【作者】 王恬；

【导师】 谢鼎华； Alan Gi-lun Cheng；

【作者基本信息】 中南大学 ， 耳鼻咽喉头颈外科学， 2011， 博士

【摘要】 第一部分小鼠内耳Wnt信号的基因表达研究目的：Wnt信号通道可调控干细胞的自我更新和分化。目前研究表明,Wnt信号通路可调节中枢神经系统,胃肠道系统,心脏系统,骨骼系统,乳腺干细胞,皮肤干细胞,造血干细胞,胚胎干细胞以及其他系统器官干细胞功能。为了解经典Wnt信号通道在调控内耳毛细胞再生中所起的作用,本章先检测Wnt信号通道下游靶基因Axin2在小鼠内耳的表达,为进一步研究提供基础。方法：通过利用野生型和作为经典Wnt信号通道激活表现模型的杂合子Axin2-LacZ转基因小鼠,1.以新生和成年小鼠作为研究对象,无菌条件下取出听泡,X-gal染色后行冰冻切片,以观察Axin2基因在内耳中表达的部位及表达部位的细胞种类,或者直接做冰冻切片,用各种不同细胞标志物(毛细胞,支持细胞,上皮细胞,间质细胞,增殖细胞)行免疫组化染色以鉴定细胞表型。2.分别在野生型小鼠出生1天(P1)、P7、P21、P28体内注射Edu (50mg/kg),2天后取出听泡冰冻切片或者整个椭圆囊观察Edu阳性细胞位置和数目以了解耳蜗内具自我增殖潜力的细胞种类和不同年龄的增殖能力。3.分别取出耳蜗和椭圆囊,以X-gal染色,或行RT-PCR和酶联免疫吸附实验,以观察Axin2基因表达随年龄的变化。结果：实验发现Wnt信号在新生小鼠耳蜗中基底膜以下的Tympanic border cells (TBCs)中表达,TBCs不表达成熟上皮细胞,毛细胞和支持细胞标志。在新生小鼠有活跃增殖细胞标志,间质细胞标志。Wnt信号表达与增殖能力一致随年龄迅速下降,出生3周后消失。而在前庭器官椭圆囊中,Wnt信号在间质细胞(stromal cells)中表达,具有间质细胞,和增殖细胞标志并随年龄增大,Axin2基因表达水平和细胞增殖能力下降缓慢,但在成年小鼠中仍有表达。结论：Wnt信号在小鼠耳蜗TBCs,椭圆囊间质细胞中均有表达。与在新生小鼠耳蜗中表达相比,成年小鼠椭圆囊中Wnt信号持续存在。目前研究已经证实成年小鼠椭圆囊可再生毛细胞,提示Wnt信号的表达对毛细胞再生有着重要的意义。第二部分新生小鼠耳蜗Axin2表达细胞自我增殖和分化功能及Wnt信号通道对其调控的研究目的：在哺乳动物耳蜗中,毛细胞仅在胚胎时期和新生小鼠中具有非常有限的再生能力。在耳蜗内寻找有干细胞潜能的细胞从而替代失去的毛细胞,恢复听力损失迫在眉睫。我们通过体内和体外实验,研究新生小鼠耳蜗Wnt信号表达细胞TBCs的自我增殖和分化功能,及Wnt信号通道对其的调控作用。方法：通过利用杂合子Axin2-LacZ转基因小鼠和野生型小鼠,1.无菌条件下取40-50只P0-P3Axin2lacZ/+小鼠耳蜗,通过β-galatosidase,3-carboxyumbeliferyl P-D-galactopyranoside的酶联免疫吸附反应将Axin2高表达细胞标记上CasBlue染料,再通过流式细胞仪鉴定分离Axin2高表达细胞和低表达细胞,以RT-PCR, q-PCR确定Axin2基因在分离后的高表达和低表达细胞中的表达。2.将分离出的细胞分别在体外无血清非粘附的含生长因子EGF, IGF-1, bFGF, and heparin sulfate的完全培养液中培养,5天后比较Axin2高表达细胞和低表达细胞形成的克隆球以观察自我增殖能力。将分离出的细胞在完全培养液和饲养细胞条件下体外培养10天后用毛细胞,支持细胞,增殖细胞标志行免疫组化染色以鉴定细胞的分化及分化细胞的表型。3.无菌条件下取P3野生型小鼠耳蜗于完全培养基中,分别加入纯化的Wnt信号通道的激动剂Wnt3a(200ng/ml), R-spondin1(1ug/ml)和抑制剂Fz8CRD(25ug/ml)体外培养3-5天,最后12小时于培养基中加入Edu (25ng/ml),免疫组化实验观察Wnt对耳蜗细胞自我增殖的作用。与增殖能力相一致,在Wnt信号通道激动剂和抑制剂培养3天后行酶联免疫吸附实验观察Axin2的表达水平。结果：通过流式细胞术分离的耳蜗内Axin2高表达细胞在体外培养中形成上皮细胞岛的数目是Axin2低表达细胞的3倍,并且其细胞岛内的增殖标志阳性细胞较低表达细胞明显活跃表达。同时Axin2局表达细胞可分化为新生的有毛细胞表达标志和支持细胞表达标志的细胞。而酶联免疫吸附实验显示Wnt信号通道激活剂和抑制剂可增加或减少β-galatosidase在新生小鼠耳蜗中的表达。且Wnt3a, R-spondin1可使TBCs区域的Edu阳性细胞数目为对照组的2倍,Fz8CRD则使之减少至对照组的1/2。结论：新生小鼠耳蜗中Wnt信号的表达细胞—TBCs,具有自我增殖和分化的能力,提示TBCs具有干/祖细胞的特性,并且为Wnt言号通路所调控。第三部分小鼠椭圆囊Axin2表达细胞增殖分化功能及Wnt信号通道对其调控的研究目的：目前已经证实,在成年哺乳动物中区别于耳蜗,前庭器官毛细胞仍然有再生能力,并存在有干细胞潜能的细胞。我们通过对椭圆囊中Wnt信号表达细胞的研究,以探索Wnt信号通路在新生和成年小鼠椭圆囊中对其增殖分化的调控。方法：通过利用野生型小鼠,杂合子Axin2-LacZ转基因小鼠和Axin2-cre杂交mTmG(membrane-Tdtomato-membrane-GFP)小鼠,1.无菌条件下取P3和P30Axin2LacZ/+小鼠椭圆囊,分别加入Rspondin-1(lug/ml), Fz8CRD(25ug/ml)在完全培养液中体外培养3天,通过酶联免疫吸附实验观察β-galatosidase浓度和q-PCR实验观察Axin2基因表达水平,以了解Wnt信号通路激动剂和抑制剂在内耳椭圆囊中的作用。2.上述条件体外培养椭圆囊,在最后12小时于培养基中加入Edu (25ng/ml),免疫染色实验观察Wnt信号对椭圆囊细胞自我增殖的影响。3.体内谱系追踪Axin2表达细胞。通过利用Axin2-Cre小鼠与mTmG (membrane-Tdtomato-membrane-GFP)小鼠的杂交后代,cre-loxp重组酶系统在没有激活的情况下所有细胞为红色,表达Tdtomato。在新生和成年小鼠中注射枸橼酸他莫昔芬(4mg/25g)激活Cre-loxp系统后,所有的Axin2表达细胞被激活,其中GFP基因代替Tdtomato表达,仅在Axin2阳性细胞中表达GFP的绿色信号。P3小鼠注射他莫昔芬32天后,成年小鼠50天后取出小鼠听泡,冰冻切片后免疫组织化学染色,谱系追踪Axin2表达细胞,以观察Axin2表达细胞是否具有干细胞/祖细胞特性。结果：体外完全培养液中培养P3和P30小鼠椭圆囊发现均有明显的增殖能力。在P3小鼠中增殖细胞平均分布在椭圆囊中,P30小鼠中增殖细胞仅仅在strialor区域表达。酶联免疫吸附和q-PCR实验均显示Wnt信号通道激活剂R-spondin1和抑制剂Fz8CRD可调节β-galatosidase及Axin2基因在新生和成年小鼠中的表达。经加入R-spondin1和Fz8CRD体外培养椭圆囊可以明显增加和减少其增殖能力,说明其增殖为Wnt系统调控。通过谱系追踪Axin2阳性细胞,追踪32天和50天后我们发现在P35和P80小鼠中,Axin2基因不仅在Stromal Cells区域表达,并可见在大量毛细胞,和少量支持细胞中有Axin2-GFP信号。说明在新生和成年小鼠椭圆囊中的Axin2阳性细胞具分化为毛细胞和支持细胞的能力。结论：新生和成年小鼠椭圆囊中Wnt信号的表达细胞一间质细胞,具有自我增殖和分化的能力,提示椭圆囊中的间质细胞具有干／祖细胞的特性,并且为Wnt信号通路所调控。更多还原

