【作者】 俞亦龄；

【导师】 周国民；

【作者基本信息】 复旦大学 ， 人体解剖与组织胚胎学， 2010， 博士

【摘要】 耳蜗毛细胞可将声波的震动转化为生物电信号,是听觉系统的关键组成部分。但毛细胞极易因噪音、药物和年龄等因素受损,而哺乳动物的耳蜗毛细胞没有天然的再生能力,一旦受到损伤,即可造成永久性的听力丧失。但是在低等的脊椎动物中,毛细胞的损伤可诱导周围的支持细胞分裂增殖和向毛细胞转分化,最终替代受损毛细胞,维持和恢复感觉上皮的功能,提示支持细胞可能是再生毛细胞的潜在干细胞。最近研究结果显示体外分离培养的小鼠耳蜗支持细胞也能够增殖并向毛细胞转分化,并且该过程中出现了细胞周期抑制因子的下调,表明在哺乳动物耳蜗内,支持细胞也可能具有毛细胞再生潜力,且细胞周期抑制因子的下调很可能对其重新进入细胞周期的启动起到重要推动作用。视网膜母细胞瘤蛋白(Rb)是细胞周期调控中起关键作用的抑制因子,可以抑制完成细胞周期所需基因的表达。在感觉前体细胞中定向敲除Rb基因,可使前体细胞长时间处于增殖状态,并分化出大量毛细胞和支持细胞。但Rb基因在小鼠耳蜗毛细胞内定向敲除后,虽然毛细胞也能进入细胞周期,但并不能完成分裂并引起细胞的增殖,反而启动了细胞凋亡。目前,Rb在耳蜗支持细胞的表达和作用并不明确。本课题通过在出生后的小鼠耳蜗支持细胞内定向敲除Rb基因,对哺乳动物耳蜗支持细胞在毛细胞再生中的潜能进行研究,以期为耳蜗毛细胞再生的机制研究和临床治疗提供更有效的方案。(1)建立检测Cre重组酶活性的指示模型小鼠。①将Prox1-CreERT2小鼠与Rosa-LacZ指示小鼠相杂交,获取Prox1-CreERT2/ROSA-LacZ小鼠,从而在具有Cre重组酶活性的细胞中表达β-半乳糖酐酶(β-gal)。②通过X-gal染色,检测耳蜗中的β-gal活性,从而显示Prox1-CreERT2小鼠耳蜗中具有Cre重组酶活性的细胞。(2)诱导Cre重组酶活性。分别在P0和P1,按每日1次,每次3mg/40g体重剂量,对新生的Prox1-CreERT2/ROSA-LacZ小鼠进行腹腔注射tamoxifen。(3)检测经tamoxifen诱导后的Prox1-CreERT2小鼠耳蜗内,Cre重组酶活性的分布情况。①对新生的Prox1-CreERT2/ROSA-LacZ小鼠进行腹腔注射tamoxifen,时间和剂量同上,以诱导Cre重组活性。②对接受过tamoxifen注射的Prox1-CreERT2/ROSA-LacZ小鼠,在P6采集其耳蜗进行冰冻切片和耳蜗铺片,利用X-gal染色并结合毛细胞特异性标记抗体Myo7a的双重染色,以显示具有Cre重组酶活性的细胞及其相对于耳蜗毛细胞的位置。(4)Cre重组酶活性在不同支持细胞亚型中的分布情况。①将Prox1-CreERT2小鼠与Rosa-EYFP指示小鼠相杂交,获取Prox1-CreERT2/ROSA-EYFP小鼠,从而在具有Cre重组酶活性的细胞中表达增强的黄色荧光蛋白(EYFP).②对Prox1-CreERT2/ROSA-EYFP小鼠进行tamoxifen腹腔注射,时间和剂量同上,以诱导Cre重组活性。③对接受过tamoxifen注射的Prox1-CreERT2/ROSA-EYFP小鼠,在P4采集耳蜗,制作铺片,利用免疫荧光染色和共聚焦显微镜的三维重建技术,观察耳蜗内表达EYFP的细胞,并分析其与毛细胞和支持细胞的关系,使EYFP阳性细胞的细胞类型得到进一步确定。(1)建立耳蜗支持细胞中定向敲除Rb基因的小鼠模型。①将Prox1-CreERT2小鼠与RbloxP/loxP小鼠杂交,获取Prox1-CreERT2/RbloxP/loxP小鼠。②分别于P0和P1腹腔注射tamoxifen,剂量同前,以诱导Prox1-CreERT2/RbloxP/loxP小鼠体内发生Cre重组酶介导的Rb基因敲除。(2)检测耳蜗支持细胞失去Rb作用后是否重新进入了细胞周期。①接受过tamoxifen注射的Prox1-CreERT2/RbloxP/loxP小鼠(即Prox1-Rb-/-小鼠),在P4或P6对其进行BrdU或EdU的腹腔注射,以标记此时重新进入细胞周期的耳蜗支持细胞。②通过免疫荧光染色(适用于BrdU)或共价叠氮反应(适用于EdU),并结合毛细胞或支持细胞特异性标记蛋白的双重荧光染色,检测重新进入细胞周期的耳蜗支持细胞。③在Prox1-Rb-/-耳蜗顶、中、底回的相应位置,分别对BrdU阳性支持细胞进行计数统计。④将Prox1-Rb-/-耳蜗内增殖性的支持细胞分布,与Prox1-CreERT2小鼠耳蜗内Cre重组酶的分布情况进行比较与分析。(3)检测耳蜗支持细胞失去Rb作用后是否可以完成细胞周期并增殖。①检测Prox1-Rb-/-耳蜗内BrdU阳性细胞的染色质凝集现象,以及其支持细胞pH3的分子表达情况,以研究通过限制点和S期的Prox1-Rb-/-耳蜗支持细胞能否进入M期。②在Prox1-Rb-/-耳蜗内进行BrdU和FISH(可检测DNA处于已被复制状态还是未被复制状态)的双重染色,以研究重新进入细胞周期的支持细胞是否可以完成有丝分裂。③对Prox1-Rb-/-小鼠进行BrdU和EdU的双重标记,以研究Rb基因缺陷的耳蜗支持细胞是否具有多次进入细胞周期的潜能。④通过外指细胞和柱细胞的计数统计,以研究失去Rb作用的耳蜗支持细胞是否真正产生了增殖反应。(1)观察Prox1-Rb-/-小鼠耳蜗中的细胞凋亡情况。①对不同日龄的Prox1-Rb-/-小鼠耳蜗进行TUNEL染色,以检测最早出现凋亡的时间和支持细胞亚型。