【作者】 薛涛；

【导师】 邱建华； 乔莉； 卢连军；

【作者基本信息】 第四军医大学 ， 耳鼻咽喉科学， 2008， 博士

【摘要】 感音神经性聋是临床常见病和多发病,严重影响患者的生活质量,临床上尚无理想的治疗方法。目前关于感音神经性聋的防治研究已成为听力学和耳科学研究的热点和难点之一。近年来,运用胚胎或神经干细胞(neural stem cells,NSCs)以及(neural progenitor cells,NPCs)的替代治疗应运而生,并受到密切的关注。目前大量的研究致力于干细胞/前体细胞移植促进听觉功能恢复,研究方向主要集中于外源性NSCs的直接或定向诱导后移植,或者将NSCs作为外源性基因的载体进行移植。虽然国内外学者在此方面已经进行了很多工作,取得了一定的进展,但仍有很多难题亟待解决。其关键问题之一是:对于NSCs/NPCs在听功能的形成与发育过程中的作用,尤其是听觉中枢神经核团内是否存在多向分化潜能的NSCs/NPCs还不甚清楚。实验一:新生大鼠脑干耳蜗核神经前体细胞的分离、培养目的:目前听觉系统是否存在NSCs/NPCs的研究主要集中在内耳感受神经元的干细胞/前体细胞上,近年研究证实哺乳动物内耳中存在着一定数量毛细胞的前体细胞,而听觉中枢神经核团内是否存在多向分化潜能的NSCs/NPCs目前尚未见报道。本研究拟分离、培养新生大鼠脑干耳蜗核NPCs,观察其增殖及传代特性,建立新生大鼠脑干耳蜗核NPCs的培养方法,为进一步研究打下基础。方法:P1、P3、P5、P7、P9、P11以及成年SD成年大鼠麻醉后,75%酒精消毒,无菌条件下开颅切取脑干耳蜗核,经剪碎、消化、离心、洗涤、吹打、过滤、计数,接种于无血清培养液DMEM/F12 (1∶1)培养,内含B27 (1∶50)、100 U/ ml青霉素、100μg/ml链霉素、20ng/ml EGF、20ng/mlbFGF,置于5 % CO2培养箱(37℃)内培养。细胞每5～7 d传代1次。取次代神经球离心后消化,吹打成单细胞悬液,通过“有限稀释法”获得从一个单细胞克隆扩增出的大量均质的脑干耳蜗核“神经前体细胞”。采取免疫荧光、免疫组织化学的方法,对NSCs/NPCs的特异性标记物Nestin、Musashi1的表达进行鉴定;通过台盼蓝染色技术检测不同日龄组细胞活力;CCK-8法绘制不同日龄组生长曲线,观察不同生长因子对于细胞增殖的影响。结果:新生5-7日龄大鼠脑干耳蜗核可以分离培养“神经前体细胞”,体外培养可以形成神经球,并保持旺盛的增殖活力,可持续传代100代以上;这些细胞经稀释纯化、传代后可以继续形成细胞克隆。细胞克隆呈nestin、musashil1免疫反应阳性;5-7日龄大鼠脑干耳蜗核中“神经前体细胞”数量较多,增殖能力旺盛,而在小于5日龄与大于9日龄大鼠中该细胞数量很少,而且增殖能力差,很难连续传代。表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF)联合应用可以明显促进耳蜗核中的“神经前体细胞”的传代、增殖。结论:从新生5-7日龄大鼠脑干耳蜗核可以分离、培养出具有长期自我更新、维持自身数量稳定、保持未分化状态进行传代分裂增殖的具有部分NSCs特性的“神经前体细胞”。实验二:新生大鼠脑干耳蜗核神经前体细胞体外分化特性的对比研究目的:在实验一中,我们在新生5-7日大鼠的脑干耳蜗核中分离出了具有体外连续传代增殖能力的“神经前体细胞”,这些细胞体外培养可以形成神经球,并表达NSCs/NPCs的特异性抗体,但还没有证实其是否存在NSCs/NPCs的另一个重要的特性——多向分化潜能。本实验拟通过免疫组织化学、免疫荧光、分子生物学的方法,验证该细胞的体外多向分化能力,观察分析其分化特性,并比较其与嗅球NSCs、嗅上皮NSCs分化特性的差异。方法:三种细胞分别取材培养,进行体外诱导分化后免疫细胞化学、免疫荧光化学以及分子生物学的鉴定;进行细胞的BrdU的体外标记,观察增殖与分化情况。结果:我们的实验结果提示大部分细胞分化为NeuN免疫反应阳性的神经元和GFAP免疫反应阳性的星形胶质细胞,少部分细胞分化为GalC免疫反应阳性的少突胶质细胞;标记后大部分细胞均为BrdU阳性,表明其处于增殖期,通过对三种来源细胞BrdU阳性率的比较可以发现,嗅球来源与耳蜗核来源的细胞BrdU的阳性率相近,均高于嗅上皮来源的NSCs。这可能由于不同脑区的发育特性导致其干细胞增殖能力不同。结论:新生大鼠脑干耳蜗核中的这种“神经前体细胞”具有了体外多向分化的潜能。实验三:新生大鼠脑干耳蜗核神经前体细胞的超微结构研究目的:了解大鼠脑干耳蜗核“神经前体细胞”的超微结构、细胞之间的联系以及细胞的增殖和发育状态,有助于阐明该细胞在体外培养条件下的生长、发育和分化等生物学行为,为该细胞的研究和应用奠定基础。方法:本研究在成功分离、培养新生大鼠脑干耳蜗核“神经前体细胞”的基础上,应用扫描及透射电子显微镜观察新生大鼠脑干耳蜗核“神经前体细胞”的超微结构,进一步研究新生大鼠脑干耳蜗核“神经前体细胞”的生物学特征,以期为更深层次的研究和应用提供依据。结果:体外培养的NSCs呈圆形或椭圆形,表面有微突起或微绒毛,细胞核浆比例高,稀少的细胞浆中有多少不等的未成熟线粒体、核糖体及高尔基复合体。不同培养时期的神经球内未分化NSCs的结构大致相同,均有分裂增殖细胞及少数凋亡细胞。分化后细胞伸出树突、轴突样结构,相邻细胞之间有突触样组织。结论:电镜提示耳蜗核“神经前体细胞”具有原始细胞的形态和结构,分化后的细胞均有神经细胞的形态学特点。实验四大鼠脑干耳蜗核神经前体细胞的冻存与复苏目的:NSCs在神经组织中含量少,获取大量纯化NSCs是研究工作的基础。NSCs在体外的合理冻存与复苏及无损保存是NSCs研究的关键。建立一种能够长期储存并维持NSCs生物学特性的方法对促进神经科学的基础和临床移植的研究具有重要的意义。方法:我们应用不同冻存保护液对新生大鼠脑干耳蜗核“神经前体细胞”进行冻存,研究低温冻存对该细胞的增殖及多向分化潜能的影响,并以胚胎大鼠嗅球神经干细胞作为对照进行研究,分析冻存与复苏对于新生大鼠脑干耳蜗核“神经前体细胞”生物学特性的影响。结果:采取正确的冻存与复苏方法,对新生大鼠脑干耳蜗核“神经前体细胞”的生物学特性无影响。结论:新生大鼠脑干耳蜗核“神经前体细胞”经低温长期冻存,复苏后对于该细胞的基本生物学特性没有明显影响,可以长期低温冻存。更多还原

