulcer, etc. Salivary gland biopsies showed lymphocytic infiltrate and destruction of acinus.Recent discovery of a family of water-specific membrane channel proteins, the aquaporins(AQPs), provided in sight into the molecular mechanism of membrane water permeability in a variety of tissues. AQPs provide the rapid and selective molecular pathway for water reabsorption through cellular membrane. Among the ten aquaporins identified to date, it was confirmed that AQP1, AQP2 and AQP5 were distributed in human salivary gland tissues, and close related to saliva secretion.Evidence showed that they played important role in pathological process of some diseases of salivary glands. AQP1 is the first identified aquaporin.It is expressed in human tissues abroadly, especially in the epithelia related to fluid absorption or secretion, and the endothelia cooperated with water transportation. AQP1 has been found localized in capillary vessels, ducts and acinus of salivary gland to participate saliva secretion. In this study, we try to investigate the in situ gene therapy effect to Sj?gren's Syndrome by adenovirus vector. First,recombinant adenovirns vector carrying AQP1 was constructed by a new simplified bacterial homologous recombinant system. The pCHIPev vector and shuttle plasmid pAdTrack-CMV were enzymatic digestion to generate recombinant shuttle plasmid. After cotransformed lineared recombinant shuttle plasmid and adenovirus backbone plasmid pAdEasy-l into BJ5183 E.coli.bacteria,recombinant adenovirus vector was generated by homologous recombination.After that,we transfected 293 cells with recombinant adenovirus plasmid by lipofectamine,and then recombinant adenovirus was generated and amplified. Titer was determined by the expression of report gene GFP in infected 293 cells under fluorescence microscope. After repeated infection一collection一freeze/thaw,the lysis supernatant of 293 was purified by CsCl gradient ultraspeed centrifuge. 48 hours after that ECV-304 cells were infected with pAdEasy-1/AQP1, the percentage of GFP positive cells was 75% under fluorescence microscope.The recombinant adenovirus can facilitate further animal researches in vivo.In our in vivo experiments,We first established a SS animal model, and then treated the mice with pAdEasy- 1/AQP1, Ad-empty vector (no foreign gene control vector ) and saline respectively. After that,dinking quantity and change of flow rate of saliva were investigated, and the expression of AQP1 of submandibular glands in each group was determined.The results showed: In SS mice, submandibular glands were destructed, and acinus were atrophy, which lead to glandular hypofunction. After deliver AQP1 gene into SS mice submandibular glands through recombinant adenovirus vector, the saliva secretion incresed significantly, the symptom of dry-mouth was improved and the drinking quantity was decreased. There were no significant differences in drinking quantity and flow rate of saliva between Ad-empty vector group and saline group. This indicated that Ad-empty vector had no effects on saliva secretion, and the treatment effects of pAdEasy-1/AQP1 to SS were accomplished through the gene AQP1, not the adenovirus vector itself. In conclusion, the recombinant AQP1 adenovirus vector that we generated in this study was effective to treat murine Sj?gren syndrome, which provides initial research foundation for further studies and applications.