Establishment Of The Primary Culture Systems Of Inner Ear Sensory Epithelial Cell And The Immortalized Utricular Epithelial Cell Lines In Rat Associated With Hair Cell Regeneration.

Over the past several years,it has been well demonstrated that lower vertebrate and avian animals have a capacity to regenerate new inner hair cell .A certain degree of hair cell regeneration or repair also has been reported in mammals inner ear,But hair cell regeneration occurs with a much lower frequency than in the avian system.Upon acoustic trauma or aminoglycoside insults,significant proliferation of supporting cells occurs in the avian inner ear epithelium.Autoradiography and immunocytochemistry clearly indicate that new hair cells incorporate DNA synthesis markers including tritiated thymidine and bromodeoxyuridin (BrdU).Thus, supporting cells can be induced to proliferate and differentiate into new hair cells following injury . supporting cells inner ear are most likely hair cell progenitors.Proliferation of sppporting cells and a direct conversion of some sppporting cells into hair cells in both lower vertebrates and mammalian inner ear epithelia seems to be the first major step during hair cells regeneration.Identification of the molecular and cellular mechanisms underlying the development , differentiation and regeneralion of hair cells has been hampered by the small tissue size,the complicated in vivo bony structures of the inner ear. One way to solve this problem is to establishment cochlear sensory epithelial cell cultures, pure utricular sensory epithelial cell cultures and immortalized supporting cell lines from the inner ear sensory epithelium.These can used to studies of the effects of growth factors on supporting cell proliferation and hair cell regeneration.In this experiment, The primary culture systems of cochlear sensory epithelial cell and utricular sensory epichlial cell of the rats were successfully established in vitro. we have used gene transfer technique to insert the SV40 large T antigen oncogene into the genome of rat utricular epithelialcells.The infected cells contiune proliferating. The immortalized rat utricular epithelial cell lines (USEC-1) display many features of primary sensory epithelial cells. We also studied the morphogical and founctional characteriztion of USEC-1 cell line. The cell lines may provide a convenient in vitro model system to study hair cell differentiation and regeneration.Part IThe primary culture of rats cochlear sensory epithlial cell and the significance of expression of hair cell characteristic markersObjectives:To investigate the culture method of inner ear sensory epithlial cell and themechanisms of hair cell generation or regeneration,the primary culture systems of cochlear sensory epithelial cell of the rats were established.Methods: Cochlear sensory epithelial (containing the organ of Corti) of postnatal day 1 rats was isolated by mechanical dissociation,the explants were grown on sterilized disposable plastic dishes,culture in Dulbecco modified Eagle medium(DMEM), and observed daily by inverted microscope.The culture medium was changed twice a week.The pure cochlear sensory epithelial cell (CSEC) was harvested by the limiting dilution method.Immunocytochemical method with cytokeratin 18 , vimentin, Brn3.a , Calretinin, BrdU,ultrastrctural examination with transmission electron microscope.The markers of epichlial cell and the markers of hair cell were used identify the origin and characteriztion of CSEC.BrdU stained the DNA of the CSEC mitotic division.ResuIts:The fresh explants were light yellow.The morphology of CSEC couldn't be seen clearly under the inverted microscope, for the complex structures of cochlear sensory epichlial.CSEC grew out of the explants usually at 2 nd culture day. Fibroblast like cells (FLC) of around the CSEC grow faster than CSEC, but could be easily excluded.Pure CSEC may grow into monolayer, " cobblestone- like " appearance and showed a large,flat,polygonal epithelial morphotype with big,round neuclei. Some cell showed "Dome" formation,probably due to fluid collection underneath the cell monolayer.The culture CSEC coexpressed cytokeratin 18 and vimentin,has rich microvilli and complex tight junction,which indicated the epithelial origination of CSEC.Coexpressed of the Brn3.a and Calretinin of the hair cell characteristic markers identified the culture cell may represent rat progenitor hair cell.BrdU staining showed CSEC producted new hair cell-like (progenitor hair cell) by the mitotic division . Conclusions:The primary culture systems of cochlear sensory epithelial cell of the rats were successfully established in vitro.CSEC coexpressed the characteristic markers of the immature hair cell.which identified the culture cell may represent progenitor cell of hair cell in rat.It may be a suitable model for in-depth investigation the molecular mechanisms of hair cell generation or regeneration.Part II Establishment and characterization of primary culture of rats utricularsensory epithelial cellObjectives:To further investigate the mechanisms of hair cell generation or regeneration,the primary culture systems of utricular sensory epithelial cell of the rats were established.Methods: Utricular sensory epithelial of postnatal day 1 rats was isolated by mechanical dissociation.The explants were digested by thermolysin,then transfrred to an aliquot containing 0.125% trypsin and 0.125% collagenase for incubation to harvest the pure utricular sensory epichlial cell(USEC). USEC wes cultured in Dulbecco modified Eagle medium(DMEM), and observed daily by inverted microscope.The culture medium was changed twice a week.Immunocytochemical method with cytokeratin 18 ,vimentin, Calretinin and Brn3a;ultrastrctural