本文選題：嗅感覺神經(jīng)元 + β3微管蛋白��； 參考：《中國醫(yī)科大學(xué)》2010年博士論文

【摘要】： 紫杉醇和神經(jīng)生長因子在嗅感覺神經(jīng)元分化過程中的作用 目的 微竹作為細(xì)胞骨架的重要組成部分,與細(xì)胞形態(tài)的維持、細(xì)胞內(nèi)物質(zhì)的運(yùn)輸、細(xì)胞運(yùn)動(dòng)及細(xì)胞分裂增殖等生命活動(dòng)密切相關(guān)。通常認(rèn)為,有絲分裂過程中需要胞質(zhì)微管的解聚以形成紡錘體微管,染色體在紡錘體微管的牽引下方能向兩極移動(dòng),有絲分裂后,這些微管又重新解聚成微管蛋白。β3微管蛋白特異性地存在于神經(jīng)細(xì)胞、滋養(yǎng)細(xì)胞中,在神經(jīng)發(fā)生早期即有表達(dá)的唯一微管異構(gòu)形式而常用于標(biāo)志于細(xì)胞的神經(jīng)分化,在成熟及未成熟的嗅感覺神經(jīng)元中都有表達(dá)。紫杉醇通過與β3微管蛋白N端的31個(gè)氨基酸殘基和中段的217-231氨基酸殘基結(jié)合,增加組成微管的13根原絲的相互作用,穩(wěn)定微管蛋白構(gòu)象,從而抑制微管解聚,使細(xì)胞復(fù)制中止在M期,進(jìn)而引起細(xì)胞凋亡。 嗅感覺神經(jīng)元是唯一直接與外界環(huán)境相接觸,并終身具有再生能力的雙極神經(jīng)元,其特有的再生和分化能力。它的重要意義在于使嗅覺功能的治療和恢復(fù)以及中樞神經(jīng)系統(tǒng)的移植修復(fù)成為可能。 本研究通過紫杉醇處理孕鼠,應(yīng)用免疫熒光、透射電鏡和實(shí)時(shí)熒光定量PCR(real-time quantitative polymerase chain reaction, RT-Q-PCR)方法檢測β3微管蛋白或嗅標(biāo)記蛋白(olfactory marker protein,OMP)的表達(dá)變化,探討紫杉醇通過抑制微管蛋白的表達(dá)在嗅感覺神經(jīng)元分化過程中的作用；并通過用神經(jīng)生長因子(nerve growth factory, NGF)處理孕鼠,初步探討其對(duì)嗅覺的保護(hù)作用。 材料和方法 SPF級(jí)小鼠,以雄：雌1:2比例合籠,取查看有陰栓雌鼠30只。隨機(jī)分為兩組,實(shí)驗(yàn)組和對(duì)照組各15只。實(shí)驗(yàn)組給予E0.5天起每日尾靜脈注射紫杉醇1ml(濃度為10nmol/L),對(duì)照組每日給予生理鹽水1ml尾靜脈注射。每組再分成E9.5天、E14.5天及E17.5天孕鼠各5只。應(yīng)用免疫組化、免疫熒光和RT-Q-PCR方法檢測β3微管蛋白或嗅標(biāo)記蛋白的表達(dá)變化；應(yīng)用透射電鏡觀察嗅細(xì)胞形態(tài)學(xué)變化。應(yīng)用免疫熒光和RT-Q-PCR方法觀察紫杉醇及NGF孕期給藥后小鼠嗅黏膜中嗅標(biāo)記蛋白的表達(dá)變化。 結(jié)果 免疫熒光結(jié)果顯示,在E9.5天嗅基板剛開始發(fā)育時(shí)可以見到β3的特異性染色,隨著孕期的延長,β3微管蛋白的特異性染色明顯增多。在E9.5天未見OMP陽性表達(dá),E14.5天可見OMP陽性表達(dá),表達(dá)強(qiáng)度小于同期β3微管蛋白,隨著孕期的延長,OMP的特異性染色明顯增多。紫杉醇處理后,β3微管蛋白和OMP的表達(dá)明顯弱于正常對(duì)照組,但隨著孕期的延長,β3微管蛋白和OMP的表達(dá)也是逐漸增多。RT-Q-PCR檢測的OMP表達(dá)結(jié)果與免疫熒光結(jié)果相同。透射電鏡顯示紫杉醇處理后,小鼠嗅細(xì)胞內(nèi)結(jié)構(gòu)紊亂,嗅纖毛減少或消失,成空泡樣改變。小鼠嗅黏膜免疫熒光染色結(jié)果顯示紫杉醇處理組β3微管蛋白和OMP表達(dá)減少,而NGF給藥后,兩者表達(dá)有所增強(qiáng),和RT-Q-PCR結(jié)果相同。 討論 微管作為細(xì)胞骨架的重要組成部分,與細(xì)胞形態(tài)的維持、細(xì)胞內(nèi)物質(zhì)的運(yùn)輸、細(xì)胞運(yùn)動(dòng)及細(xì)胞分裂增殖等生命活動(dòng)密切相關(guān)。