Expression Patterns And Implication Of Cav1.3 And LaminB1 In Developmantal Stages Of The Rat Cochleae

Part one: Expression of Cav1.3 and LaminB1 in marginal cells of the neonatal SD rat stria vascularis in primary culture Objective: To establish stable methods for primary culture of marginal cells in neonatal rat cochleae,and to investigate whether Cav1.3 calcium channel and LaminB1 were expressed in the marginal cells of the neonatal rats in primary culture.Methods: The lateral wall was dissected from postnatal zero or three-day-old SD rats and cut into tiny pieces.After enzymatic digestion,the cell suspension was plated in a six-well plate,cultured with animal epithelial medium supplemented with 2% fetal bovine serum.The culture medium was changed every other day.Examine the marginal cells in primary culture with antibody of CK-18,Cav1.3 calcium channels,and LaminB1 by immunofluorescence staining.Results: Under the inverted light microscope,most of the cell masses were well adhered to the wall after being plated for 24 hours.At 4-5 days,the polygonal marginal cells and spindle fibroblasts grew together.The MCs were varied in size,dark in color,well-defined,closely connected,and formed a typical “cobblestone-like” appearance.At 7-9 days,"dome" structures can be observed.After 14 days,the volume of the MCs became larger,the cytoplasmic particulate matter was increased,multiple nuclei or large nuclei appeared,and the cytoskeleton became obvious.CK-18,Cav1.3 calcium channel protein,and LaminB1 were positive in MCs.Conclusion: The stable method for primary culture of marginal cells in neonatal rat cochleae was successfully established.Meanwhile,CK-18,Cav1.3 calcium channel protein,and LaminB1 were demonstrated expressed in MCs of the neonatal rats for the first time,preliminarily revealing that Cav1.3 and LaminB1 play a role during the development of the cochleae,and providing the theoretical foundation for subsequent experiments.Part two: Expression of Cav1.3 calcium channels in cochleae of the developing rats Objective: To investigate the temporal expression profile of Cav1.3 calcium channels in the lateral wall and basilar membrane of the developing rat cochlea and explore the role of Cav1.3 in auditory system.Methods: Sprague-Dawley rats from postnatal day(p)0 to 21 were used and divided randomly into 4 groups(p0,p7,p14,p21)according to development stage.Immunofluorescence,quantitative real-time PCR and reverse transcription-polymerase chain reaction were applied to detect the expression of Cav1.3 calcium channels in the cochlear lateral wall and basilar membrane at protein and mRNA levels.Results: Immunofluorescence and reverse transcription-polymerase chain reaction determined that Cav1.3 calcium channels was present in hair cells,stria vascular and stria ligment.Quantitative real-time PCR revealed that the expression of Cav1.3 calcium channels was up-regulated during development.Conclusion: The expression of Cav1.3 calcium channels was increased in the cochleae of rats with development.The up-regulation of Cav1.3 calcium channels may be closely associated with the development of the cochlea,onset of the hearing,the homeostasis of the calcium,and the regulation of the endocochlear potential(EP).Our result provided a theoretical basis for further investigation of the role of Cav1.3 in the auditory system.Part three: Differential expression of Lamin B1 in the developing rat cochleaObjective: Lamin B1 is a potent regulator of cellular proliferation and differentiation and also known to be essential for neuronal migration and brain development.We aimed to explore the cochlear morphology changes and temporal expression profile of Lamin B1 in postnatal rat,and whether the Lamin B1 is associated with cochlear development and auditory function.Methods: Sprague-Dawley rats ranging from birth to Postnatal(p)21 were used.The tissues of stria vascularis(STV,including spiral ligament),basilar membrane(BM,including the organ of Corti),and spiral ganglion cell(SGC)were dissected respectively.The staining of Hematoxylin and eosin(HE)was employed to detect the cochlear morphology changes.Immunofluorescence,quantitative real-time PCR and western blot were applied to detect the expression of Lamin B1 in individual cochlear tissues at both m RNA and protein levels along with postnatal time.Results: HE staining showed the morphological changes of rats' cochlea development.Immunofluorescence revealed that Lamin B1 was localized in the outer hair cells(OHCs),inner hair cells(IHCs),kolliker's organ,Reissner's membrane(RM),spiral ganglion cell(SGC),stria vascularis and spiral ligament.The intensity of staining surrounding the scala media decreased during cochlear development.The expression of Lamin B1 m RNA and protein in STV,SGC,and BM was at a maximum level at p0 but gradually declined to a minimum level at p21.Conclusion: Cochlear development was involved in many typical morphology changes.Lamin B1 was also altered in cochlear tissues along with postnatal time.This implicated possible functions for Lamin B1 in the development and formation of auditory function.What's more,the distribution of Lamin B1 shared similarity with Cav1.3 in the cochlea,and expression of two proteins were all altered during development,suggesting that Lamin B1 may also played an important role in the cochlea like Cav1.3.Part four: Selective expression Patterns of Lamin B1 in Rat CochleaeObjective: Lamin B1,a major component of the nuclear lamina,is a potent regulator of cellular proliferation and differentiation.We aimed to explore the expression patterns and implication of Lamin B1 in the individual rat cochlear tissues.Methods: Sprague-Dawley rats at different development stages were used.Immunofluorescence,Western blot,and quantitative real time-PCR were applied to detect the distribution and expression of Lamin B1 at m RNA and protein levels in the individual tissues(BM,SGC,and STV)of rat cochlea.Results: Immunofluorescence staining indicated that Lamin B1 was mainly localized in the auditory hair cells(HCs),spiral ganglion cells(SGC),stria vascularis(STV,including spiral ligament),Reissner's membrane(RM),and limbus laminae spiralis(LLS).Western blot analysis illustrated that the distribution of Lamin B1 in rat cochlea was characterized by tissue specificity at p21.The Lamin B1 protein was expressed more in SGC and basilar membrane(BM)than in STV.Meanwhile,the expression of Lamin B1 m RNA at p21 displayed difference in cochlear tissues.The expression of Lamin B1 at m RNA level level showed no difference between cochlear tissues during p0-p14,while the expression of Lamin B1 protein in SGC and BM were more than that in STV which is lack of neurons at p14.Conclusion: These observations preliminarily revealed that the expression of Lamin B1 in cochlea was characterized by tissue specificity when the cochlea was mature,implicating a role of Lamin B1 in the development of auditory neurons and hearing functions.Moreover,the selective expression pattern of Lamin B1 in the cochlea is the same as that of Cav1.3,further demonstrating the close connection between them,and also implicating the important role of Lamin B1 in the cochlea...