【作者】 宋楠；

【导师】 贾心善；

【作者基本信息】 中国医科大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 大鼠气管干细胞增殖分化过程中Oct3/4、Nanog和Sox2的表达及意义前言干细胞是存在于胚胎和成体中的一类特殊细胞,它能长期的自我更新,在特定的条件下具有分化形成多种终末细胞的能力。成体干细胞为存在特定组织中的干细胞,由于无伦理学方面的困扰,成体干细胞的应用越来越受到人们的关注。随之成体干细胞增殖分化调控的机制也成为研究的重点,有学者提出胚胎干细胞相关基因的概念,包括:Oct3/4、Nanog和Sox2等。它们在胚胎干细胞中有表达,而在成熟组织细胞中无表达。这些胚胎干细胞相关基因可以维持胚胎干细胞未分化状态,保持干细胞的多向分化潜能。另外,Oct3/4、Nanog和Sox2的激活能使体细胞发生再编程,表现出未分化细胞特征,使其具有干细胞多潜能分化的能力,为成体干细胞应用提供理论基础。气管上皮在正常情况下更新缓慢,但在损伤修复时能再生以维持其完整性,这说明气管上皮中存在干细胞,只有在损伤的过程中才能激活其潜能。我们实验室已经建立了5-FU引发离体气管损伤修复模型,这一模型成功再现了气管干细胞的增殖分化过程,使气管干细胞增殖分化机制的初步研究成为可能。因5-FU属抗嘧啶类代谢药,经5-FU作用后,气管上皮增殖期细胞脱落,基底膜上残留的细胞为G0期细胞,并出现了干细胞标记物ABCG2的表达,撤除5-FU后气管上皮经扁平细胞、立方细胞,直至48小时恢复到假复层纤毛柱状上皮,证明气管干细胞存在于对5-FU抗性G0期细胞中。但对这一过程的调控机制还所知甚少。本研究利用5-FU引发离体气管损伤修复模型,观察在气管干细胞增殖分化过程中Oct3/4、Nanog和Sox2的动态变化,并探讨其维持气管干细胞未分化状态的分子机制。材料和方法一、离体大鼠气管损伤修复模型的制备及5-azaC处理取约200克左右的Wistar大鼠,雌雄不限,水合氯醛麻醉,无菌条件下取出气管,PBS冲洗,置于DMEM/F12培养液中（含10%胎牛血清）。取正常气管,剪取一气管环固定,留做石蜡切片,灌入蛋白酶ⅩⅣ（0.5mg/ml）,结扎另一端,4℃消化过夜,收集消化液,加FBS至终浓度为2.5%终止酶反应收集正常气管上皮细胞于2mlEppendorf管中,-70℃冻存,留做mRNA提取。其余气管组织分为5-FU作用组和5-azaC处理组。5-FU作用组为5-FU作用12小时后,置于DMEM/F12培养液中（含10%胎牛血清）,分别于换液后0、3、6、12、24、48小时取出气管组织分别固定,留做石蜡切片;5-azaC处理组在5-FU作用后,当换液后24小时,换为含有1μM 5-azaC DMEM/F12（含10%胎牛血清）继续培养,方法同上,6小时后取出气管组织分别固定,留做石蜡切片;消化,收集细胞于2mlEppendorf管中,-70℃保存,以备DNA,RNA和蛋白提取。二、RT-PCR检测按Trizol^TM试剂使用说明提取气管上皮细胞总RNA,检测它的浓度,纯度和完整性。按逆转录试剂盒使用说明操作,选用Oligo-dT做引物合成第一链cDNA,逆转录条件为:30℃10min,40℃40min,99℃5min,5℃5min;PCR反应条件为:94℃变性2min后,94℃30s,退火,72℃1min,进行35个循环,最后于72℃延伸7min。扩增产物使用2%琼脂糖凝胶电泳,溴化乙啶染色分析表达结果,测灰度值,统计。相同条件下用β-actin作为内对照。三、蛋白印迹检测按蛋白抽提试剂盒说明抽提气管上皮细胞总蛋白,SDS-PAGE电泳,50V恒压湿转120mins,BSA室温封闭2hrs,一抗4℃孵育过夜,二抗37℃孵育2hrs,DAB显色。转膜后各步之间均用洗膜液洗膜3次,每次5mins。相同条件下用β-actin作为内对照。四、间接免疫荧光检测气管上皮Oct3/4,Nanog和Sox2的表达石蜡切片脱蜡至水,抗原修复,非免疫动物血清封闭,一抗分别为山羊抗Oct3/4,兔抗Nanog和山羊抗Sox2;二抗分别TRITC标记兔抗羊IgG和FITC标记羊抗兔IgG。