【作者】 陈俊；

【导师】 邱建华；

【作者基本信息】 第四军医大学 ， 耳鼻咽喉科学， 2009， 博士

【摘要】 在成年哺乳动物耳蜗中,听觉毛细胞和神经元在损伤后不能自然再生,这是感音神经性聋的主要原因。近年来,成功从新生鼠耳蜗分离出耳蜗前体细胞,也有人称之为耳蜗干细胞,在体外培养的耳蜗前体细胞能分化为毛细胞和螺旋神经元样细胞。研究耳蜗前体细胞的分化机制及微环境将为诱导损伤后毛细胞和螺旋神经元再生提供依据。我们通过实验发现细胞周期调节分子cyclin A2和CRP2可能参与了耳蜗的发育以及前体细胞的增殖、分化。实验一新生大鼠耳蜗前体细胞的分离、培养目的对新生大鼠耳蜗前体细胞进行分离、培养。方法从P2大鼠耳蜗分离培养前体细胞,血清诱导分化后用免疫细胞化学染色鉴定多向分化潜能,通过扫描电镜观察分化细胞表面的超微结构,进一步了解分化细胞的特性。结果原代培养的细胞第3天即可见“细胞球”,随着培养天数的增加,可见细胞球体积增大。细胞球内绝大部分细胞呈nestin和BrdU阳性,表明其具有自我更新的能力。细胞球经诱导分化14 d后,对分化细胞行免疫荧光化学染色,发现部分细胞表达毛细胞标志物myosin VIIA,部分细胞表达星形胶质细胞标志物GFAP或者成熟神经元标志物NeuN,证明其具有多向分化潜能。扫描电镜下可见扁平的三角形或不规则形细胞,表面具有纤毛样结构,其纤毛顶端的结构与正常耳蜗毛细胞纤毛顶端相似,此外还可见双极神经元形态的细胞。结论本部分实验为研究耳蜗前体细胞的分化机制奠定基础。实验二cyclin A2在大鼠耳蜗组织发育和耳蜗前体细胞分化过程中的表达变化目的细胞周期分子cyclin A2具有调控G1/S转换和G2/M转换的双重作用。目前对于cyclin A2在内耳中的功能尚未见任何报道。我们拟通过检测cyclin A2在大鼠耳蜗组织发育及耳蜗前体细胞分化过程中的表达情况,探讨其是否参与了哺乳动物耳蜗的发育。方法一、cyclin A2在大鼠耳蜗组织发育过程中的表达情况:P2、P10和P42的SD大鼠取耳蜗组织,RT-PCR、Western blot和免疫组织化学染色的方法检测cyclin A2的表达。二、cyclin A2在大鼠耳蜗前体细胞分化过程中的表达情况:用RT-PCR、免疫细胞化学染色和Western blot的方法检测cyclin A2在未分化、分化6天及分化14天耳蜗前体细胞的表达情况。结果cyclin A2表达于P2大鼠耳蜗的螺旋缘,在P10后的耳蜗未见表达。在前体细胞的分化过程中cyclin A2的表达量逐渐下降。结论在大鼠耳蜗组织发育及耳蜗前体细胞分化的过程中cyclin A2表达量逐渐下降,这表明cyclin A2可能参与了大鼠出生后耳蜗的发育。实验三CRP2及CRIP2在大鼠耳蜗组织发育和耳蜗前体细胞分化过程中的表达变化目的我们实验室在以往研究中通过RT-PCR及Western blot首次证实LIM分子CRP2及CRIP2表达于大鼠的内耳。其中CRP2表达于胚胎期耳蜗,在成年耳蜗中无表达;而CRIP2在不同年龄耳蜗中(包括胚胎期)表达量无明显差异。为进一步探讨CRP2及CRIP2是否参与了哺乳动物耳蜗的发育,我们拟通过免疫组织化学和免疫细胞化学的方法检测CRP2及CRIP2在不同年龄大鼠耳蜗组织中的分布及耳蜗前体细胞分化前后的表达变化。方法一、CRP2及CRIP2在大鼠耳蜗组织发育过程中的表达情况:免疫组织化学染色的方法检测其在P2、P6和P10大鼠耳蜗中的表达。二、CRP2及CRIP2在大鼠耳蜗前体细胞分化过程中的表达情况:用免疫细胞化学染色的方法检测其在未分化和分化7天耳蜗前体细胞中的表达情况。结果免疫组化结果显示,CRP2主要表达于螺旋神经节,并且在耳蜗发育过程中,其阳性细胞数逐渐减少。CRIP2主要表达于耳蜗组织中的螺旋神经节、螺旋缘和Corti器,而且在P2、P6和P10各组中阳性细胞数量无明显差异。另外,在P2组前庭的感觉上皮部位也可见CRIP2和CRP2的表达。免疫细胞化学染色显示,CRIP2和CRP2均可表达于未分化的前体细胞,在胞核和胞质中均呈阳性表达,CRIP2可表达于分化7天细胞的胞质。分化细胞未见CRP2的表达。结论CRP2可能参与了耳蜗的发育以及毛细胞和螺旋神经元的增殖;CRIP2在耳蜗不同发育时期以及前体细胞分化前后均稳定表达,表明其可能在维持耳蜗细胞基本功能中发挥一定作用。实验四耳蜗前体细胞分化的微环境对神经干细胞分化影响的初步研究目的耳蜗前体细胞向毛细胞和螺旋神经元定向分化的具体机制尚不清楚,前体细胞所处的微环境在分化过程中起重要作用。为探讨微环境在细胞分化中的作用,我们在体外将GFP标记的神经干细胞和耳蜗前体细胞按比例混合,并诱导其分化,观察耳蜗前体细胞分化的微环境对神经干细胞分化的影响。方法将耳蜗前体细胞和GFP标记的神经干细胞按10:1的比例混合后接种于同一培养皿中,诱导分化后进行贴壁培养。21天后采用免疫荧光的方法对分化细胞进行鉴定。一抗采用抗myosin VIIA(毛细胞标志物)和抗peripherin(神经元标志物)。结果耳蜗前体细胞分化的微环境促进神经干细胞的存活,并诱导其向myosin VIIA阳性细胞及双极神经元形态细胞分化。结论耳蜗前体细胞的成功分离、培养为研究其分化的机制提供了一个极佳的体外模型,对于耳蜗前体细胞分化的微环境需要更加深入的研究。实验五干细胞向雏鸡和成年豚鼠耳蜗移植方法的建立目的研究耳蜗前体细胞分化的机制,目的是诱导外源性干细胞定向分化为听觉毛细胞及神经元,或刺激体内内源性耳蜗前体细胞再生,从而修复受损的听觉细胞。将外源性干细胞或治疗基因表达载体(如腺病毒等)植入耳蜗是治疗的前提,为此我们建立了干细胞向动物耳蜗移植的方法。方法雏鸡耳蜗的干细胞移植是经外耳道剥离鼓膜,暴露中耳腔的蜗窗,再经蜗窗膜造孔注入干细胞。成年豚鼠耳蜗鼓阶的干细胞移植是行耳后切口暴露听泡,在耳蜗底转近镫骨动脉处钻孔,并注入GFP标记的干细胞。中阶的移植是经耳蜗第三转的血管纹外侧壁注入干细胞。细胞移植1天后,分离雏鸡和豚鼠的耳蜗作冰冻切片,在荧光显微镜下观察移植细胞的分布。结果移植后的干细胞可分布于雏鸡耳蜗及豚鼠耳蜗的鼓阶、中阶。结论成功建立了干细胞向雏鸡和成年豚鼠耳蜗移植的方法,为进一步将干细胞应用于耳聋的治疗及机制研究奠定基础。更多还原

