胚胎大鼠神经干细胞体外培养及分化为类毛细胞的研究

目的探讨体外培养和鉴定胚胎大鼠神经干细胞(neural stem cells,NSCs)及诱导其分化为类毛细胞。方法从SD系胚鼠大脑分离NSCs,在无血清培养基中培养,用含10%胎牛血清的DMEM/F12培养基诱导其分化为神经元和星形胶质细胞:将NSCs球放到含鼠尾胶原包被的盖玻片6孔培养板中培养,然后将乳鼠基底膜体外培养后汲取其上清液与NSCs一起培养,14～21天后通过免疫荧光和免疫组化法检测毛细胞标志物肌球蛋白(myosin)Ⅶa和钙视网膜蛋白(calretinin)。结果培养的NSCs胞体透亮,折光性好,分化后的细胞免疫组化示神经元特异性烯醇化酶、胶原纤维酸性蛋白、myosinⅦa和calretinin阳性。结论在无血清条件下能培养出活性很好的NSCs,并且能诱导其分化为神经元、星形胶质细胞和类毛细胞。     

体外诱导大鼠骨髓间充质干细胞向内耳毛细胞样细胞分化的研究

目的 探讨体外定向诱导大鼠骨髓间充质干细胞向耳蜗毛细胞样细胞分化的可行性.方法 分离培养大鼠骨髓间充质干细胞,并加以鉴定.分离出生1～3 d的乳鼠耳蜗Corti器,与骨髓间充质干细胞在体外共培养14 d.通过反转录聚合酶链反应(RT-PCR)和免疫细胞化学染色对分化细胞的特异性分子指标(myosinⅦa、math1及calretinin)进行鉴定.结果 免疫细胞化学染色显示诱导分化后的细胞myosinⅦa、math1和calretinin表达阳性,RT-PCR提示分化后的细胞可表达毛细胞的标志物myosinⅦa和math1.结论 体外培养的骨髓间充质干细胞可诱导分化为具有毛细胞分子标志物的毛细胞样细胞.     

胎鼠神经干细胞的原代培养及体外诱导分化研究

目的探讨神经干细胞的原代培养以及体外诱导分化为毛细胞样细胞的方法 ,为神经干细胞移植治疗感音神经性聋奠定基础。方法取胎龄为14.5天的SPF级昆明小鼠脑组织,采用机械吹打的方法在无血清培养基中原代培养,传代培养,诱导分化为毛细胞样细胞,采用免疫细胞化学方法鉴定神经干细胞以及毛细胞的免疫表型。结果①在含成纤维细胞生长因子-2（FGF-2）和表皮生长因子（EGF）的无血清培养液中,神经干细胞在体外培养6～8代后,其细胞呈指数级增加,增殖的细胞表达神经上皮干细胞蛋白（nestin）;②在诱导分化培养基中培养3周后它们能被诱导分化为神经胶质细胞、毛细胞样细胞,4周后表达支持细胞表型。结论神经干细胞在体外可以诱导分化为毛细胞样细胞。     

大鼠耳蜗大上皮嵴细胞体外培养的实验观察

目的建立大鼠耳蜗大上皮嵴（greater epithelial ridge，GER）细胞体外培养体系，探讨其在离体培养条件下的生物学特性、生长模式及超微结构。方法取生后1d大鼠耳蜗，利用机械分离和酶消化相结合的方法分离出纯GER细胞，分别于无血清和含10％胎牛血清的DMEM培基中进行培养，观察细胞生长特性，并对培养物进行溴脱氧尿苷（BrdU）、鬼笔环肽（phalloidin）、Z01、钙视网膜蛋白（calretinin）及肌球蛋白VIIa（myosin VIIa）免疫组化鉴定及扫描电镜观察。结果GER体外培养细胞呈现典型的上皮样多角形外观，细胞间连接紧密，具有明显的增殖能力，在含血清培养基中易形成贴壁细胞岛，而在无血清培养条件下则易形成悬浮细胞球。GER细胞呈现BrdU、phalloidin以及Z01染色的阳性反应，而calretinin及myosinVIIa则表现为阴性。扫描电镜显示GER细胞表面可见微绒毛和中心体，但未见毛细胞特异性结构纤毛束。结论GER体外培养细胞具有增殖能力，在不同培养条件下可分别呈现贴壁细胞岛及悬浮细胞球生长特性，GER细胞表达上皮细胞来源标记物，但不表达毛细胞特异性标记物。     

