Investigation of the role of AIP gene in pituitary adenoma pathogenesis on animal and human models

[M. Stojanović]

86 listova

Medicina - Interna medicina - endokrinologija / Medicine - Internal medicine - endocrinology

Naučno interesovanje za aryl hydrocarbon receptor-interacting protein (AIP) gen poteklo je od saznanja da su pacijenti sa inaktivirajućim AIP mutacijama predisponirani za razvoj velikih, invazivnih tumora hipofize, somatotropinoma ili prolaktinoma, koji se javljaju u mlađem dobu i otporni su na lečenje. Zebrice (Danio rerio) - (ZF) kao eksperimetalni životinjski model pružaju anatomske i fukcionalne sličnosti sa ljudskim neuroendokrinim sistemom. AIP gen je evoluciono visoko konzerviran i široko eksprimiran u organizmu. Fiziološka uloga AIP proteina u zdravoj hipofizi nije poznata. Zapažena je kolokalizacija AIP proteina sa hormonom rasta (HR) u sekretornim vezikulama somatotrofa. Prisustvo proteina AIP u serumu pokazano je proteomičkom analizom. Ciljevi: Na životinjskom modelu ZF ispitivan je uticaj inaktivacije AIP gena na tumorigenezu na nivou hipofize. Na ljudskom modelu ispitivana je fiziološka uloga AIP proteina na nivou hipofize. Pretpostavljena kosekrecija AIP i HR je ispitivana analizom serumskog nivoa AIP i HR bazno i u toku testova stimulacije ili supresije sekrecije HR kod ispitanika sa očuvanom sekrecijom HR, pacijenata sa deficitom HR (GHD) i pacijenata sa akromegalijom – nekontrolisanom hipersekrecijom HR. Studija na životinjskom modelu – Materijal i metode: Inaktivacija AIP gena - knock down (AIP KD) na embrionima ZF ostvarena je primenom antisens morfolino oligonukleotida u jednoćelijskom stadijumu razvoja. Kontrolni embrioni su tretirani neaktivnim oligonukleotidima sa 5 promenjenih baznih parova (KMo). Neinjektirani embrioni iz istog legla - wild type (WT) analizirani su kao netretirana kontrola. Svi embrioni (N=307) su inkubirani pod istim uslovima po 5 dana tokom kojih je morfometrijski analiziran njihov razvoj. Whole mount in situ hibridizacija za AIP korišćena je za ispitivanje ekspresije AIP gena kod normalnih zebrica (WT) i za potvrdu inaktivacije kod AIP KD. 120h nakon oplodnje, sprovedeno je whole mount imunobojenje ispitivanih i kontrolnih embriona primenom anti-PRL antitela (zečija antilosos poliklonalna 1:2000). Po 5 embriona iz svake grupe je slučajnim izborom izdvojeno za analizu digitalnom mikroskopijom. Površina bojenja na nivou hipofize analizirana je pomoću softvera za analizu slike (NIH ImageJ 1.48v). Studija na životinjskom modelu - Rezultati: Kod ZF embriona sa inaktivacijom AIP gena (AIP KD) zapažen je ukupni zastoj u rastu i razvoju u odnosu na kontrolne grupe WT i KMo...

Scientific interest for aryl hydrocarbon receptor-interacting protein (AIP) gene originates from the fact that patients with inactivating AIP mutations are predisposed to large, invasive, GH- or PRL-secreting pituitary tumours, occurring at a younger age and poorly responsive to treatment. The zebrafish (ZF) model provides anatomical and functional similarities to human neuroendocrine system. AIP is evolutionarily well conserved and widely distributed throughout organism. The role of AIP in normal pituitary function is largely unknown. AIP is co-localized with growth hormone (GH) in somatotroph secretory vesicles. Serum AIP protein was proteomically identified. We aimed at investigating the role of AIP inactivation on pituitary tumorigenesis in ZF model and investigating the physiological role of AIP at pituitary level in human model. We investigated whether AIP and GH are co-secreted by measuring serum AIP and GH levels at baseline and after GH stimulation and suppression tests using in vivo human models of normal GH secretion, patients with GH deficiency (GHD) and patients with GH hypersecretion - acromegaly. Animal model study - Material and Methods: AIP knock down (KD) ZF embryos were generated using antisense morpholino oligonucleotides injected at one-cell stage. Control embryos were injected with 5-base mispaired oligonucleotide as control morpholinos (CM). Wild type (WT) embryos from the same batch served as uninjected controls. All embryos (N=307) were incubated in the same conditions for 5 days, and assessed during development. Whole mount in situ hybridization for AIP was used to confirm the inactivation in AIP KD and to asses AIP expression in WT. At 120 hours post fertilization (hpf) whole mount immunostaining of all embryos was performed with anti-PRL antibodies (rabbit anti-salmon polyclonal 1:2000). A total of 15 embryos (5 from each group) were randomly selected for digital microscopy. Pituitary staining was assessed by image analysis software (NIH ImageJ 1.48v). Animal model sudy - Results: Overall developmental delay and retardation was observed in the AIP KD compared to WT and CM control groups. KD embryos exhibited reduced total body length, transitory midbrain enlargement, pericardium enlargement and swim bladder underdevelopment...

adenomi hipofize, sekrecija HR, hipopituitarizam, ITT, OGTT, akromegalija, FIPA, AIP, ARA9, XAP2

pituitary adenoma, GH secretion, hypopituitarism, ITT, OGTT, acromegaly, FIPA, AIP, ARA9, XAP2

616.432-006(043.3)

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