人胚胎脊髓发育过程中NTs家族及其受体的表达及变化

发布时间：2018-02-10 20:33

  本文关键词： 神经营养素 受体 人胚胎 脊髓 发育　出处：《昆明医学院》2007年博士论文　论文类型：学位论文

【摘要】： 目的探讨神经营养素家族及其受体与人胚胎脊髓发育的关系。 方法应用免疫组织化学、原位杂交、蛋白质印迹和逆转录酶多聚酶链反应等方法系统研究神经营养素家族及其受体在人胚胎3w～8m脊髓内的表达及变化。 结果1．NGF蛋白在3w末和4w的神经管上皮中已有表达，在5～7w，随胚胎发育在套层、基板、翼板的神经细胞前体表达，阳性反应随胚龄增加。8～9w，套层大量的阳性细胞，翼板较基板更为密集。逐渐出现分化中的各种形态的成神经细胞。3m后，阳性细胞密度下降，但灰质内可见形态逐渐趋于成熟的神经细胞，数量逐渐增加。6m后脊髓形态趋于成体化，灰质内阳性神经细胞的形态更趋典型。6w时边缘层可见阳性纤维，3m时白质内可见少量阳性胶质细胞，6m时增多。通常胶质细胞的分化晚于神经细胞，白质内阳性纤维的出现早于胶质细胞；Western blotting蛋白定量分析显示：6～8w，蛋白含量随胚龄逐渐增加，8、9w时达到高峰(P＜0.05)，3m后下降(P＜0.05)，6m后又有所增加，并保持相对平稳。蛋白含量测定的结果与免疫组化定位表达的变化相吻合；原位杂交显示NGFmRNA在4w的神经管上皮呈弱阳性，晚于蛋白表达出现的时间。之后随脊髓的发育，阳性表达增加，至8w、9w时，可见分化中各种形态的成神经细胞，3m时，细胞密度下降。至4m时，可见阳性的多极神经元。6～8m，阳性神经细胞形态更趋成熟。白质内纤维和胶质细胞的阳性反应晚于神经细胞，表达模式与NGF蛋白的相似；RT-PCR的结果显示，NGFmRNA的表达在6～8w是逐渐上调的，8w时达到峰值(P＜0.05)，从第9w后表达下调，在第4m时下调到最低值(P＜0.05)，以后又逐渐上调。NGFmRNA含量测定的结果与原位杂交定位表达的变化相吻合；2．TrkA蛋白在3w末4w的神经管上皮、内界膜表达阳性，5～7w随神经管的发育相继在套层、基板、翼板的部分细胞胞浆表达，8～9w，套层阳性细胞密集，翼板较基板更多，细胞核膜和核仁明显，逐渐出现分化中的各种形态的阳性成神经细胞。10w～3m，多突起的大神经元逐渐增多，白质内胶质细胞阳性。4～5m，背角、腹角内较多阳性神经元，白质内可见阳性胶质细胞和纤维。6m后趋于成熟；Western blotting显示：TrkA蛋白在6w已有一定的含量，但比较低，6w到3m之间含量缓慢增加，4m时明显增加(P＜0.05)，5m，含量有所下降，7m略有升高。与免疫组化定位表达的变化基本吻合：原位杂交显示TrkAmRNA在4w的神经管上皮呈弱阳性，晚于蛋白表达出现的时间。之后相继在套层、基板、翼板表达，阳性反应逐渐增加，阳性神经细胞从以幼稚的无极成神经细胞为主转为以多种形态为主，9w可见少数多极成神经细胞，3～4m逐渐增多，并渐趋成熟。表达模式与TrkA蛋白的相似：RT-PCR的结果显示：9w起可检测到TrkAmRNA的表达，3m时达到最高(P＜0.05)，从4m时开始下调，但幅度不大；3．BDNF蛋白在3w末4w的神经管内界膜、外界膜表达，阳性反应较强，随脊髓的发育，BDNF表达的部位与NGF的相似，但早期在神经上皮细胞突起的表达较为明显，晚期，在6m时除在灰质内见到各种形态的神经细胞外，还可见深染的白质纤维束；Western blotting显示：BDNF蛋白含量测定的结果与免疫组化定位表达的变化基本相吻合，变化曲线与NGF的相似；原位杂交显示BDNFmRNA在3w末4w的神经管上皮即有明显表达，表达模式与蛋白的相似，阳性反应较NGF强；BDNFmRNA含量测定的结果与原位杂交定位表达的变化相吻合；4．