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  本文關鍵詞： 神經營養(yǎng)素 受體 人胚胎 脊髓 發(fā)育　出處：《昆明醫(yī)學院》2007年博士論文　論文類型：學位論文

【摘要】： 目的探討神經營養(yǎng)素家族及其受體與人胚胎脊髓發(fā)育的關系。 方法應用免疫組織化學、原位雜交、蛋白質印跡和逆轉錄酶多聚酶鏈反應等方法系統(tǒng)研究神經營養(yǎng)素家族及其受體在人胚胎3w～8m脊髓內的表達及變化。 結果1．NGF蛋白在3w末和4w的神經管上皮中已有表達，在5～7w，隨胚胎發(fā)育在套層、基板、翼板的神經細胞前體表達，陽性反應隨胚齡增加。8～9w，套層大量的陽性細胞，翼板較基板更為密集。逐漸出現(xiàn)分化中的各種形態(tài)的成神經細胞。3m后，陽性細胞密度下降，但灰質內可見形態(tài)逐漸趨于成熟的神經細胞，數(shù)量逐漸增加。6m后脊髓形態(tài)趨于成體化，灰質內陽性神經細胞的形態(tài)更趨典型。6w時邊緣層可見陽性纖維，3m時白質內可見少量陽性膠質細胞，6m時增多。通常膠質細胞的分化晚于神經細胞，白質內陽性纖維的出現(xiàn)早于膠質細胞；Western blotting蛋白定量分析顯示：6～8w，蛋白含量隨胚齡逐漸增加，8、9w時達到高峰(P＜0.05)，3m后下降(P＜0.05)，6m后又有所增加，并保持相對平穩(wěn)。蛋白含量測定的結果與免疫組化定位表達的變化相吻合；原位雜交顯示NGFmRNA在4w的神經管上皮呈弱陽性，晚于蛋白表達出現(xiàn)的時間。之后隨脊髓的發(fā)育，陽性表達增加，至8w、9w時，可見分化中各種形態(tài)的成神經細胞，3m時，細胞密度下降。至4m時，可見陽性的多極神經元。6～8m，陽性神經細胞形態(tài)更趨成熟。白質內纖維和膠質細胞的陽性反應晚于神經細胞，表達模式與NGF蛋白的相似；RT-PCR的結果顯示，NGFmRNA的表達在6～8w是逐漸上調的，8w時達到峰值(P＜0.05)，從第9w后表達下調，在第4m時下調到最低值(P＜0.05)，以后又逐漸上調。NGFmRNA含量測定的結果與原位雜交定位表達的變化相吻合；2．TrkA蛋白在3w末4w的神經管上皮、內界膜表達陽性，5～7w隨神經管的發(fā)育相繼在套層、基板、翼板的部分細胞胞漿表達，8～9w，套層陽性細胞密集，翼板較基板更多，細胞核膜和核仁明顯，逐漸出現(xiàn)分化中的各種形態(tài)的陽性成神經細胞。10w～3m，多突起的大神經元逐漸增多，白質內膠質細胞陽性。4～5m，背角、腹角內較多陽性神經元，白質內可見陽性膠質細胞和纖維。6m后趨于成熟；Western blotting顯示：TrkA蛋白在6w已有一定的含量，但比較低，6w到3m之間含量緩慢增加，4m時明顯增加(P＜0.05)，5m，含量有所下降，7m略有升高。與免疫組化定位表達的變化基本吻合：原位雜交顯示TrkAmRNA在4w的神經管上皮呈弱陽性，晚于蛋白表達出現(xiàn)的時間。之后相繼在套層、基板、翼板表達，陽性反應逐漸增加，陽性神經細胞從以幼稚的無極成神經細胞為主轉為以多種形態(tài)為主，9w可見少數(shù)多極成神經細胞，3～4m逐漸增多，并漸趨成熟。表達模式與TrkA蛋白的相似：RT-PCR的結果顯示：9w起可檢測到TrkAmRNA的表達，3m時達到最高(P＜0.05)，從4m時開始下調，但幅度不大；3．BDNF蛋白在3w末4w的神經管內界膜、外界膜表達，陽性反應較強，隨脊髓的發(fā)育，BDNF表達的部位與NGF的相似，但早期在神經上皮細胞突起的表達較為明顯，晚期，在6m時除在灰質內見到各種形態(tài)的神經細胞外，還可見深染的白質纖維束；Western blotting顯示：BDNF蛋白含量測定的結果與免疫組化定位表達的變化基本相吻合，變化曲線與NGF的相似；原位雜交顯示BDNFmRNA在3w末4w的神經管上皮即有明顯表達，表達模式與蛋白的相似，陽性反應較NGF強；BDNFmRNA含量測定的結果與原位雜交定位表達的變化相吻合；4．