【Abstract】 In adult mammalian cochlea, the hair cells cannot regenerate spontaneously after damaged. This is the major cause of permanent hearing loss. Cochlear stem cells have been isolated from the early postnatal rat and mouse in previous studies. These stem cells cultured in vitro can differentiate into neurons, astrocytes, hair cells and supporting cells. Differentiation of cochlear stem cells is critical to the development of cochlea tissue. Study of cochlear stem cells may help to learn more about cochlear development.Microenvironment is critical to differentiation of stem cells. But little is known about microenvironment of cochlear stem cell differentiation. Microenvironment of differentiation consists of all molecules in molecular network. We need to know more molecules involved in this network. Cell cycle exit participates in stem cell differentiation. Several cell cycle regulators, including cyclin A2 and CRP2, may be involved in this network. Hence, we isolated cochlear stem cells from newborn rats and induced differentiation of these cells. The level of cyclin A2 and CRP2 were assessed in stem cells and differentiated cells for better understanding of cochlea development mechanism. Then we cultured neural stem cells in the microenvironment of cochlear stem cell differentiation to see the effect of microenvironment on neural stem cell differentiation.1. Isolation and culture of cochlear stem cells from newborn ratStem cells were isolated from the P2 rat cochlea tissues. Stem cells were incubated at 37°C in 5% CO2 atmosphere. The culture medium consisting of DMEM/F12 supplemented with B27, N2, EGF and FGF-2. For cell expansion and testing for self-renewal, we plated the cells at very low density to ensure that cell spheres were formed from single cells. For analysis of cell differentiation, we transferred cell spheres into 6-well dishes with poly-l-lysine treated coverslips and filled with 10% FCS. After 16 h, we replaced the medium with serum-free DMEM/F12 supplemented with N2 and B27. Half of the medium was replaced every second day. The differentiated cells were identified after 14 days in culture by immunocytochemistry. The cell spheres and differentiated cells were fixed with 4% paraformaldehyde, and incubated at 4°C overnight with primary antibodies: mouse anti-nestin monoclonal antibody, mouse anti-BrdU monoclonal antibody, rabbit anti-myosin VIIA (hair cell marker) polyclonal antibody, rabbit anti-GFAP (astrocyte marker) polyclonal antibody, mouse anti-NeuN (neuron marker) monoclonal antibody. Cy3 conjugated second antibodies were used to detect primary antibodies. Hoechst 33342 was used to visualize nuclei. For BrdU detection, stem cells were incubated with 10μM BrdU in the culture medium for 24 h, fixed for 30 min, treated with 2 N HCl for 30 min before antibody incubation. The differentiated cells cultured for 21 d were analyzed by SEMWe found sphere-forming capacity of isolated cells after 7 days in culture. These cells were further identified with nestin and BrdU antibodies. The stem cell-derived differentiated cells expressed myosin VIIA, GFAP and NeuN. SEM was performed in order to determine the shape and surface structure of hair cells. Hair cells in culture were identified with stereocilia of cell surface. We found that hair cells with stereocilia were flat and irregularly shaped. We also found bipolar neuron-like