②在P9对Prox1-Rb-/-小鼠耳蜗铺片中的TUNEL阳性细胞进行统计,并与对照组比较以了解Rb基因缺陷的支持细胞凋亡情况。③对不同日龄Prox1-Rb-/-小鼠耳蜗铺片进行Myo7a或Myo6免疫荧光染色,以检测毛细胞数量是否受到影响。(2)研究Rb基因缺陷的耳蜗支持细胞在增殖后的细胞分化命运。①分别在P4-P5或P8-P14每日1次对Prox1-Rb-/-小鼠进行EdU的腹腔注射,通过EdU染色结合毛细胞和/或支持细胞特异性标记蛋白Myo6、Prox1和Sox2的多重荧光染色,以检测Rb基因缺陷的支持细胞在增殖过程中的分化命运。②研究在耳蜗内出现继发性毛细胞丢失的情况下,其分化是否会受到影响,探讨如何对哺乳动物耳蜗支持细胞进行适当的调控,可以使其对毛细胞再生发挥最大的潜能。分别在P0和P1,按3mg/40g体重剂量腹腔注射tamoxifen, Prox1-CreERT2小鼠耳蜗中Cre重组酶的活性局限于外指细胞和柱细胞内。而且,具有重组酶活性的细胞数量从耳蜗底回至顶回呈逐渐上升的趋势。在Prox1-CreERT2/ROSA-EYFP小鼠耳蜗顶回的EYPF阳性细胞中,外指细胞和柱细胞分别为72.77%±1.12%和27.23%±1.12%,即具有Cre重组酶活性的支持细胞大多为外指细胞。在Prox1-Rb-/-小鼠耳蜗内,发现外指细胞和柱细胞部位有BrdU阳性细胞出现。在毛细胞分布的位置有时也会发现BrdU阳性细胞,但这些细胞都表达支持细胞抗原Prox1,而不表达毛细胞抗原Myo7a。从耳蜗底回至顶回,BrdU阳性支持细胞的数量呈渐增的趋势。在P4时,BrdU阳性的柱细胞数量明显多于外指细胞。至P6时,与对照组相比,Rb基因缺失后支持细胞数量明显增多。Rb基因缺失的外指细胞和柱细胞都可进入细胞有丝分裂期(M期),表达M期特有的蛋白和具有M期特征性的凝集染色质。在P6的BrdU阳性柱细胞,发现其仅含2个FISH信号点,显示G1期或早期S期的特征,表明其重新进入细胞周期后已完成了细胞分裂。在Prox1-Rb-/-耳蜗中还发现有BrdU/EdU双重染色阳性的柱细胞存在,提示其具有进行多次细胞分裂的潜能。在P5之前的Prox1-Rb-/-耳蜗切片上,并未见到Corti器内出现TUNEL阳性的支持细胞,而在P7的Prox1-Rb-/-耳蜗铺片上,我们首先观察到了Corti器中的凋亡细胞。从TUNEL染色阳性细胞位置推测,这些最早出现死亡信号的细胞很可能是那些细胞核迁移至毛细胞层的外指细胞。之后Prox1-Rb-/-小鼠耳蜗继发毛细胞死亡,其毛细胞数量在P9并未发生显著改变,但在P12的耳蜗顶回可见零星的毛细胞丢失,在出生后7周的耳蜗铺片上可见更严重的毛细胞丢失。并且在P12和P21耳蜗顶回的毛细胞丢失比底回更严重。5周龄的Prox1-Rb-/-小鼠听性脑干反应(ABR)测试显示听阈上升,听觉能力明显低于对照组。在P4时,Peox1-Rb-/-耳蜗所有EdU阳性细胞均可被Prox1和Sox2所标记,证实了增殖中的Rb基因缺陷的支持细胞在重新进入细胞周期后仍保持支持细胞的分化命运。在有毛细胞丢失的位置,增殖约1周的外指细胞和柱细胞也仍保持支持细胞命运,所有EdU阳性的耳蜗支持细胞在P14均可表现Sox2的双重标记,并且未发现任何EdU和Myo6双重阳性的细胞。更多还原

【Abstract】 Cochlear hair cells (HCs) are mechanosensory receptors that transduce sound into electrical signals. But HCs are very sensitive and can be easily damaged by age, adverse reaction of certain medications and noise. Once HCs are damaged, it will cause permanent hearing loss, because mammalian HCs cannot regenerate. HC damage in non-mammalian vertebrates induces surrounding supporting cells (SCs) to divide, transdifferentiate and replace lost HCs, suggesting that SCs are the origin of HC regeneration. Isolated SCs from the postnatal mouse cochlea can proliferate and transdifferentiate into HCs in vitro, and the down-regulation of the cell cycle inhibitor, p27Kip1, was observed in the process. Thus, postmitotic mammalian cochlear SCs may be good candidates for HC regeneration and the down-regulation of cell cycle inhibitors may play important roles in initiating cell cycle reentry. Rb is a key cell cycle inhibitor which suppresses genes required for