紫杉醇和神经生长因子在嗅感觉神经元分化过程中的作用

发布时间：2018-05-12 20:13

本文选题：嗅感觉神经元 + β3微管蛋白　；参考：《中国医科大学》2010年博士论文

【摘要】： 紫杉醇和神经生长因子在嗅感觉神经元分化过程中的作用目的微竹作为细胞骨架的重要组成部分,与细胞形态的维持、细胞内物质的运输、细胞运动及细胞分裂增殖等生命活动密切相关。通常认为,有丝分裂过程中需要胞质微管的解聚以形成纺锤体微管,染色体在纺锤体微管的牵引下方能向两极移动,有丝分裂后,这些微管又重新解聚成微管蛋白。β3微管蛋白特异性地存在于神经细胞、滋养细胞中,在神经发生早期即有表达的唯一微管异构形式而常用于标志于细胞的神经分化,在成熟及未成熟的嗅感觉神经元中都有表达。紫杉醇通过与β3微管蛋白N端的31个氨基酸残基和中段的217-231氨基酸残基结合,增加组成微管的13根原丝的相互作用,稳定微管蛋白构象,从而抑制微管解聚,使细胞复制中止在M期,进而引起细胞凋亡。嗅感觉神经元是唯一直接与外界环境相接触,并终身具有再生能力的双极神经元,其特有的再生和分化能力。它的重要意义在于使嗅觉功能的治疗和恢复以及中枢神经系统的移植修复成为可能。本研究通过紫杉醇处理孕鼠,应用免疫荧光、透射电镜和实时荧光定量PCR(real-time quantitative polymerase chain reaction, RT-Q-PCR)方法检测β3微管蛋白或嗅标记蛋白(olfactory marker protein,OMP)的表达变化,探讨紫杉醇通过抑制微管蛋白的表达在嗅感觉神经元分化过程中的作用；并通过用神经生长因子(nerve growth factory, NGF)处理孕鼠,初步探讨其对嗅觉的保护作用。材料和方法 SPF级小鼠,以雄：雌1:2比例合笼,取查看有阴栓雌鼠30只。随机分为两组,实验组和对照组各15只。实验组给予E0.5天起每日尾静脉注射紫杉醇1ml(浓度为10nmol/L),对照组每日给予生理盐水1ml尾静脉注射。每组再分成E9.5天、E14.5天及E17.5天孕鼠各5只。应用免疫组化、免疫荧光和RT-Q-PCR方法检测β3微管蛋白或嗅标记蛋白的表达变化；应用透射电镜观察嗅细胞形态学变化。应用免疫荧光和RT-Q-PCR方法观察紫杉醇及NGF孕期给药后小鼠嗅黏膜中嗅标记蛋白的表达变化。结果免疫荧光结果显示,在E9.5天嗅基板刚开始发育时可以见到β3的特异性染色,随着孕期的延长,β3微管蛋白的特异性染色明显增多。在E9.5天未见OMP阳性表达,E14.5天可见OMP阳性表达,表达强度小于同期β3微管蛋白,随着孕期的延长,OMP的特异性染色明显增多。紫杉醇处理后,β3微管蛋白和OMP的表达明显弱于正常对照组,但随着孕期的延长,β3微管蛋白和OMP的表达也是逐渐增多。RT-Q-PCR检测的OMP表达结果与免疫荧光结果相同。透射电镜显示紫杉醇处理后,小鼠嗅细胞内结构紊乱,嗅纤毛减少或消失,成空泡样改变。小鼠嗅黏膜免疫荧光染色结果显示紫杉醇处理组β3微管蛋白和OMP表达减少,而NGF给药后,两者表达有所增强,和RT-Q-PCR结果相同。讨论微管作为细胞骨架的重要组成部分,与细胞形态的维持、细胞内物质的运输、细胞运动及细胞分裂增殖等生命活动密切相关。目前在真核生物中已经确定的微管蛋白有7种,分别称为α、β、γ、δ、ε、ζ和η微管蛋白。β微管蛋白由455个氨基酸残基组成。根据C末端的10-15个氨基酸残基不同将β微管蛋白分为7种β同型(β1、β2、β3、β4a、β4b、β5、β6)。这些β微管蛋白同型同属于一个高度同源的蛋白家族,其中β3微管蛋白是特异性酪氨酸磷酸化,在神经发生早期即有表达的唯一微管异构形式而常用于标志干细胞的神经分化。β3微管蛋白的表达起始于神经母细胞有丝分裂中期或末期,在成熟及未成熟的嗅感觉神经元中都有表达。嗅感觉神经元位于嗅上皮的中间层,构成上皮的绝大部分,是气味识别的起始结构,顶层为单层的支持细胞,底层是靠近基底膜的基底细胞,分为2种亚型：水平基底细胞和球形基底细胞。本实验采用常规化疗剂量的紫杉醇和常规治疗剂量的NGF处理孕鼠,干扰微管蛋白的形成,探讨其在嗅感觉神经元的分化过程中的作用。结果显示,在E9.5天嗅基板刚开始发育时可以见到β3的表达,但在E9.5天未见OMP阳性表达,提示嗅感觉神经元前体在嗅基板发育早期即开始形成,但未形成成熟细胞。随着孕期的延长,β3微管蛋白的表达明显增多。E14.5天可见OMP阳性表达,提示胚胎中期出现成熟的嗅感觉神经元,但其表达强度小于同期β3微管蛋白,提示此时期还有一定量的前体细胞存在。随着孕期的延长,OMP的表达明显增多,提示嗅感觉神经元随着胚胎的逐渐成长,不断分化成熟。紫杉醇处理后,β3微管蛋白和OMP的表达明显弱于正常对照组,提示紫杉醇通过抑制微管蛋白的解聚,使微管束不能与微管组织中心相互连接,抑制纺锤体的正常形成,由此抑制了神经元的轴浆运输,阻碍了嗅感觉神经元的发育成熟。但随着孕期的延长,β3微管蛋白和OMP的表达也是逐渐增多,说明紫杉醇并不能完全使嗅感觉神经元分化和发育停滞。NGF通过促进神经系统的发育,维持神经元的生长、存活、分化,并影响突触的可塑性。病理状态下,通过诱导神经生长因子基因的表达,抑制神经元的死亡加强自由基的清除,促进神经元功能的恢复,达到保护神经元的目的。实验结果显示NGF给药后紫杉醇处理组β3微管蛋白和OMP表达有所增加,提示NGF对紫杉醇导致的嗅觉功能障碍有一定的保护作用。结论 1、β3微管蛋白在小鼠胚胎发育早期就有表达,随着胚胎的发育成熟,其的表达明显增强。 2、嗅感觉神经元在小鼠胚胎发育早期无表达,在胚胎中期出现表达,且随着胚胎的发育成熟,其表达明显增强。 3、紫杉醇通过作用于微管蛋白,抑制嗅感觉神经元的分化,阻碍其发育,但不能使发育完全停滞。 4、神经生长因子对紫杉醇所致的小鼠嗅感觉神经元损伤有一定的保护作用。
[Abstract]:The role of paclitaxel and nerve growth factor in the differentiation of olfactory sensory neurons
objective
As an important part of the cytoskeleton, microbamboo is closely related to the maintenance of cell morphology, the transport of substances in cells, cell movement and cell division and proliferation. It is generally believed that the depolymerization of the cytoplasmic microtubules is needed to form a spindle microtubule during mitosis, and the chromosomes can be directed to the poles under the traction of the spindle microtubule. After mitosis, these microtubules are again depolymerize into tubulin. Beta 3 microtubulin exists specifically in nerve cells. In the trophoblastic cells, the only microtubule isomerization that is expressed in the early stage of neurogenesis is often used to mark the differentiation of neurons in cells, and is expressed in mature and immature olfactory sensory neurons. By combining the 31 amino acid residues at the N end of the beta 3 microtubulin and the 217-231 amino acid residues in the middle section, alcohol increases the interaction between the 13 precursors of the microtubule and stabilizes the conformation of microtubules, thus inhibiting the depolymerization of microtubules, making the cell replication terminating in the M phase and causing the cell death.