examination with transmission electron microscope,reverse transcription PCR with mRNA of AchRa9 and Myosin VIIa.The markers of hair cells were used identify the origin and characteriztion of USEC. Results: USEC showed a large,flat,polygonal epithelial morphotype with big,round neuclei.USEC grew into monolayer, " cobblestone- like " apearance. Some cell showed "dome" formation,probably due to fluid collection underneath the cell monolayer.The culture USEC expressed cytokeratin 18 and not expressed vimentin,has rich microvilli and complex tight junction,which indicated the epithelial origination of USEC. Coexpressed of the Calretinin, Brn3.a and mRNA of AchR a9,Myosin VIIa of the hair cell characteristic markers identified the culture cell may represent rat progenitor hair cell. Conclusions:The primary culture systems of utricular sensory epithelial cell of the rats were successfully established in vitro.USEC coexpressed the characteristic markers of the hair cell , which identified the culture cell may represent progenitor cell of rats hair cell.It may be a suitable model for in-depth investigation the mechanisms of hair cell generation or regeneration.Part IIIEstablishment and characterization of immortalized rat utricular epithelialcell lines using a simian virus 40 by gene transfer techniqueObjectives:To further investigate the mechanisms of hair cell generation or regeneration,gene expression feature,and signal transduction involved in the development of hair cells. The immortalized utricular sensory epithelial cell line ( USEC-1 ) of the rat was established.Methods: Utricular sensory epithelial of postnatal day 1 rats was isolated by mechanical dissociation.the explants were digested by thermolysin,then transfrred to an aliquot containing 0.125% trypsin and 0.125% collagenase for incubation to harvest the pure utricular sensory epichlial cell (USEC). USEC wes cultured in Dulbecco modified Eagle medium (DMEM) for 3 days, the pure utricular sensory epichlial cell co-cultured with SV40 in serum-free DMEM for 3 days ,then was chosen by 2% new bron calf serum in DMEM for other 3 days.The identification of USEC line (USEC-1) depended on the morphogical and founctional characteriztion.So the inverted microscope and transmission electron microscope was undertaken. Anti-cytokeratin 18 of epichlial cells markers and Anti-vimentin were used in the immunocytochemical method.Reverse transcription PCR was used to study the mRNA expressed of SV40 large T antigen oncogene.These markers of epichlial cells was used identify the origin of USEC-1 line.Number of cell,Growth factor respose and flow cytometry method was used to detect the proliferation capable. Soft agar medium and Null mice were used to test the ability of USEC-1 line inducing neoplasm. Results: The cell line has retained many characteristics of the parental primary culture.The USEC-1 line showed a large,flat,polygonal epichlial morphotype with big,round neuclei.USEC-1 cell grew into monolayer, " cobblestone-like " appearance. Some cell showed "Dome" formation,probably due to fluid collection underneath the cell monolayer.The culture USEC-1 cell expressed cytokeratin 18 and not expressed vimentin,has rich microvilli and complex tight junction,which indicated the epithelial origination of USEC-1. Expressed of mRNA of SV40 large T antigen oncogene identified the SV40 DNA was inserted into DNA of USEC-1 cell.The inserted gene can remain stably in daughter cells.The cell lin has been passaged 25 passages,and the passage interval was 5-6 days,passage ratio 1:2-3. USEC-1 cell grew well in serum free DMEM,and the proliferation ability to bFGF with a dose-dependent. The USEC-1 was not capable of anchorage- independent growth.The test for neoplasm induced ablitily of USEC-1 was negative.Different passage USEC-1 was kept at -196°C. Conclusions: The immortalized utricular sensory epithelial cell line USEC-1 from the rats were successfully established in vitro.It possesses many structure features and immunocytochemical characteristics of the primary USEC cell, and does not result in tumorigenicy. It may be a suitable for in-depth investigation the mechanisms of hair cell generation or regeneration,molecular and genetic studies of the inner ear,especially in electric physiology and biomolecular aspect. Part IVThe significance of expression of hair cell characteristic markers of rats utricular sensory epithelial cell line USEC-1Objectives:Investigate the mechanisms of hair cell generation or regeneration by study to the expression of hair cell characteristic markers of Utricular sensory epithelial cell line USEC-1 in rats and the possiblity of USEC-1 differentiate into hair cell. Methods: USEC-1 wes cultured in serum free Dulbecco modified Eagle medium (DMEM) with 100ng/ml bFGF. Immunofluorescence and Western blot method with Calretinin and Brn3a; reverse transcription PCR with mRNA of AchRa9 and Myosin VIIa.The markers of hair cells were used identify the characteriztion of USEC-1. Results: USEC-1 expressed the Calretinin, Brn3.a , mRNA of AchR a9 and Myosin VIIa of the hair cell characteristic markers under certain culture condition,which identified the cell may represent rat progenitor hair cell.The USEC-1 cell with a large,flat,polygonal epichlial morphotype shrank in size,became elongated into " Fibroblast-like cell " appearance under condition with bFGF, which identified USEC-1 differentiation and possiblity of converte into hair cell. Conclusions: USEC-1 cell expressed the characteristic markers of the hair cell under certain culture condition, which identified the cell may represent progenitor cell of hair cell.It may be a suitable model for in-depth investigation the mechanisms of hair cell generation or regeneration.