Study Of Type Ⅲ Adenylate Cyclase(AC3) Locates On Non-neuronal Cells

Cyclic AMP is one of the most important intracellular second messenger，by regulatingthe activity of the enzyme to achieve regulation of many cellular metabolic processes，including but not limited to oogenesis，embryogenesis，larval development，hormonesecretion，glycogen breakdown，smooth muscle relaxation，cardiac contraction，olfaction，and learning and memory．Adenylyl cyclase (AC) is an ATP-pyro-phosphate lyase that converts ATP to cAMP andpyro-phosphate．AC3is the third cloning of AC isoforms，one of nine transmembrane ACsthat have been separated and recognized．AC3is not only found expression in the mousehippocampus，hypothalamus and epidermal tissue and other parts of the olfactory nerve cellcilia，still widely present in the spinal cord，adrenal medulla and cortex，atria，aortic smoothmuscle，lung，kidney，pancreatic，testes，ovaries and other tissues and organs，which is closelyrelated to a variety of physiological functions．Cilia are microtubule-based organelles like“hair” that extend from the surface of cellsand can receive both mechanical and chemical signals from other cells and theenvironment．be classified into two categories，motile cilia and non-motile cilia．The primarycilium is present in almost every mammalian cell type．The primary cilia contain a variety ofspecific receptors，ion channel proteins and signal transduction proteins．In recent years，found that primary cilia involved in a variety of biological processes such as cell intracellularsignal transduction， regulation of animal development and maintenance of normalphysiological function of various tissues and organs play an important role．Despite the large number of experiments show that AC3expressed in a variety ofnon-neuronal cells and had important physiological functions，but which part of thesenon-neuronal cells that expressed remains to be determined．This study aims to clear AC3expression in these cells，and expression sites．In this thesis，rat adrenal medulla cells (PC12)，human human astrocytes cells(U251)，inner medullary collecting duct3cells (IMCD3)，human kidney epithelial cells (293T)，bone marrow stromal cells (BMSC)，osteoblasts (OB)，HeLa cells and human lung cancercells(A549)as research material．The main content of the study is divided into three parts:Part1Cloning and expression of AC3from cells by RT-PCR technology．Cloningproducts were measured sequence，and the resulting cDNA sequences，respectively，and AC3human and murine genes were compared with the biological analysis softwareDNAMAN6．0．Part2In this study，immunofluorescence staining was used to detected AC3site inPC12，U251，IMCD3，293T，OB，BMSC，A549and HeLa cells．The results are as follows：1． Using specific primers to amplify and detect AC3gene derived cells．Comparativeanalysis showed that AC3endogenous expression in these cells derived from human andmouse．2． Immunofluorescence staining showed that the AC3labeled antibodies were foundand overlaped with Acetylated α-Tubulin in PC12and U251cells．Most of IMCD3and293Tcells had primary cilia and AC3located the cilia site．BMSC cells can be visually observedAC3located on the primary cilia．Although OB also has positive AC3，but there were noco-localization relationships with primary cilia．In A549and HeLa cells，AC3was expressedexclusively in the primary cilia．Conclusion:1AC3gene endogenous expression in PC12, U251, IMCD3,293T, BMSCs, OB, HeLaand A549cells.2AC3expressed in neurons primary ciliary can be used as a marker of primary cilia.3Except for OB cells, AC3is located in non-neuronal cells cilia of the five detectedkinds.