The olfactory sensory neurons are the only bipolar neurons that have direct contact with the external environment and have the ability to regenerate for life, their unique ability to regenerate and differentiate. Its important significance lies in the possibility of the treatment and recovery of the olfactory function and the transplantation of the central nervous system.
In this study, the expression of beta 3 microtubulin or olfactory labeled protein (olfactory marker protein, OMP) was detected by taxol treatment in pregnant mice by immunofluorescence, transmission electron microscopy and real-time fluorescent quantitative PCR (RT-Q-PCR polymerase chain reaction, RT-Q-PCR). The expression of paclitaxel was investigated by inhibiting the expression of microtubule protein in the olfactory. The role of the sensory neurons in the differentiation process and the treatment of pregnant rats by using nerve growth factory (NGF) and their protective effects on the olfactory sense are preliminarily discussed.
Materials and methods
SPF mice, male: female 1:2 ratio cage, 30 female rats with negative suppository were randomly divided into two groups, the experimental group and the control group were 15. The experimental group was given E0.5 day tail vein injection of paclitaxel 1ml (10nmol/L), and the control group was given a daily saline 1ml tail vein injection. Each group was then divided into E9.5 days, E14.5 days and E17.5 days pregnant rats. The changes in the expression of beta 3 microtubulin or olfactory labeling protein were detected by immunohistochemistry, immunofluorescence and RT-Q-PCR, and the morphological changes of olfactory cells were observed by transmission electron microscopy. The changes in the expression of olfactory labelling protein in the olfactory mucosa of the mice after paclitaxel and NGF were observed by immunofluorescence and RT-Q-PCR.
Result
The immunofluorescence results showed that the specific staining of beta 3 could be seen when the olfactory base was first developed on E9.5 days. With the prolongation of pregnancy, the specific staining of beta 3 microtubule increased obviously. No OMP positive expression was found on E9.5 days, OMP positive expression was found on E14.5 days, and the expression intensity was less than that of beta 3 microtubulin. With the prolongation of pregnancy, OMP was special. The expression of heterosexual staining was significantly increased. After paclitaxel treatment, the expression of beta 3 microtubulin and OMP was significantly weaker than that in the normal control group, but with the prolongation of pregnancy, the expression of beta 3 microtubule protein and OMP was gradually increased by.RT-Q-PCR detection, and the result of OMP expression was the same as that of immunofluorescence. Transmission electron microscopy showed that the structure of olfactory cells in mice after paclitaxel treatment The results of olfactory mucous membrane immunofluorescence staining showed that the expression of beta 3 microtubulin and OMP in the paclitaxel treatment group decreased, and the expression of the two groups was enhanced after NGF administration, and the results were the same as that of RT-Q-PCR.
discuss
Microtubules, as an important part of the cytoskeleton, are closely related to the maintenance of cell morphology, the transport of substances in cells, cell movement and cell division and proliferation. At present, 7 kinds of tubulin have been identified in eukaryotes, called alpha, beta, gamma, Delta, epsilon, ETA and ETA microtubulin. The beta microtubulin is 455 amino acid residues. Based on the 10-15 amino acid residues of the C terminal, beta microtubulin is divided into 7 beta homotypes (beta 1, beta 2, beta 3, beta 4a, beta 4b, beta 5, beta 6). These beta microtubule homologous proteins belong to a highly homologous protein family, in which beta 3 microtubulin is specific tyrosine phosphorylation and is expressed at the early stage of neurogenesis.
