Expression And Significance Of Oct3/4, Nanog And Sox2 In The Proliferation And Differentiation Of Rat Tracheal Stem Cells

IntroductionStem cells are specialized cell types in the embryonic and adult tissue.They have the ability of long term self-renewal and could give rise to various terminal differentiated cell types in specific condition.Adult stem cells exist in various adult tissues and organs.The application of adult stem cells has attracted much attention because of no ethical consideration.Recently,much attention has been focus on the mechanism of proliferation and differentiation in adult stem cells.Some researchers have pointed out a new definition:the correlative genome of embryonic stem cells, including Oct3/4,Nanog and Sox2 gene,and so on.This genome is expressed only in the embryonic stem(ES)cells but not in the mature somatic cells,and plays an important role to maintain the undifferentiated state and pluripotency of stem cells. Remarkably,Oct3/4,Sox2 and Nanog have also shown to participate in the reprogramming of differentiated cells back to pluripotent states.It could provide theoretical foundation of application of adult stem cells.The tracheal epithelium is slowly renewed under normal circumstances,but tracheal epithelial cells can also proliferate extensively to repair an injury.It has been demonstrated that tracheal stem cells exist in tracheal epithelium,they are indispensable for damage repair of tracheal epithelium.We constructed a tracheal regeneration model induced by fluorouracil(5-FU)ex vivo which revived the process of proliferation and differentiation of tracheal stem cells successfully and made it possible to explore the mechanism involved in the regulation of this process.5-FU is a member of the antimetabolite drug family,after treatment with 5-FU,the normally proliferating tracheal epithelial cells desquamated,leaving only a few cells in G0 phase in the basement membrane,these cells were ABCG2 positive.Thereafter,the tracheal epithelium turned flat then cuboidal and restored to pseudostratified epithelium at about 48h after removal of 5-FU.It suggested that that tracheal stem cells exist in G0 phase cells with resistance to 5-FU.However,the precise molecular mechanisms involved in the regulation of this process has been elusive.In this study,we use tracheal regeneration model induced by 5-FU,detected the dynamic changes of Oct3/4,Nanog and Sox2 in the process of proliferation and differentiation of tracheal stem cells,and it provided a theoretical evidence for advanced study.Materials and methods1.Preparation of Tracheal Epithelium Regeneration Model and 5-azaC TreatmentTracheas were excised sterilely from male and female Wistar rats(～200 g)and cultured in DMEM/F12 containing 120 mg/ml 5-FU and 10%FBS for 12 h at 37℃. Following removal of 5-FU,tracheas were cultured in DMEM/F12 containing 10% FBS.Tracheas were removed at 0,3,6,9,12,24,and 48 h after removing 5-FU and analyzed by one of several methods.Another group of tracheas were firstly cultured in DMEM/F12 containing 120 mg/ml 5-FU and 10%FBS for 12 h and cultured for another 24h in DMEM/F12 containing 10%FBS without 5-FU,then the tracheas were cultured in DMEM/F12 containing 10%FBS with or without 1μm 5-azaC for 6h.For RT-PCR,MSPCR and western blot analyses,tracheal epithelial cells were digested and stored at-70℃until use.For immunofluorescent analysis,tracheas were fixed in 4% paraformaldehyde,and prepared as paraffin-embedded tissue sections for hematoxylin-eosin(HE)stain and immunofluorescent staining.Untreated tracheas were used as controls.2.RT-PCR analysisRT-PCR was performed with the TaKaRa RNA PCR Kit(AMV)version 3.0, according to the manufacturer's protocol.β-actin was used as an endogenous control. PCR conditions were as follows:94℃for 2 min,94℃for 30 s,variable temperature for 40 s,and 72℃for 1 min,for 35 cycles.Reverse transcription reactions lacking reverse transcriptase served as negative controls.PCR products were visualized by ethidium bromide staining on 2%agarose gels on a gel scanner. 3.Western blot analysisTotal cell homogenates were prepared by lysing cells in NP40 lysis buffer.Total protein was subjected to SDS-PAGE,followed by blotting to PVDF.Membranes were blocked with nonfat dried milk in PBS,incubated with primary antibody in overnight at 4℃with shaking,then incubated with secondary antibodies for 2 hours at room temperature.Membranes were washed again and then incubated with DAB at room temperature.When bands reached the desired intensity,membranes were washed in water,followed by PBS.Finally,membranes were dried and photographed and scanned. After scanning,the densitometric analysis was performed using Image J 1.33 software.4.Indirect ImmunofluorescenceIndirect immunofluorescence staining was performed to the serial tissue sections from tracheas during the recovery from injury with Oct3/4,Nanog and Sox2 antibodies respectively.Briefly,goat anti-Oct3/4,rabbit anti-Nanog,and goat anti-Sox2(dilution 1:100) were used as primary antibodies.Fluorescein isothiocyanate(FITC)-conjugated goat antirabbit immunoglobulin G(IgG) and Rhodamine isothiocyanate (TRITC)-conjugated rabbit antigoat IgG(dilution 1:100) were used as secondary antibodies.Both antibodies were diluted with 1%bovine serum albumin-PBS.After sections were treated with the secondary antibody,they were incubated with DAPI for nuclear counterstaining.Specimens were examined with an