entering and progressing through the cell cycle. Deletion of Rb in cochlear HC and SC progenitors produces supernumerary progenitors, which subsequently acquire features of differentiated HCs and SCs and later undergo cell death. The acute elimination of Rb in postmitotic HCs causes cell cycle reentry and mitosis; however, HCs die at different stages of the cell cycle before division is complete. The effect of inactivating Rb in postmitotic SCs remains unknown. Here, we induce the acute elimination of Rb in postnatal SCs and examine their capability to proliferate and transdifferentiate in vivo, in order to understand the potential use of SCs in the regeneration of mammalian HCs.(1) Creating mouse line that can visualize cells with Cre recombinase activity.①Cross Prox1-CreERT2 mice with Rosa-LacZ reporter mice, to create Prox1-CreERT2/ROSA-LacZ mice that expressβ-galactosidase (β-gal) in the cells with Cre recominase activity.②Use X-gal staining to detect cells with Cre activity in the Prox1-CreERT2 cochlea. (2) Inducing Cre activity in Prox1-CreERT2 mice. Administrate intraperitoneal (IP) injection of tamoxifen once daily at P0 and P1 to Prox1-CreERT2/ROSA-LacZ mice at 3mg/40g body weight, to induce Cre activity.(3) Detecting induced Cre activity in the Prox1-CreERT2 cochlea.①Administrate IP injection of tamoxifen to neonatal Prox1-CreERT2/ROSA-LacZ mice, following the dosage above.②Collect cochlea at P6 from Prox1-CreERT2/ROSA-LacZ mice that has received tamoxifen injection at P0 and P1, and use X-gal staining in combination with co-staining of a HC marker, Myo7a, to observe the localization of cells with Cre activity in the Prox1-CreERT2 cochlea.(4) Study in detail the subtype of Cre positive SCs.①Cross Prox1-CreERT2 mice with Rosa-EYFP reporter mice, to create Prox1-CreERT2/ROSA-EYFP mice that express enhanced yellow fluorescent protein (EYFP) in the cells with Cre recominase activity.②Administrate IP injection of tamoxifen to neonatal Prox1-CreERT2/ROSA-EYFP mice, following the dosage above.③Collect cochlea at P4 from Prox1-CreERT2/ROSA-EYFP mice that has received tamoxifen injection at P0 and P1, and then observe the relative localization of EYFP-positive cells to the HCs and SCs by immunofluerecent staining under the confocal microscopy.(1) Creating mouse line that can ablate Rb gene specifically in the neonatal cochlear SCs.