cells.2. Decreased level of cyclin A2 in rat cochlea development and cochlear stem cell differentiationIn order to detect the distribution of cyclin A2 protein in the rat cochlea, the immunohistochemical staining method was used in our study. P2 (postnatal day 2), P10 and adult (P42) rats were deeply anesthetized and exsanguinated, then decapitated. The cochlea tissues were excised carefully and then fixed in 4% paraformaldehyde in PBS at 4°C overnight. The P2 samples were decalcified for 1 day. The P10 and P42 samples were decalcified for 10 days. All samples were then immersed in 30% sucrose in PBS for 1 day and embedded carefully in optimal cutting temperature (OCT) compound. Sections (10μm thick) were cut on a cryostat at -27°C and mounted on poly-l-lysine treated glass slides. The immunohistochemistry of cyclin A2 in the rat cochlea was performed by streptavidin–biotin complex (SABC) staining. The primary antibody was mouse anti-cyclin A monoclonal antibody, and the second antibody was biotinylated horse anti-mouse IgG. For negative control, an equivalent dilution of normal mouse IgG was used in place of the primary antibody. We used immunocytochemistry to detect cyclin A2 protein expression in differentiated cells and cell spheres.We used RT-PCR to detect cyclin A2 mRNA expression in cochlea tissues, cochlear stem cells and differentiated cells. The cochlea tissues were excised from P0, P2, P14 and P42 rats respectively. The differentiated cells cultured for 6 or 14 days and cell spheres were collected for further assay. Total RNA was extracted. Then cDNA was amplified by PCR. The cyclin A2 PCR product was sequenced.Western blotting was used to detect cyclin A2 protein expression in cochlear stem cells, differentiated cells cultured for 14 days, and cochlea tissues (P0 and P14). Each protein sample was electrophoresed onto 12% SDS-polyacrylamide gel and transferred onto the nitrocellulose membranes. The membranes were incubated with mouse anti-cyclin A monoclonal antibody. The membranes were then incubated with the HRP-conjugated goat anti-mouse IgG. The membranes were also probed with anti-β-actin as internal control. The reaction product was visualized with enhanced chemiluminescence.With immunohistochemical staining, cyclin A2 was observed localizing in the spiral limbus of the P2 rat cochlea, but not in the cochlea of P10, P42 and the negative control. Cyclin A2 mRNA and protein levels in cultured cells fell down after differentiation of cochlear stem cells. Cyclin A2 level in cochlea tissues decreased from newborn to adult. The sequence of PCR product was determined to be identical to the originally published sequence of rat cyclin A2.3. Expression of CRP2 and CRIP2 in rat cochlea development and cochlear stem cell differentiationIn order to detect the distribution of CRP2 and CRIP2 proteins in the rat cochlea of P2, P6 and P10, the immunohistochemical staining method was used. We used immunocytochemistry to detect CRP2 and CRIP2 proteins in differentiated cells