【Abstract】 Hearing loss is one of the most frequent diseases that disable people. Options for improvement of hearing of patient with sensorineural deafness are limited to hearing aid and cochlear implants. The effect of aid and cochlear implants depends on the number of live hair cell and spiral ganglion neuron. It’s new strategy for restoring hearing to improve inner ear hair cell and spiral ganglion neuron regeneration and to prevent necrosis of spiral ganglion neuron after hair cell injury. Cell therapy may replace damaged and lost hair cell and spiral ganglion neuron and hence restore hearing pathway. Stem cells are undifferentiated cells, which have the ability to undergo numerous divisions and self-renewal in culture, as well as to differentiate into multilineage, functionally specialized cells. The replacement of lost or damaged neurons by neural stem cells (NSCs) or neural progenitor cells(NPCs) represents a great promise for clinical treatment for hearing loss. NSCs, with the capacity for unlimited self renewal and the production of none-restricted lineage committed progenitors in contrast to neural‘progenitor’or‘precursor’cells, have long been thought of as central to the repair and regeneration processes to replacing cells lost in hearing loss diseases. Although hair cell replacement occurs spontaneously following injury in birds and lower vertebrates, the mature mammalian cochlea is unable to regenerate new hair cells. As a matter of fact, cochlear hair cell loss is one of the major causes of hearing impairment. Hair cell replacement, either by stimulation of regeneration or by transplantation of progenitor cells capable of differentiating into hair cells, remains the ultimate goal in the development of treatment applications to reconstruct damaged inner ears. An alternative way to replace lost hair cells is by introducing exogenous stem cells into the inner ear. Recent work by Heller and co-workers has demonstrated the existence of progenitor/stem cells in the sensory epithelium of a mature mouse vestibular end organ, which is responsible for balance. These cells are not only able to differentiate into cells that express hair cell markers in vitro, but also differentiate into hair cell-like cells in developing chick ears. The recent discovery of stem cells in the adult inner ear that is capable of differentiating into hair cells, as well as the finding that embryonic stem cells can be converted into hair cells, raise hope for the future development of stem-cell-based treatment regimens. While stem cells/progenitors may exist in adult vestibular end organs, stem cells/progenitors from the cochlear nucleus have not been isolated.We have for the first time isolated neural precursor cells from the newborn rat cochlear nucleus. Under our optimized conditions, precursor cells isolated from the cochlear nucleus proliferate in culture in the same way as neural stem cells after epigenetic stimulation and retain all of the typical characteristics of neural stem cells: they proliferate in response to mitotic factors (basic fibroblast growth factor, bFGF, and epidermal growth factor, EGF) and have the ability to give rise to neurons, astrocytes, and oligodendrocytes. Part I Isolation and culture of NPCs from the newborn rat cochlear nucleus.NPCs were isolated and cultured from P3, P5, P7, P9, P11 and adult rat cochlear nucleus using DMEM/F12 (1:1) containing 10% heat-inactivated fetal bovine serum. Cell clones were formed 8 days after culture. Cell clones and most single round cells were semi-suspending. Cell clones can be formed again after dilution, purification and passage. Observe the growth of NPCs every day in inverted microscope, 24h later, in experiment group, we can observe many living cell homogeneous distribution and many collagenoblast sink in the base of culture flask, part of cells grow floating in culture fluid, cell appear conjugation usually and some cells begin to division. 