①Cross Prox1-CreERT2 mice with RbloxP/loxP mice, to create Prox1-CreERT2/RbloxP/loxP mice.②Administrate tamoxifen injection to Prox1-CreERT2/RbloxP/loxP mice following the description above, to induce Rb gene knock-out specifically in the neonatal SCs mediated by Cre recombinase activity.(2) Investigating cell cycle reentry of cochlear SCs after Rb ablation.①Administrate IP injections of BrdU or EdU at P4 or P6 to the Prox1-Rb-/- mice that has received P0-P1 tamoxifen injection.②Co-stain BrdU or EdU with HC or SC markers, to investigate whether postmititic SCs reentered the cell cycle.③Count the number of BrdU-positive cells in the basil, middle and apical turn of Prox1-Rb-/- cochleae.④Compare the distribution of proliferating cells with that of Cre activity in the Prox1-CreERT2 mice.(3) Investigating the completion of cell division and proliferation of Prox1-Rb-/- SCs.①Immunostaining of pH3, a marker of M phase nuclei, to determine whether Prox1-Rb-/ SCs can enter mitosis (M) phase.②Stain BrdU in combination with FISH, which allows discrimination between cells with unduplicated or duplicated chromosomes, to test whether Prox1-Rb-/- SCs could complete the cell cycle.③Double label BrdU and EdU in Prox1-Rb-/- cochleae, to confirm that Prox1-Rb-/- SCs progressed through more than one round of S phase.④Quantify SC number, to confirm that Prox1-Rb-/- SCs proliferated.(1) Cell death in Prox-Rb-/- cochleae.①Use TUNEL staining to observe cell death in Prox1-Rb-/- cochlea at different ages.②Analyze the TUNEL-positive SC number in the P9 Prax1-Rb-/- cochleaer wholemount.③Stain Myo7a or Myo6 on Prox1-Rb-/- cochlear wholemounts at different ages, to detect the change of HC number.(2) The cell fate decision of Prox1-Rb-/- SCs during proliferation.①Administrate IP injection of EdU once daily into Prox1-Rb-/- mice at P4-P5 or P8-P14, and then co-stained with HC or SC markers, like Myo6, Prox1 and Sox2, to determine the cell fate of Prox1-Rb-/- SCs during proliferation.②Investigate the influence of HC death to the cell fate decision of the proliferating SCs, and discuss a best way to manipulate SCs to regenerate HCs.(1) Inducible Cre activity in DCs and PCs of postnatal Prox1-CreERT2 cochlea. After injection of tamoxifen at P0 and P1, Prox1-CreERT2;Rosa-LacZ cochleae showed restricted Cre activity in DCs and PCs, and the number of cells with Cre activity increased from basal to apical turn. In the apical turn of Prox1-CreERT2;Rosa-EYFP cochlea,72.77%±1.12% of YFP positive SCs are DCs while 27.23%±1.12% are PCs, i.e. the majority of Cre-positive SCs are DCs.