and cell spheres.CRP2 protein level fell down during the development of cochlea tissues and differentiation of cochlear stem cells. While we found no difference of CRIP2 protein level in cochlea development and cochlear stem cell differentiation.4. Microenvironment of cochlear stem cell differentiation induces neural stem cells into myosin VIIA-positive cells and bipolar neuron-like cellsCocultures were performed on poly-L-lysine-coated coverslips in 6-well plates mixed with 1×10~5 CPCs and 1×10~4 NPCs per well. Cell spheres of NSCs and CPCs were dissociated by 0.025% trypsin and mechanical procedures. Then dissociated cells were mixed and allowed to attach for 16 h in wells filled with DMEM/F12 containing 10% FCS. After the cells were attached, we replaced the medium with serum-free DMEM/ F12 supplemented with N2 and B27 solutions. Half of the medium was replaced every second day. Cocultured cells were analyzed after 21 d by immunocytochemistry. Primary antibodies were as follows: rabbit anti-myosin VIIA polyclonal antibody, rabbit anti-peripherin polyclonal antibody. Cy3 conjugated second antibodies were used to detect primary antibodies. Hoechst 33342 was used to visualize nuclei. For negative controls, 1×10~5 NPCs were dissociated and cultured in 6-well plates with the same medium and methods as above.After 21 d in coculture, about 8% differentiated GFP-labeled NSCs expressed myosin VIIA. Myosin VIIA-positive cells were flat and irregularly shaped. About 1% differentiated NSCs expressed peripherin, a marker of peripheral neuron. Some of them were bipolar neuron-like cells. We also observed that spontaneous neural cell apoptosis which occurred in adherent cultures was delayed in the presence of differentiated CPCs. After 21 d in culture, no differentiated NPC was alive in negative controls, while the number of living differentiated NSCs among cocultured cells was about 8×103 per well.5. Methods for transplantation of stem cells into the cochlea of chickens and mature guinea pigsNSCs labeled with GFP were transplanted into chicken’s cochlea via cochlear window after exposure of middle ear cavity. For cell transplantation into cochlear perilymphatic space of guinea pigs, NSCs were inoculated via a small hole drilled on the bony wall of scala tympani in the basal turn of the cochlea. For cell transplantation into scala media, a small hole was made to penetrate through the stria vascularis in the third turn of the cochlea. NSCs were then injected into the scala media through the fenestra. One day after transplantation, the transplant cells were examined with fluorescent microscope.After transplantation, NSCs labeled with GFP were observed in chicken’s cochlea. In the cochlea tissues of guinea pigs, NSCs were detected in scala tympani and scala media, respectively. The methods for transplantation of stem cells into the cochlea of chickens and mature guinea pigs were established, which provides a basis for application of stem cells in studies of sensorineural hearing loss.更多还原