免疫組化顯示TrkB蛋白在5w時的神經管內界膜、神經上皮和套層呈弱陽性反應，之后在發(fā)育中脊髓的表達部位與BDNF蛋白的相似，但陽性反應明顯弱于BDNF；蛋白定量顯示：TrkB從第6w至4m呈現(xiàn)逐漸上調的趨勢，但未見明顯上調的時間段，4m后下調，6m后略有上調，總體水平很低。與免疫組化定位表達的變化相吻合；原位雜交顯示TrkBmRNA在5w的神經管有表達，表達模式與TrkB蛋白的表達相似。RT-PCR結果顯示，早期TrkBmRNA隨發(fā)育逐漸上調，，8w時達到峰值，從9w開始表達下調，類似于BDNF的變化；5．NT-3蛋白的表達很具特征性，陽性表達以細胞突起和纖維最為突出。NT-3在第3、4w的神經管上皮即呈強陽性反應，各期細胞的陽性突起呈放射狀，5～8w，神經上皮層及套層大量放射狀纖維，8～11w，陽性神經細胞前體也較豐富。陽性反應出現(xiàn)的部位與NGF和BDNF相似；Westernblotting分析顯示：NT-3從第6w開始即達一定水平，并且逐漸上升到第9周達峰值(P＜0.05)，之后下降并維持在一定水平，與定位表達的變化吻合；原位雜交顯示NT-3mRNA在4w的神經管上皮呈弱陽性反應，晚于蛋白的表達，且陽性弱。表達模式與蛋白的相似，但在細胞突起內的表達不如蛋白的突出；RT-PCR的結果顯示，NT-3mRNA的變化與定位的表達變化相吻合；6．免疫組化定位表達顯示TrkC蛋白在3w末的神經管內界膜呈弱陽性反應，表達模式與NT-3相似，但在細胞突起內的表達不如NT-3那么突出，但較其它兩種受體TrkA和TrkB的明顯；Western blotting的測定結果與定位表達的變化相符；TrkCmRNA的定位表達：在4w的神經上皮部分細胞表達，5w起其表達部位與TrkC蛋白的相似，但陽性較弱；RT-PCR結果顯示：TrkCmRNA含量的變化與定位表達的相符；7．NT-4蛋白在3w末的神經管上皮呈弱陽性反應，表達模式與NGF、BDNF相似，但6m時，腹角神經元非常明顯，中間帶、后角各板層均可見陽性神經元。白質的后索、外側索比前索的陽性膠質細胞多：Western blotting結果顯示：NT-4于第6w已存在，之后緩慢升高到第8w后快速增加，第9w達高峰(P＜0.05)，以后又緩慢下降，第5、6m在一定水平保持平穩(wěn)。NT-4蛋白含量的變化基本與免疫組化顯示的形態(tài)學變化一致；NT-4mRNA的定位表達于4w的神經管上皮可見，其表達模式與蛋白的相似；RT-PCR的實驗結果顯示，NT-4mRNA含量的變化基本與原位雜交顯示的形態(tài)學變化相似；8．在檢測時段內，神經管上皮不同細胞周期的室細胞或室管膜上皮細胞始終有部分表達各種因子。 結論1．神經營養(yǎng)素家族各因子(NGF、BDNF、NT-3、NT-4)廣泛分布于人胚胎脊髓發(fā)育各個時期的各種結構中，并且在早期脊髓的表達更強，各因子的表達既有重疊又有差異，提示神經營養(yǎng)素家族在人胚胎脊髓發(fā)育的各個階段特別是胚期脊髓的發(fā)育中發(fā)揮著重要的作用，但在不同的區(qū)域和細胞又各自發(fā)揮著不同的作用；2．神經營養(yǎng)素家族各因子的mRNA廣泛地存在于各個發(fā)育時期神經細胞和膠質細胞中，提示胚胎發(fā)育時期，神經管或脊髓的神經細胞和膠質細胞具有自身合成神經營養(yǎng)素的功能；3．神經營養(yǎng)素家族的高親和受體廣泛分布在人胚胎發(fā)育的各個時期的神經細胞和膠質細胞，提示人胚胎脊髓發(fā)育時期神經營養(yǎng)素家族各因子還可通過自分泌和旁分泌方式發(fā)揮其各種生理功能；4．神經營養(yǎng)素家族可能具有誘導脊髓室管膜上皮神經干細胞分裂增殖的能力。
[Abstract]:Objective to investigate the relationship between the neurotrophin family and its receptor and the development of human embryonic spinal cord.