【Abstract】 Part1Expression of Wnt signal gene in the mouse inner earSpecific Aim:The canonical Wnt signaling pathway is critical in maintaining the stem cell populations in several organ systems, including the central nervous system (CNS), skin appendages, hematopoietic system, mammary glands, and gastrointestinal system, heart, embryonic stem cell, bone system and other system and organs. To figure it out how the canonical Wnt signal pathway promotes hair cells regeneration in the mammalian inner ear, an in-depth characterization of the expression and function of the canonical Wnt pathway in the context of mouse inner ear, we plan to characterize Axin2-a Wnt target gene expression in the mouse inner ear firstly.Method:The heterozygous Axin2-LacZ reporter mouse was used as a read-out of active canonical Wnt signaling in the postnatal cochlea.1. X-gal staining for the temporal bone from neonatal and adult mice before cryosection or the cryosection directly was done. Inner ear cryosection with different cell marker including hair cell marker, supporting cell marker, epithelial marker, mensenchymal marker were analyzed to determine where was the Axin2expressed and what was the cell types of Axin2positive cells by using immunohistochemistry.2. Edu (50mg/kg) was injected in postnatal1day wild type mouse (P1), P7, P21, P28in vivo2days before sacrificing mouse and dissecting the temporal bone. Then cryosection and whole organ utricle was analysed by using proliferative marker (Edu dye) to see if the Axin2positive cells have proliferative capacity in different ages.3. Meanwhile, the X-gal staining and RT-PCR, ElISA for the whole cochleas and utricles from different age mice were done to characterize Axin2expression in different age mouse inner ear.Result:Cryosection of cochlea from the axin2lacZ/+mouse demonstrated the most intense axin2expression to be in tympanic border cells (TBCs) beneath the basement membrane. These cells were uniformly devoid of markers of supporting cells, hair cells and sensory epithelium and were highly proliferative, expressed the mesenchymal marker. Axin2expression and proliferative capacity sharply declined with age, little axin2expressions was detected in the3weeks old mice. While Axin2was expressed in the stromal cells in the utricle both neonatal and adult mice vestibular organ. These cells highly proliferative, expressed the mesenchymal marker not supporting cells, hair cells and sensory epithelium marker. Axin2expression and proliferative capacity decreased slowly with age, and were still detected even in the adult mice.Conclusion:Axin2as a Wnt target gene is expressed in TBCs of the cochlea and stromal cells in utricle in the postnatal mouse inner ear. Compared to