新生大鼠耳蜗前体细胞的培养及鉴定

目的对新生大鼠耳蜗前体细胞进行分离、培养和鉴定。方法从出生7天的大鼠耳蜗中分离、培养前体细胞,用免疫细胞化学的方法对前体细胞进行鉴定;血清诱导分化后鉴定分化潜能,进一步了解其多向分化特性。结果原代培养的细胞,培养1d后即可见"细胞球"。细胞球内大部分细胞呈nestin、musashi1、pax2和BrdU阳性,表明其具有自我更新及有丝分裂的能力。细胞球经诱导分化14d后,对分化细胞行免疫细胞化学鉴定,发现分化细胞表达毛细胞标志物myosin VIIA和phalloidin,表达成熟神经元标志物NeuN,表达不成熟神经元标志物Tuj1,表达星形胶质细胞标志物GFAP,表达少突胶质细胞标志物galactocerebroside,以及谷氨酸能神经元标志物GluR-1,证明其具有多向分化潜能。结论本实验证明耳蜗前体细胞具有神经干细胞的多向分化潜能,为研究通过基因治疗的方法促进耳蜗神经细胞增殖提供了一种体外模型。     

新生豚鼠海马神经干细胞分化为类毛细胞的体外实验

目的体外定向诱导新生豚鼠海马神经干细胞分化为内耳毛细胞。方法将原代培养的新生豚鼠海马来源的神经干细胞进行体外培养,分别置入含有10%胎牛血清和5%～15%人工外淋巴液培养基使其分化,并用免疫荧光化学,蛋白免疫印迹扫描电镜对分化的细胞进行鉴定。结果胎牛血清和人工外淋巴液均能诱导新生豚鼠海马神经干细胞分化为表达毛细胞特异抗体的细胞。结论神经干细胞在胎牛血清和人工外淋巴液中可以存活并分化出表达毛细胞特异抗体的细胞,为神经干细胞移植内耳治疗提供理论依据。     

大鼠神经干细胞生长及增殖规律的体外研究

目的:体外无血清条件下培养胚鼠神经干细胞,观察其生长及分化情况.方法:取孕16～18 d SD系大鼠的胚胎海马组织,在含EGF、bFGF和B27的DMEM/F12培养基中培养,电子显微镜下观察其增殖分化过程,并通过免疫荧光方法鉴定分化后的细胞类型.结果:神经干细胞在无血清培养基中生长旺盛,8 d左右就可以形成胞体透亮、折光性好的干细胞球,分化后的细胞显示NSE、GFAP免疫阳性.结论:无血清培养条件下神经干细胞生长良好,而在含血清的培养基中可以分化为神经元和星形胶质细胞.     

人脂肪源性间充质干细胞向内耳毛细胞定向诱导分化的实验研究

目的 验证在体外将人脂肪源性间充质干细胞(human adipose-derived mesenchymalstem cells,hAD-MSC)定向分化为内耳毛细胞的可行性.方法 用特定的培养体系配合多种细胞因子定向诱导hAD-MSC向神经干/祖细胞样细胞分化,而后进一步将诱导后细胞与发育期鸡胚听泡细胞在体外进行共培养,以促使其向内耳毛细胞分化,通过免疫组化等方法对分化不同阶段的细胞特异性指标进行鉴定.结果 hAD-MSC诱导后呈现神经干/祖细胞样的形态并表达其特异性标志,与发育期鸡胚听泡细胞共培养后表达内耳毛细胞特异性标志.结论 hAD-MSC在体外可定向诱导分化为具有内耳毛细胞特异性标志的毛细胞样细胞.     

miR-182可以促进耳蜗前体细胞分化为毛细胞

目的探讨miR-182诱导耳蜗前体细胞向毛细胞分化的作用及机制。方法从新生大鼠耳蜗中分离培养耳蜗前体细胞并用血清诱导分化,BrdU、Nestin免疫荧光染色鉴定细胞自我增殖能力;myosinⅦa和phalloidin免疫荧光染色鉴定其是否具有分化为毛细胞的潜能。分别利用miR-182 mimics和siRNA在新生大鼠耳蜗前体细胞中过表达和低表达miR-182,然后在细胞诱导分化7天后,用流式细胞仪检测分化细胞中myosinⅦa阳性细胞比例,用Western-blot检测支持细胞标志分子中Sox2的变化。结果使用miR-182 mimics进行过表达后,耳蜗前体细胞分化为myosinⅦa阳性表达的细胞比例显著高于对照组,使用miR-182 siRNA进行低表达后此比例显著低于对照组。Western-blot检测显示,Sox2在miR-182 mimics组较对照组降低,miR-182 siRNA组较对照组增加。结论过表达miRN182可以促进耳蜗前体细胞向毛细胞方向分化,ox2可能是此过程中的靶基因。     