(2) Inactivation of Rb resulted in cell cycle reentry and proliferation of cochlear SCs. BrdU-positive DCs and PCs were frequently detected in the Prox1-Rb-/- cochlea, but never in the cochlea of control mice. Although BrdU-positive nuclei were also observed in the HC layer, no Myo7a-positive cells were labeled by BrdU and all EdU-positive SCs were labeled by Proxl. The number of BrdU-positive cells displayed a gradient from basal to apical turn, consistent with the gradient of Cre-positive cells in Prox1-CreERT2 cochlea. More PCs than DCs were labeled with BrdU in the apical turn at P4. Prox1-Rb-/-cochleae showed a significant increase in SC number at P6. PCs and DCs with strong pH3 labeling, or with BrdU-positive condensed chromosome, were abserved in Prox1-Rb-/- cochlear whole mounts at various ages. BrdU-positive PCs showed G1 or early S phase features (i.e., two FISH signals) at P6. BrdU/EdU double positive PCs were also observed in Prox1-Rb-/- cochleae.(3) Cell death of Prox1-Rb-/- SCs. In Prox1-Rb-/- cochleae, no TUNEL-positive SCs were observed by cross section before P5. At P7, we first detected dying cells in the organ of Corti using TUNEL staining. These dying cells are likely DCs whose nuclei have migrated into the HC layer. HC numbers were not significantly changed at P9. At P12 scattered HC loss was clearly evident in the Rb-/- cochlea and was more severe at 7 weeks of age. Moreover, HC loss was more prominent in the apical turn than in middle and basal turns at P12 and P21. Furthermore, ABR thresholds of Rb-/- mice at 5 weeks of age were significantly increased from 6-32 kHz compared to controls.(4) Proliferating Prox1-Rb-/- DCs and PCs still maintain SC fate. All EdU-positive cells were also labeled with Proxl and Sox2 at P4, demonstrating that proliferating cells still maintained their SC fate during cell cycle reentry. In the area where HCs were lost, EdU-positive SCs were also found co-labeled with the SC marker, Sox2. No EdU/Myo6 double positive cells were observed, suggesting that Prox1-Rb-/-SCs did not transdifferentiate into HCs even after the loss of neighboring HCs in one week.更多还原