72h later, cell aggregation and the cell colony consist of about 4-6 cells; 5 days later, cell colony is consisted of about ten to hundred cells; we can observe lots of cell colony. The cell sphere growth floating and arrange tight among cells, some cell colony appear cellular necrosis because of alimentary deficiency. In control group, we can not observe cell proliferate. Cell clones were nestin immuno-positive and Musashi1 immuno- positive. The Cell viability of P5 and P7 rat cochlear nucleus was higher than the others. The viability of NPCs had significant difference between P5、P7 and the others (P>0.05). Made cell sphere into mono-cell suspension, add dispase during dispersive process. The method is to obtain neural cell spheres that growth good in culture flask first\digest them for 2 hours\then blow lightly and get out supernatant after sink nature and obtain mono-cell suspension. Divide into 4 groups, Add EGF in experiment group 1; add bFGF in experiment group 2; add EGF and bFGF together in experiment group 3; add base serum-free medium only in control group. Count cell amount of cell spheres in micro after cultured for 3 days and measure the diameter of cell spheres at 7 days. EGF and bFGF can significantly promote proliferation of cochlear nucleus NPCs. EGF can excite neural stem cells proliferate lonely; bFGF can not excite neural stem cells proliferate lonely but can enhance the proliferation function of EGF. The results showed that NPCs with self-renewal capacity could be cultured in vitro from cochlear nucleus of newborn rat.Part II Cell differentiation biologic characteristics of NPCs from cochlear nucleus of newborn rat.NPCs and NSCs were isolated and cultured from P7 rat cochlear nucleus, E14 rat olfactory bulb and adult rat olfactory epithelium using serum free media with mitogen and neurosphere forming method. Cultured NPCs and NPCs were round or oval without process. Cells from P7 cochlear nucleus, E14 olfactory bulb and adult olfactory epithelium divided 2 days after primary culture, looked like bean sprout and at 4th day formed suspending small neurospheres, which grew gradually. After omitting mitogen and adding fetal bovine serum, most of NSCs/NPCs from P7 rat cochlear nucleus, E14 olfactory bulb and adult rat olfactory epithelium differentiated into Neun and Tuc-4 immunopositive neuron and GFAP immunopositive astrocyte, some of them differentiated into GalC immunopositive oligodendrocyte. RT-PCR and In-cell Western also show the same result. The proliferation of P7 rat cochlear nucleus, E14 olfactory bulb and adult rat olfactory epithelium NSCs/NPCs depended on EGF and bFGF. The results showed that NSCs with self-renewal capacity and potential multi-differentiation could be cultured in vitro from P7 rat