The expression of a microtubule isomerism often used to mark the nerve differentiation of the stem cells. The expression of beta 3 microtubule begins at the metaphase or end of the mitosis of the neuroblastoma, and is expressed in the mature and immature olfactory sensory neurons. The olfactory sensory neurons are located in the middle layer of the olfactory epithelium, the vast majority of the epithelium, and the starting junctions of odor identification. The top layer is a single layer of supporting cells. The underlying cells are basal cells near the basement membrane. They are divided into 2 subtypes: horizontal basal cells and spherical basal cells.
In this experiment, pregnant rats were treated with paclitaxel and conventional therapeutic dose of NGF to interfere with the formation of microtubule protein and explore its role in the differentiation of olfactory sensory neurons. The results showed that the expression of beta 3 could be seen when the olfactory substrate was first developed on E9.5 days, but no OMP positive expression was found on E9.5 days, suggesting the olfactory sensation. The precursor was formed at the early stage of the olfactory base, but no mature cells were formed. With the prolongation of the pregnancy, the expression of beta 3 microtubule was obviously increased on.E14.5 days and OMP positive expression was visible, suggesting that the mature olfactory sensory neurons appeared in the middle of the embryo, but the expression intensity was less than that of the same phase beta 3 microtubule. The expression of OMP was significantly increased with the prolongation of pregnancy, suggesting that the olfactory sensory neurons gradually grow and mature with the gradual growth of the embryo. After paclitaxel treatment, the expression of beta 3 microtubulin and OMP is obviously weaker than that of the normal control group. It suggests that the microtubule can not be used in microtubule tissue by inhibiting the depolymerization of microtubule protein. The heart is interconnected to inhibit the normal formation of the spindle, which inhibits the axonal transport of the neurons and hinders the maturation of the olfactory sensory neurons. However, the expression of the beta 3 microtubulin and OMP is gradually increased with the prolongation of pregnancy, indicating that taxol does not completely cause the differentiation and development of the olfactory sensory neurons to stagnate.NGF through the promotion of nerve. The development of the system maintains the growth, survival, differentiation, and the plasticity of the synapses. Under the pathological state, the expression of the nerve growth factor gene is induced to inhibit the death of the neurons to strengthen the free radical scavenging, to promote the recovery of neuronal function and to protect the neuron. The experimental results show that NGF is given to paclitaxel after the drug is given. The expression of beta 3 tubulin and OMP increased, suggesting that NGF has a protective effect on olfactory dysfunction induced by paclitaxel.
conclusion
1, beta 3 tubulin was expressed in the early stage of mouse embryo development, and its expression increased significantly as the embryo developed and matured.
2, olfactory sensory neurons were not expressed in the early stage of mouse embryonic development, and were expressed in the middle stage of embryo development.
3, paclitaxel inhibits the development of the olfactory sensory neurons by inhibiting the differentiation of olfactory sensory neurons by acting on tubulin.
4, nerve growth factor has a protective effect on the injury of olfactory sensory neurons induced by taxol in mice.

【学位授予单位】：中国医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

【引证文献】