DAPI复染细胞核。50%缓冲甘油封片,荧光显微镜OlympasBX51下观察并照相。阴性对照实验:用等量的0.01mol/L PBS代替一抗,其余步骤同前。五、免疫组化检测气管上皮CK14和P63的表达石蜡切片脱蜡至水,高温高压抗原修复,非免疫动物血清封闭,一抗分别为山羊抗CK14和山羊抗P63,4℃孵育过夜;二抗37℃孵育30min。DAB显色。显微镜下观察并照相。阴性对照实验:用等量的0.01mol/L PBS代替一抗,其余步骤同前。六、甲基化特异性PCR检测以酚-氯仿抽提法提取气管上皮细胞DNA,参见试剂盒说明进行甲基化修饰,然后进行甲基化特异性PCR（MSPCR）反应,MSPCR反应条件为:95℃变性3min后,98℃10s,退火,72℃1min,进行40个循环,最后于72℃延伸7min。扩增产物使用2%琼脂糖凝胶电泳,溴化乙啶染色分析表达结果,测灰度值,统计。七、统计分析所有的RT-PCR实验和蛋白印迹实验均独立重复三次,所得数据的值用均数（means）±标准偏差（SD）来表示。用SPSS 11.5软件对所得数据进行单因素方差分析,当P＜0.05认为有统计学意义。结果1、5-FU诱导损伤修复过程中CK14、P63和Oct3/4的表达变化正常气管上皮几乎没有Oct3/4的表达,去除5-FU作用后0h,Oct3/4少量表达,随后表达量逐渐增高,至6h达到峰值,随后逐渐降低,至48小时几乎恢复至正常水平;正常气管上皮CK14和P63高表达,去除5-FU作用后0h,CK14和P63几乎不表达,之后表达逐渐增加,至48h几乎恢复正常水平。2、间接免疫荧光检测5-FU作用后大鼠气管上皮Oct3/4、Nanog和Sox2的表达正常气管上皮几乎没有Oct3/4、Nanog和Sox2的表达,5-FU打击后0h,出现Oct3/4、Nanog和Sox2阳性细胞,随后逐渐增多,至6h达到高峰,随后逐渐降低,至48h几乎恢复至正常水平。Oct3/4、Nanog和Sox2的定位相同,均位于相同的细胞中。3、5-FU诱导损伤修复过程中Oct3/4、Nanog和Sox2的表达变化蛋白印迹检测大鼠气管上皮损伤修复过程中Oct3/4、Nanog和Sox2的蛋白水平发现,正常气管上皮没有Oct3/4、Nanog和Sox2表达。5-FU打击后0h,出现Oct3/4、Nanog和Sox2的表达,并随着气管上皮的修复逐渐增加,在6h达到高峰,随后逐渐降低,至48h降至正常水平。RT-PCR的结果与蛋白印迹的结果相同。4、MSPCR检测5-FU作用后大鼠气管上皮Oct3/4、Nanog和Sox2的启动子甲基化状态选取几个有代表性的时间点,分别选取正常气管上皮细胞、5-FU作用后0h的细胞、5-FU作用后恢复6h的细胞和恢复48h的气管上皮细胞进行检测。结果显示在正常气管上皮细胞中仅存在三种基因的甲基化条带,而5-FU作用后0h和6h出现了这三种基因的甲基化和未甲基化条带,48h也只有三种基因的甲基化条带。5、5-azaC处理对大鼠气管上皮损伤修复的影响HE染色显示,5-FU诱导损伤后,气管上皮细胞恢复24h出现分化时用5-azaC作用,与单纯5-FU作用对照组相比,气管上皮的形态出现差异。单纯5-FU作用对照组,气管上皮于30h后出现双层,而5-azaC作用组,气管上皮呈现扁平细胞。免疫荧光结果检测Oct3/4、Nanog和Sox2的表达,5-azaC作用组Oct3/4、Nanog和Sox2阳性的细胞数明显高于单纯5-FU作用组。RT-PCR与荧光结果相同。结论1、5-FU作用后,经48小时气管上皮可基本恢复正常形态。2、Oct3/4可作为气管干细胞标志物,此细胞CK14阴性、P63阴性,可向基底细胞、纤毛细胞、粘液细胞分化。3、Oct3/4、Nanog和Sox2在气管干细胞增殖分化的过程中发挥了重要的作用。4、在气管干细胞的增殖分化过程中,Oct3/4、Nanog和Sox2表达的动态变化与其启动子甲基化调控有关。更多还原

【Abstract】 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.更多还原