目前在真核生物中已經(jīng)確定的微管蛋白有7種,分別稱為α、β、γ、δ、ε、ζ和η微管蛋白。β微管蛋白由455個(gè)氨基酸殘基組成。根據(jù)C末端的10-15個(gè)氨基酸殘基不同將β微管蛋白分為7種β同型(β1、β2、β3、β4a、β4b、β5、β6)。這些β微管蛋白同型同屬于一個(gè)高度同源的蛋白家族,其中β3微管蛋白是特異性酪氨酸磷酸化,在神經(jīng)發(fā)生早期即有表達(dá)的唯 一微管異構(gòu)形式而常用于標(biāo)志干細(xì)胞的神經(jīng)分化。β3微管蛋白的表達(dá)起始于神經(jīng)母細(xì)胞有絲分裂中期或末期,在成熟及未成熟的嗅感覺神經(jīng)元中都有表達(dá)。嗅感覺神經(jīng)元位于嗅上皮的中間層,構(gòu)成上皮的絕大部分,是氣味識(shí)別的起始結(jié)構(gòu),頂層為單層的支持細(xì)胞,底層是靠近基底膜的基底細(xì)胞,分為2種亞型：水平基底細(xì)胞和球形基底細(xì)胞。 本實(shí)驗(yàn)采用常規(guī)化療劑量的紫杉醇和常規(guī)治療劑量的NGF處理孕鼠,干擾微管蛋白的形成,探討其在嗅感覺神經(jīng)元的分化過程中的作用。結(jié)果顯示,在E9.5天嗅基板剛開始發(fā)育時(shí)可以見到β3的表達(dá),但在E9.5天未見OMP陽性表達(dá),提示嗅感覺神經(jīng)元前體在嗅基板發(fā)育早期即開始形成,但未形成成熟細(xì)胞。隨著孕期的延長,β3微管蛋白的表達(dá)明顯增多。E14.5天可見OMP陽性表達(dá),提示胚胎中期出現(xiàn)成熟的嗅感覺神經(jīng)元,但其表達(dá)強(qiáng)度小于同期β3微管蛋白,提示此時(shí)期還有一定量的前體細(xì)胞存在。隨著孕期的延長,OMP的表達(dá)明顯增多,提示嗅感覺神經(jīng)元隨著胚胎的逐漸成長,不斷分化成熟。紫杉醇處理后,β3微管蛋白和OMP的表達(dá)明顯弱于正常對(duì)照組,提示紫杉醇通過抑制微管蛋白的解聚,使微管束不能與微管組織中心相互連接,抑制紡錘體的正常形成,由此抑制了神經(jīng)元的軸漿運(yùn)輸,阻礙了嗅感覺神經(jīng)元的發(fā)育成熟。但隨著孕期的延長,β3微管蛋白和OMP的表達(dá)也是逐漸增多,說明紫杉醇并不能完全使嗅感覺神經(jīng)元分化和發(fā)育停滯。NGF通過促進(jìn)神經(jīng)系統(tǒng)的發(fā)育,維持神經(jīng)元的生長、存活、分化,并影響突觸的可塑性。病理狀態(tài)下,通過誘導(dǎo)神經(jīng)生長因子基因的表達(dá),抑制神經(jīng)元的死亡加強(qiáng)自由基的清除,促進(jìn)神經(jīng)元功能的恢復(fù),達(dá)到保護(hù)神經(jīng)元的目的。實(shí)驗(yàn)結(jié)果顯示NGF給藥后紫杉醇處理組β3微管蛋白和OMP表達(dá)有所增加,提示NGF對(duì)紫杉醇導(dǎo)致的嗅覺功能障礙有一定的保護(hù)作用。 結(jié)論 1、β3微管蛋白在小鼠胚胎發(fā)育早期就有表達(dá),隨著胚胎的發(fā)育成熟,其的表達(dá)明顯增強(qiáng)。 2、嗅感覺神經(jīng)元在小鼠胚胎發(fā)育早期無表達(dá),在胚胎中期出現(xiàn)表達(dá),且隨著胚胎的發(fā)育成熟,其表達(dá)明顯增強(qiáng)。 3、紫杉醇通過作用于微管蛋白,抑制嗅感覺神經(jīng)元的分化,阻礙其發(fā)育,但不能使發(fā)育完全停滯。 4、神經(jīng)生長因子對(duì)紫杉醇所致的小鼠嗅感覺神經(jīng)元損傷有一定的保護(hù)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.

【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李榮榮;憨鑌;陳晨;霍介格;;紫杉醇致周圍神經(jīng)毒性研究進(jìn)展[J];遼寧中醫(yī)藥大學(xué)學(xué)報(bào);2014年06期

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本文編號(hào)：1879997