Hath1基因诱导新生大鼠大上皮嵴细胞形成毛细胞样细胞

目的 探讨Hathl(human atonal homolog 1)基因对大鼠耳蜗大上皮嵴(greater epithelialridge,GER)细胞的诱导分化作用.方法 取出牛后1 d大鼠耳蜗,利用酶消化和机械分离相结合的方法分离出GER细胞,并行体外培养.以腺病毒为载体,对GER细胞培养物进行Hath1基因和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因(ad-Hath1-EGFP)的转染,单纯转染EGFP(ad-EGFP)作为对照组,对感染后不同灭数的标本行毛细胞特异性标记物myosin Ⅶa免疫组化染色鉴定.结果 ad-Hathl-EGFP组中的GER细胞中出现了myosin Ⅶa和EGFP双标记细胞,并且从感染后第5天开始出现myosin Ⅶa阳性细胞,随后阳性细胞的数量有所增加,但增加不明显;而ad-EGFP组感染后3～12 d的GER标本均未见myosin Ⅶa阳性细胞.结论 GER细胞可能是耳蜗毛细胞的前体细胞,Hath1基冈过表达可以诱导GER细胞向毛细胞样细胞(myosin Ⅶa阳性)分化.     

Effects of glucocorticoids on infiltrating cells and epithelial cells of nasal polyps

Glucocorticoids are known to be effective in the treatment of nasal polyps (NPs). To examine the mechanisms of their effect, we evaluated 1) the ability of glucocorticoids to induce the apoptosis of eosinophils and T lymphocytes in NPs, and 2) the ability of dexamethasone to down-regulate epithelial cell functions that relate to eosinophilic inflammation. In vitro and in vivo, glucocorticoids increased the apoptosis of both eosinophils and T lymphocytes in NPs. Dexamethasone inhibited the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) from both NP epithelial cells that were unstimulated and NP epithelial cells that were stimulated with interleukin-4 or tumor necrosis factor alpha. These results suggest that the clinical efficacy of glucocorticoids on NPs may be due to 1) induction of apoptosis in both eosinophils and T lymphocytes that infiltrate NPs, and 2) down-regulation of epithelial GM-CSF production, which prolongs eosinophil survival.     

内耳毛细胞再生的前体细胞及其发育调控基因

自从在鸟类等非哺乳脊椎动物中发现毛细胞再生现象至今已有20年,人们对毛细胞再生的研究也取得了丰硕的成果,而且有可能从试验性研究向临床应用性研究发展。目前对内耳毛细胞再生方面的研究．尤其是再生毛细胞的前体细胞和再生机制的探讨日益增多。     

内耳毛细胞再生的前体细胞及其发育调调控基因

自从在鸟类等非哺乳脊椎动物中发现毛细胞再生现象至今已有20年,人们对毛细胞再生的研究也取得了丰硕的成果,而且有可能从试验性研究向临床应用性研究发展.目前对内耳毛细胞再生方面的研究,尤其是再生毛细胞的前体细胞和再生机制的探讨日益增多.本文将对毛细胞再生的前体细胞进行简单介绍,并对参与内耳毛细胞形成的相关蛋白,Notch信号途径及Math1基因进行综述,以进一步了解毛细胞产生的机制.     

Distribution of canalicular reticulum in Deiters cells and pillar cells of gerbil cochlea

Postfixation with an osmium tetroxide-potassium ferrocyanide solution revealed in supporting cells in the organ of Corti a network of canaliculi termed canalicular reticulum (CR). In Deiters cells (DCs), the CR filled cytosol at the base of the phalanx and under plasmalemma apposed to either the outer hair cells' (HCs) basal surface or nerve terminals. From these locations the CR, accompanied by dense fibrillar substance, descended along microtubule bundles and terminated by surrounding the rosette complex in the apical cytosol. Canalicular profiles protruding from the reticulum penetrated the loose meshwork comprising the periphery of the rosette complex to contact at intervals branches of the dense trabeculae that make up the core of the complex. This arrangement disclosed a structural and presumably functional relationship between outer HCs and the CR and rosette complex. Inner pillar cells (PCs) exhibited moderately abundant to sparse profiles of CR interspersed between microtubule bundles of the microtubule stalk that connected head and foot regions. More elaborate CR extended as a network upward from the top of the microtubule stalk part way into the head body and downward into a conical expansion of the stalk at the base of the cell. Cytosol on the medial side of the basal microtubule expansion contained abundant CR which in conjunction with CR between basal microtubule bundles lay situated for possible uptake of ions or neurotransmitter released from numerous adjoining nerves. CR in outer PCs resembled that in inner PCs but appeared less prevalent in the head and foot regions and did not occur in cytosol beside the basal microtubule stalk. Characteristically small Golgi complexes accompanied the reticulum in DCs and were prevalent in the upper regions but absent in the mid and lower part of inner PCs. Short cisternae in the Golgi stacks associated with CR contrasted with the lengthier cisternae in the complexes infrequently observed in cytosol outside the microtubule stalk of inner PCs.     