Methods immunohistochemistry, in situ hybridization, Western blot and reverse transcriptase polymerase chain reaction were used to study the expression and change of neurotrophin family and its receptor in 3W ~ 8m spinal cord of human embryo.
The results of 1.NGF protein in 3W and 4W at the end of the neural tube expression has epithelium, in 5 ~ 7W, with the development of embryos in the substrate layer, and the expression of wing nerve cells, the positive reaction with embryonic age increased.8 ~ 9W, set a layer of positive cells, a more intensive wing plate substrate. Gradually,.3m nerve cell differentiation in a variety of forms at the post, the density of positive cells decreased, but the gray matter seen in shape gradually mature nerve cells, a gradual increase in the number of.6m after spinal cord morphology more adult, more typical morphology of.6w positive cells in the gray matter at the edge layer visible positive fibers, 3M white a small amount of cytoplasm positive glial cells, 6m increased. Usually glial differentiation was later than nerve cell cells, appear in the white matter fibers were earlier than glial cells; Western blotting protein quantitative analysis showed: 6 ~ 8W, the protein content increased gradually with embryonic age, Reached the peak at 8,9w (P < 0.05), 3M decreased (P < 0.05), 6m increase again, and remained relatively stable. The change of protein content determination results and immunohistochemical localization of expression is consistent; in situ hybridization showed that NGFmRNA 4W in the neural tube epithelium showed weak positive expression appeared later than. Time. With the development of spinal cord, positive expression increased to 8W, 9W, neural cells, a variety of forms visible differentiation in 3M, cell density decreased. To 4m,.6 to visible multipolar neurons positive 8m positive nerve cell morphology is more mature. The positive reaction after nerve cells in the white matter fiber and glial cells, and the expression pattern of NGF protein was similar; RT-PCR showed that the expression of NGFmRNA in 6 ~ 8W is gradually increased, reached peak at 8W (P < 0.05), from the 9W expression at 4m, down to the lowest value (P < 0.05), after gradually Determination of.NGFmRNA content increase coincided with the results of changes in expression and in situ localization; 2.TrkA protein in 3W 4W at the end of the tube epithelial nerve, internal limiting membrane expression, 5 ~ 7W with neural tube development have been set in the layer, substrate, expression of cytoplasmic wing, 8 ~ 9W, a layer of positive the cell density, more than the wing plate substrate, nuclear membrane and nucleolus cells, nerve cells appeared.10w ~ 3M positive differentiation in a variety of forms, many processes of the large neurons gradually increased, positive glial cells in the white matter of.4 ~ 5m, dorsal horn neurons in the ventral horn, more positive, seen in the white matter glial cells and fibers after.6m mature; Western blotting showed that TrkA protein content in some existing 6W, but relatively low, 6W to 3m content slowly increased, 4m increased significantly (P < 0.05), 5m content decreased, 7m slightly increased. Immunohistochemistry and fixed Expression changes basically: in situ hybridization showed that TrkAmRNA 4W in the neural tube epithelium showed weak positive expression, later than the time it appeared. In succession after coating substrate, the wing plate expression, the positive reaction increased gradually, the positive cells from immature apolar neuroblast mainly to various forms. 9W, showing a few multipolar neuroblast, 3 ~ 4m gradually increased, and gradually mature. The expression pattern of TrkA protein and the similarity. The RT-PCR result showed that 9W can detect the expression of TrkAmRNA and 3M reached the maximum (P < 0.05), from 4m began to cut, but modest; 3.BDNF protein 3W 4W at the end of the neural tube internal limiting membrane, external membrane expression, strong positive reaction, with the development of spinal cord, and the expression of BDNF in NGF site is similar, but the early expression in neuroepithelial cell. Obviously, the late in the 6m except when seen in the gray matter Various types of neural cells, white matter fibers are visible hyperchromatic; Western blotting showed that the change of BDNF protein content determination results and immunohistochemical localization of expression is coincident with that curve is similar with NGF; in situ hybridization showed that BDNFmRNA in the 3W 4W at the end of the neural tube that is obviously the expression of epithelial expression. Pattern and protein is similar to that of the positive reaction was stronger than NGF BDNFmRNA; content determination results coincide with changes in expression and in situ localization; 4. immunohistochemistry showed that TrkB protein in 5W neural tube with internal limiting membrane, nerve and epithelial layer showed weak positive reaction