cochlear nucleus. The newborn rat cochlear nucleus maybe is rich source of NSCs/NPCs and thereby an ideal providing organ for the study of NSCs/NPCs transplantation.Part III Ultrastructure of NSCs from rat olfactory bulbThe ultrastructures of NPCs from newborn rat cochlear nucleus were investigated with scanning and transmission electron microscopes. Cultured NPCs were round or oval, with many tiny processes or microvilli on the surface. Neurospheres contained healthy, apoptotic, and necrotic cells. Healthy cells were attached to each other by adherens junctions. They showed many pseudopodia and occasionally a single cilium. Sphere cells showed phagocytic capability because healthy cells phagocytosed the cell debris derived from dead cells in a particular process that involves the engulfment of dying cells by cell processes from healthy cells. The nucleus/cytoplasm ratio was very high. Cells had very little cytoplasm with various numbers of immature mitochondria, ribosome and Golgi complex. Inside neurosphere, dividing cells and apoptosis can be found and the structure of pre-differentiating NSCs remains the same at different time after culture. Differentiated cells with rough endoplasmic reticulum and thick and long processes appeared 1~2 months after culture. Gap junction was found between the processes of adjacent differentiated cells. The results showed that cultured NPCs from newborn rat cochlear nucleus were primitive cells that can differentiate into nerve cells.Part IV Proliferation, Multipotency and Neuronal Differentiation of Cryopreserved Neural Progenitor Cells Derived from Newborn Rat Cochlear NucleusStem cell replacement has emerged as the novel therapeutic strategy for many diseases. These researches not only offers unique opportunities for developing new medical therapies for devastating diseases, but also provides a new mode to explore fundamental questions of biology. The study of newborn rat cochlear nucleus NPCs requires efficient recovery methods and cryopreservation procedures. The purpose of this study was to evaluate different cryopreservation techniques for NPCs derived from newborn rat cochlear nucleus. Initially, we compared the survival rates of cryopreserved NPCs treated with four different cryoprotectants: dimethylsulphoxide (DMSO), glycerol and each with or without 10% FBS, meanwhile with two different storage period at liquid nitrogen (–196℃) , that is 3 days standing for short-term storage and 3 months for long-term storage. We assessed the recovery efficiency of NPCs after freezing and thawing by viability testing, colony-forming assay as well as immunocytochemistry under different conditions. No significant difference in survival rate was observed among these different cryoprotectants. With these protocols, NPCs from newborn rat cochlear nucleus retained their multipotency, differentiated into glial (GFAP-positive), neuronal (NeuN-positive). Collectively, our results imply that, under the optimal conditions, NPCs from newborn rat cochlear nucleus might be cryopreserved for longer storage period than 3 months without losing proliferation and multipotency activity.更多还原