epi-illumination fluorescence microscope BX50.For serum controls,1%bovine serum albumin-PBS was used instead of the primary antibody as a negative control.5.ImmunohistochemistryParaffin sections were dewaxed in xylene and rehydrated in graded alcohols. Antigen retrieval was performed by heating the sections for 1.5 min in 0.01 mol/L citrate buffer(pH 6.0).Non-specific staining was reduced using a blocking serum for 30 min.The sections were then incubated with goat anti-CK14 or goat anti-P63 antibodies overnight at 4℃.The next day,the sections were incubated with primary antibodies for 30min.The reaction was visualized using the DAB plus chromogen. Specimens were examined with microscope BX50.For serum controls,1%bovine serum albumin-PBS was used instead of the primary antibody as a negative control.6.MSPCR Genomic DNA was extracted from tracheal epithelial cells by proteinase K digestion and phenolechloroform method.Sodium bisulfite treatment of the extracted DNA was performed as described previously with some modifications.The MSPCR was performed with the PrimeSTARTM HS DNA Polymerase,according to the manufacturer's protocol.MSPCR conditions were as following:95℃for 3 min followed by 40 cycles of three steps at 98℃for 10 s,variable temperature for10 s and 72℃for1 min,then 72℃for 7 min.MSPCR products were visualized with ethidium bromide staining on 2%agarose gels on a gel scanner.7.Statistical analysisData from at least three independent experiments were used for statistical analysis by SPSS 11.5.All values were expressed as mean±standard deviation(SD).Statistical analyses were preformed by one way ANOVA,where p＜0.05 was considered significant.Results1.Expression levels of CK14,P63,and Oct3/4 in rat tracheal epithelium during recovery from injury induced by 5-FUIn untreated rat tracheal epithelium,almost no Oct3/4 was detected,while levels of CK14,P63 were very high relative levels in 5-FU treated tracheal epithelium. Immediately following removal of 5-FU,levels of Oct3/4 increased and reached peak levels at about 6h after removal of 5-FU,and decreased gradually,returning to normal levels about 48h after removal of 5-FU.Expression of CK14,and P63 were lowest at 0h and increased slowly until 6h,thereafter the expression of CK14,and P63 increased sharply and nearly returned to normal level about 48h after removal of 5-FU.2.Immunofluorescence of anti-Oct3/4,anti-Nanog and anti-Sox2We observed that Oct3/4 is negative in normal tracheal epithelium.Immediately following the 5-FU treatment,very few Oct3/4-positive cells were detected. Subsequently,the number of Oct3/4-positive cells increased gradually and reached the maximal level at about 6 h after removal of 5-FU.Then,the expression began to decrease and returned to the baseline levels after 48 h after removal of 5-FU.The expression patterns of Nanog and Sox2 were similar to that of Oct3/4,and they are all localized in the same cells.3.Expressions of Oct3/4,Nanog and Sox2 in tracheal epithelium injury induced by 5-FUOct3/4,Nanog and Sox2 were not detectable in the normal rat tracheal epithelium with Western blot analysis.After treatment with 5-FU,the expression levels increased and reached the maximal level at 6 h,and decreased gradually to return to very low levels by about 48 h.The results of RT-PCR were consistent with the results obtained in the Western blot analysis.4.Methylation status of the promoter region of Oct3/4,Nanog and Sox2 assessed by MSPCRWe selected the normal tracheal epithelial cells as well as the tracheal epithelial cells at 0 h,6 h,48 h after removal of 5-FU.The result showed that in normal tracheal epithelial cells and tracheal epithelial cells at 48 h after removal of 5-FU showed methylated only,but tracheal epithelial cells at 0 h,6 h after removal of 5-FU demonstrated amplification products from both methylated and unmethylated alleles.Our results suggested that the promoter regions of Oct3/4,Nanog and Sox2 underwent methylation in the normal tracheal epithelial cells. The same regions underwent demethylation in the treated tracheal epithelial cells at 0 h and 6 h after removal of 5-FU,and returned to normal methylation state in the treated tracheal epithelial cells at 48 h after removal of 5-FU.5.The influence of recovery of the tracheal epithelium treatment with 5-azaCHE staining showed that in the 5-azaC-treated group,morphological differences with the control group.In the control group,tracheal epithelium became pseudostratified epithelium at 30h after removal of 5-FU.In the 5-azaC-treated group, tracheal epithelial cells became flat.We detected the expression of Oct3/4,Nanog and Sox2 with immunofluorescence or RT-PCR mothod.It suggested that the expression levels of Oct3/4,Nanog and Sox2 in 5-azaC-treated group were higher than that in the control group. Conclusions1.The morphology and the proliferation and differentiation state of tracheal epithelium can revert to its original appearance at about 48h after removal of 5-FU;2.Oct3/4 is the marker of tracheal stem cells.The tracheal stem cells are CK14 nagative,P63 negative,can differentiated to basal cells,ciliated cells,and mucous cells;3.Oct3/4,Nanog and Sox2 may play an important role in the proliferation and differentiation of rat tracheal stem cells;4.The change of Oct3/4,Nanog,Sox2 is correlated with methylation of the promoters of these genes,in the process of proliferation and differentiation of rat tracheal stem cells.