the neonatal mice, the adult mice utricles still have Axin2expression but not cochleas. Several reports present evidence that the adult mammalian utricles have the capacity to regenerate hair cells, which support that active canonical Wnt signaling plays an important role in hair cell regeneration. Part2Characterization of the proliferative and differentiate capacity in the cochlea Axin2positive cells and the role of canonical Wnt signaling in regulating this capacitySpecific Aim:Hair cells just have very limited regeneration in the embryonic and neonatal mouse cochlea. It is very urgent to determine if there have some potential stem/progenitor cells in the cochlea to replace lost hair cells and the future therapy to restore hearing loss. By in vivo and in vitro experiment, we plan to observe the proliferative and differentiated capacity of TBCs known as Wnt signal response cells in mouse cochlea. Meanwhile, we also focus on the role of canonical Wnt signaling in regulating this capacity.Method:The heterozygous Axin2-LacZ reporter mouse and wild type mouse were used in this study.1. Application of the fluorogenic substrate3-carboxyumbe-lliferyI β-D-galactopyranoside (CUG)(blue fluorescence) combined with Fluorescence Activated Cell Sorting (FACS) analysis, Axin2high cells and Axin2low cells were isolated from40-50Axin2-LacZ P0-P2mice cochlea. For confirming the Axin2gene expression in both Axin2high and low cells, we detected that with RT-PCR and q-PCR.2. Sorted Axin2high and low cells were cultured in serum-free media containg EGF, IGF-1, bFGF, and heparin sulfate (FM) in non-adherent conditions in vitro. Then we observed floating spheres for comparing the proliferative capacity after culturing5days. Meanwhile, to further characterize the behavior of proliferative and differentiated capacity, we cultured sorted cells10days and used different hair cell, supporting cell and proliferative marker.3. To detect how the Wnt signal pathway modulates proliferation in the TBCs layer, we cultured whole organ cochlea in FM in the presence of Wnt agonists Wnt3a (200ng/ml), R-spondin1(lug/ml) and Wnt antagonists Fz8CRD (25ug/ml)3-5days and added Edu in the last12hours. In consistent with proliferation, we used ELISA to measure the Axin2expression after culturing with Wnt agonists/antagonists. Result:Isolation of Axin2high and low cells by FACs, we found that the Axin2-high cells consistently formed approximately3times more primary spheres than Axin2low cells and labeling for other proliferation markers (EdU) further demonstrated that a subset of cells within colonies derived from Axin2-high cells was highly proliferative. We also observed that both hair cell and supporting cell marker labeled a subset of Axin2high cell island after culturing10days. After culturing the whole organ cochlea with Wnt agonists/antagonists, quantitative analysis of Edu marked cells indicated that Wnt activation induced a doubling of the baseline proliferation and Wnt antagonists significantly