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Na(+)/K(+)-ATPase in the lacrimal glands of rabbits and its changes during induced autoimmune dacryoadenitis.

ABSTRACT: To test the hypothesis that expression of Na(+)/K(+)-ATPase subunits in the lacrimal glands (LGs) of rabbits with induced autoimmune dacryoadenitis (IAD) changes.LGs were obtained from adult female rabbits with IAD and age-matched female control rabbits. The LGs were processed for laser capture microdissection (LCM), real time RT-PCR, western blot, and immunofluorescence for the detection of mRNA and proteins of the ?1, ?2, ?1, ?2, and ?3 subunits of Na(+)/K(+)-ATPase.In the rabbits with IAD, mRNA levels of ?1, ?1, and ?3 from whole LGs were significantly lower. In samples of acini and epithelial cells from various duct segments, collected by LCM, mRNA levels of ?1, ?1, ?2, and ?3 were significantly lower in the rabbits with IAD, although mRNA for ?2 could not be detected. However, western blots demonstrated that all five subunits were significantly higher in the rabbits with IAD, although their distribution patterns were similar to those of the control rabbits, as demonstrated by immunofluorescence.The data presented herein demonstrated significant changes in mRNA and protein expressions of Na(+)/K(+)-ATPase subunits in rabbits with IAD, suggesting that these changes may play a role in the pathogenesis of Sjögren's syndrome and altered LG secretion, as observed in these animals.

SUBMITTER: Ding C

PROVIDER: S-EPMC3171493 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Na(+)/K(+)-ATPase in the lacrimal glands of rabbits and its changes during induced autoimmune dacryoadenitis.

Ding Chuanqing C Lu Michael M Huang Jianyan J

Molecular vision 20110901

<h4>Purpose</h4>To test the hypothesis that expression of Na(+)/K(+)-ATPase subunits in the lacrimal glands (LGs) of rabbits with induced autoimmune dacryoadenitis (IAD) changes.<h4>Methods</h4>LGs were obtained from adult female rabbits with IAD and age-matched female control rabbits. The LGs were processed for laser capture microdissection (LCM), real time RT-PCR, western blot, and immunofluorescence for the detection of mRNA and proteins of the α1, α2, β1, β2, and β3 subunits of Na(+)/K(+)-AT ...[more]

PMID: 21921989

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Project description:BackgroundTear deficiency due to lacrimal gland (LG) dysfunction is one of the major causes of dry eye disease (DED). Therefore, LG stem cell-based therapies have been extensively reported to regenerate injured lacrimal tissue; however, the number of stem cells in the LG tissue is low, and 2D long-term cultivation reduces the differentiation capacity of stem cells. Nevertheless, 3D LG organoids could be an alternative for a DED therapy because it is capable of prolonged growth while maintaining the characteristics of the LG tissue. Here, we report the development of LG organoids and their application as cell therapeutics.MethodsDigested cells from human LG tissue were mixed with Matrigel and cultured in five different media modified from human prostate/salivary organoid culture media. After organoid formation, the growth, specific marker expression, and histological characteristics were analyzed to authenticate the formation of LG organoids. The secretory function of LG organoids was confirmed through calcium influx or proteomics analysis after pilocarpine treatment. To explore the curability of the developed organoids, mouse-derived LG organoids were fabricated and transplanted into the lacrimal tissue of a mouse model of DED.ResultsThe histological features and specific marker expression of LG organoids were similar to those of normal LG tissue. In the pilocarpine-treated LG organoid, levels of internal Ca2+ ions and β-hexosaminidase, a lysosomal protein in tear fluid, were increased. In addition, the secreted proteins from pilocarpine-treated lacrimal organoids were identified through proteomics. More than 70% of the identified proteins were proven to exosome through gene ontology analysis. These results indicate that our developed organoid was pilocarpine reactive, demonstrating the function of LG. Additionally, we developed LG organoids from patients with Sjogren's syndrome patients (SS) and confirmed that their histological features were similar to those of SS-derived LG tissue. Finally, we confirmed that the mouse LG organoids were well engrafted in the lacrimal tissue two weeks after transplantation.ConclusionThis study demonstrates that the established LG organoids resemble the characteristics of normal LG tissue and may be used as a therapy for patients with DED.

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Na(+)/K(+)-ATPase in the lacrimal glands of rabbits and its changes during induced autoimmune dacryoadenitis.

Publications

Na(+)/K(+)-ATPase in the lacrimal glands of rabbits and its changes during induced autoimmune dacryoadenitis.

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