after the expression site of the spinal cord during development and BDNF protein is similar, but the positive reaction was significantly weaker than that of BDNF; protein showed that TrkB from 6W to 4m showed a gradual upward trend, but the time had no obvious increase, 4m down, 6m after a slight increase, the total body water The flat is very low. The expression changes and immunohistochemical localization coincide; in situ hybridization showed that TrkBmRNA 5W expression in the neural tube, the expression of TrkB protein is similar to the.RT-PCR model and the results show that with the development of early TrkBmRNA increased gradually, reached peak at 8W, starting from 9W expression, changes similar to BDNF; the expression of 5.NT-3 protein was characteristic, positive expression of the cell processes and fiber is the most prominent.NT-3 in section 3,4w of the neural tube epithelial is strongly positive, positive cells in each phase of the protruding radially, 5 ~ 8W, neural epithelial layer and the cover layer large amount of radial fiber, 8 ~ 11W, positive neural precursor cells are more abundant. Parts of the positive reaction with NGF and BDNF; Westernblotting analysis showed that NT-3 reached a certain level at 6W, and gradually increased to ninth weeks and reached a peak (P < 0.05), then decreased and maintained at a certain The level of agreement change and localization expression; in situ hybridization showed that NT-3mRNA 4W in the neural tube epithelium was weakly positive, and the positive expression was later than that of protein, protein expression pattern and weak. Similar, but in the cell. The expression of RT-PCR protein as prominent; the result indicated that the expression and localization of NT-3mRNA the 6. match; immunohistochemical localization showed that TrkC protein expression was weakly positive in 3W at the end of the neural tube internal limiting membrane, and the expression pattern was similar to that of NT-3, but the expression of NT-3 in cell processes not so prominent, but compared with the other two by TrkA and TrkB. The test results of the Western blotting; change. And localization of expression; the expression of TrkCmRNA expression in cells of neuroepithelial positioning: part of 4W, 5W and the expression site of TrkC protein was similar, but weaker positive; RT-PCR results showed that the content of TrkCmRNA Consistent expression and localization of 7.NT-4 protein; epithelium showed weak positive reaction in 3W at the end of the nerve, the expression pattern of NGF, similar to BDNF, but 6m, ventral horn neurons is very obvious, the middle zone, the plate angle layer showed positive neurons. White matter after cable, cable positive lateral soapy before glial cells: Western blotting results showed that NT-4 in the 6W already exists, then slowly increased to the rapid increase of the 8W, the peak at 9W (P < 0.05), then decreased slowly, the change of 5,6m.NT-4 protein content remained stable at a certain level and the basic immune group morphological changes consistent with display the localization of NT-4mRNA expression in 4W; the neural epithelium, and its expression pattern is similar to the RT-PCR protein; experimental results show that the change of NT-4mRNA content was similar with in situ hybridization showed morphological changes; 8. in the detection period of nerve The cell or ependymal epithelial cells with different cell cycles in the tube have always expressed a variety of factors.
Conclusion the 1. neurotrophins family factors (NGF, BDNF, NT-3, NT-4) of various structures are widely distributed in the human embryonic spinal cord development in different periods, and the stronger expression of early spinal cord, expression of each factor both overlap and differences, suggesting that neurotrophins in various stages of human embryonic spinal cord development especially is playing an important role in the development of embryonic spinal cord, but in different regions and cells play different roles; the 2. neurotrophins family factors mRNA widely exist in various developmental stages of nerve cells and glial cells, suggesting that during embryonic development, neural cells and glial cells or neural tube the spinal cord has the function of self synthesis of neurotrophin 3.; neurotrophin family of high affinity receptors are widely distributed in nerve cells and glial cells in each period of embryonic development, presenting Neurotrophin family factors can also exert various physiological functions through the autocrine and paracrine mode during the development of human embryonic spinal cord. 4., the neurotrophin family may have the ability to induce the division and proliferation of neural stem cells in the ependymal epithelium of spinal cord.

【學位授予單位】：昆明醫(yī)學院
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R321

【引證文獻】

相關期刊論文 前1條

1 李力燕;;神經營養(yǎng)因子前體的研究[J];昆明醫(yī)科大學學報;2013年05期

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