decreased the proliferation coincides with Axin2expression.Conclusion:TBCs-Wnt responsive cells have the capacity of proliferati-on and differentiation, act as stem/progenitor stem cells in the postnatal mouse cochlea. Wnt agonists/antagonists modulate the proliferative capacity of these endogenous stem/progenitor cells. Part3Characterization of the proliferative and differentiate capacity in the utricle Axin2positive cells and the role of canonical Wnt signaling in regulating this capacitySpecific Aim:Compared to the adult mammalian cochlea, a lot of evidence indicated that hair cells can be regenerated in adult vestibular organ and there has a subset of potential stem/progenitor cells in utricular macula. Observation of Wnt responsive cells in utricle, we plan to figure out the role of Wnt signal pathway in regulating the proliferation and differentiate-on.Method:We used wild type, heterozygous Axin2-LacZ reporter mouse and Axin2-cre X mTmG (membrane-Tdtomato-membrane-GFP) mouse.1. P3and P30whole organ utricle were cultured in FM in the presence of Wnt agonists R-spondinl (1ug/ml) and Wnt antagonists Fz8CRD (25ug/ml) and then we used ELISA and q-PCR to quantify the Axin2expression after culturing with Wnt agonists/antagonists3days.2. P3utricles were cultured with Wnt agonists/antagonists3days and added Edu in the last12hours to count the Edu maker cells to see whether Wnt signal could increase or decrease the proliferation.3. Tracing lineage the Axin2positive cells in vivo by using Axin2-cre x mTmG mouse, all the cells were red and expressed Tdtomato. After injecting tomoxifen (4mg/25g mice) in this neonatal and adult mouse, all the Axin2expression cells were activated with cre recombinase and Tdtomato gene is disrupted following a reportor GFP gene transcribed instead in Axin2positive cells. Then analysis of cryosection after tracing the Axin2-GFP cells32days in P3mice,50days in P30mice, we observed if the Axin2positive cells in utricle have the behavior of stem/progenitor cell.Result:After culturing the neonatal and adult whole organ utricle in vitro, we found that both ages had the strong proliferative capacity and Edu marked cells were in the whole utricle in P3mice and just in the strialor region in P30utricle. Meanwhile, Wnt agonists promoted proliferation and Wnt antagonists significantly decreased the proliferation coincides with Axin2expression in ELISA and q-PCR. Surprisingly, the tracing lineage experiment indicated that all the stromal cells in P3and P30mice expressed Axin2-GFP signal and we also detected increased GFP signal in hair cells, very few in supporting cells with different tracing age. Simultaneously, the GFP signal could be observed in the gap of basement membrane.Conclusion:Stromal cells-Wnt responsive cells have the capacity of proliferation and have the behavior of stem/progenitor stem cells in the neonatal and adult mice. Wnt agonists/antagonists modulate the proliferative capacity of these endogenous stem/progenitor cells.更多还原