Mitochondrial volume density of Schwann cells associated with rat vestibular ganglion cells

The purpose of this study was to quantitate the mitochondrial volume density (MVD) within Schwann cells associated with vestibular ganglion cells in the female Wistar rat. Results show that this type of Schwann cell (SC) has a significantly higher MVD (19.4% +/- 1.9) than that reported for myelinating SC of peripheral nerve (1-5%). This large difference in SC MVD may be related to the energy requirements needed to maintain the local ion homeostasis around the ganglion cells given the environmental differences created by the different barrier systems of these regions of the nervous system.     

Retinoic acid induces differentiation of cochlear neural progenitor cells into hair cells

IntroductionInner ear progenitor cells have the potential for multi-directional differentiation. Retinoic acid is an important requirement for the development of the inner ear. Blocking the Curtyr's retinoic acid signaling pathway can significantly reduce the number of hair cells. Therefore, we believe that retinoic acid may induce the regeneration of inner ear hair cells.ObjectiveTo investigate whether the cochlear neural progenitor cells maintain the characteristics of stem cells during recovery and subculture, whether retinoic acid can induce cochlear neural progenitor cells into hair cells in vitro, and whether retinoic acid promotes or inhibits the proliferation of cochlear neural progenitor cells during differentiation.MethodsCochlear neural progenitor cells were cultured and induced in DMEM/F12 + RA (10^?6 M) and then detected the expressions of hair cell markers (Math1 and MyosinVIIa) by immunofluorescence cytochemistry and realtime-polymerase chain reaction, and the proliferation of cochlear neural progenitor cells was detected by Brdu.ResultsThe nestin of cochlear neural progenitor cells was positively expressed. The ratios of Math1-positive cells in the control group and experimental group were 1.5% and 63%, respectively; the ratios of MyosinVIIa-positive cells in the control group and experimental group were 0.96% and 56%, respectively (p < 0.05). The ratios of Brdu⁺-labeled cells in retinoic acid group, group PBS, and group FBS were 20.6%, 29.9%, and 54.3%, respectively; however, the proliferation rate in the experimental group decreased.ConclusionRetinoic acid can promote cochlear neural progenitor cells to differentiate into the hair cells.     

Neural stem cells injected into the sound-damaged cochlea migrate throughout the cochlea and express markers of hair cells, supporting cells, and spiral ganglion cells

Most cases of hearing loss are caused by the death or dysfunction of one of the many cochlear cell types. We examined whether cells from a neural stem cell line could replace cochlear cell types lost after exposure to intense noise. For this purpose, we transplanted a clonal stem cell line into the scala tympani of sound damaged mice and guinea pigs. Utilizing morphological, protein expression and genetic criteria, stem cells were found with characteristics of both neural tissues (satellite, spiral ganglion, and Schwann cells) and cells of the organ of Corti (hair cells, supporting cells). Additionally, noise-exposed, stem cell-injected animals exhibited a small but significant increase in the number of satellite cells and Type I spiral ganglion neurons compared to non-injected noise-exposed animals. These results indicate that cells of this neural stem cell line migrate from the scala tympani to Rosenthal's canal and the organ of Corti. Moreover, they suggest that cells of this neural stem cell line may derive some information needed from the microenvironment of the cochlea to differentiate into replacement cells in the cochlea.     

The protective effect of olfactory ensheathing cells on post-injury spiral ganglion cells

Conclusions: Transplantation of OECs into the cochlea may protect and increase the survival of SGCs.

Objective: To investigate the protective effect of the transplantation of olfactory ensheathing cells (OECs) on injured spiral ganglion cells (SGCs) in rats.

Methods: OECs were transplanted into the cochlea in rats with SGCs that were injured by kanamycin sulfate (KM). An equal volume of D-Hanks was injected into the cochlea of control rats. Auditory brainstem responses (ABRs) were recorded from the rats in both groups to monitor changes in hearing thresholds. Immunofluorescence was employed to examine the density and morphology of SGCs to assess the ototoxic condition of the cochlea.

Results: There was no significant difference in the ABR threshold at each frequency between the control and experimental groups. Notably, in the experimental group, a number of Hoechst 3334-labeled nuclei were detected from the apex to the basal turn of the cochlea, demonstrating that the OECs were successfully transplanted and survived in the cochlea. In the experimental group, most of the SGCs were tightly arranged, and the nuclear membrane, chromatin, and nucleolus were all clear. The SGCs in the control group were loosely arranged, and only a few normal SGCs were observed in this group.