免疫组化显示TrkB蛋白在5w时的神经管内界膜、神经上皮和套层呈弱阳性反应，之后在发育中脊髓的表达部位与BDNF蛋白的相似，但阳性反应明显弱于BDNF；蛋白定量显示：TrkB从第6w至4m呈现逐渐上调的趋势，但未见明显上调的时间段，4m后下调，6m后略有上调，总体水平很低。与免疫组化定位表达的变化相吻合；原位杂交显示TrkBmRNA在5w的神经管有表达，表达模式与TrkB蛋白的表达相似。RT-PCR结果显示，早期TrkBmRNA随发育逐渐上调，，8w时达到峰值，从9w开始表达下调，类似于BDNF的变化；5．NT-3蛋白的表达很具特征性，阳性表达以细胞突起和纤维最为突出。NT-3在第3、4w的神经管上皮即呈强阳性反应，各期细胞的阳性突起呈放射状，5～8w，神经上皮层及套层大量放射状纤维，8～11w，阳性神经细胞前体也较丰富。阳性反应出现的部位与NGF和BDNF相似；Westernblotting分析显示：NT-3从第6w开始即达一定水平，并且逐渐上升到第9周达峰值(P＜0.05)，之后下降并维持在一定水平，与定位表达的变化吻合；原位杂交显示NT-3mRNA在4w的神经管上皮呈弱阳性反应，晚于蛋白的表达，且阳性弱。表达模式与蛋白的相似，但在细胞突起内的表达不如蛋白的突出；RT-PCR的结果显示，NT-3mRNA的变化与定位的表达变化相吻合；6．免疫组化定位表达显示TrkC蛋白在3w末的神经管内界膜呈弱阳性反应，表达模式与NT-3相似，但在细胞突起内的表达不如NT-3那么突出，但较其它两种受体TrkA和TrkB的明显；Western blotting的测定结果与定位表达的变化相符；TrkCmRNA的定位表达：在4w的神经上皮部分细胞表达，5w起其表达部位与TrkC蛋白的相似，但阳性较弱；RT-PCR结果显示：TrkCmRNA含量的变化与定位表达的相符；7．NT-4蛋白在3w末的神经管上皮呈弱阳性反应，表达模式与NGF、BDNF相似，但6m时，腹角神经元非常明显，中间带、后角各板层均可见阳性神经元。白质的后索、外侧索比前索的阳性胶质细胞多：Western blotting结果显示：NT-4于第6w已存在，之后缓慢升高到第8w后快速增加，第9w达高峰(P＜0.05)，以后又缓慢下降，第5、6m在一定水平保持平稳。NT-4蛋白含量的变化基本与免疫组化显示的形态学变化一致；NT-4mRNA的定位表达于4w的神经管上皮可见，其表达模式与蛋白的相似；RT-PCR的实验结果显示，NT-4mRNA含量的变化基本与原位杂交显示的形态学变化相似；8．在检测时段内，神经管上皮不同细胞周期的室细胞或室管膜上皮细胞始终有部分表达各种因子。 结论1．神经营养素家族各因子(NGF、BDNF、NT-3、NT-4)广泛分布于人胚胎脊髓发育各个时期的各种结构中，并且在早期脊髓的表达更强，各因子的表达既有重叠又有差异，提示神经营养素家族在人胚胎脊髓发育的各个阶段特别是胚期脊髓的发育中发挥着重要的作用，但在不同的区域和细胞又各自发挥着不同的作用；2．神经营养素家族各因子的mRNA广泛地存在于各个发育时期神经细胞和胶质细胞中，提示胚胎发育时期，神经管或脊髓的神经细胞和胶质细胞具有自身合成神经营养素的功能；3．神经营养素家族的高亲和受体广泛分布在人胚胎发育的各个时期的神经细胞和胶质细胞，提示人胚胎脊髓发育时期神经营养素家族各因子还可通过自分泌和旁分泌方式发挥其各种生理功能；4．神经营养素家族可能具有诱导脊髓室管膜上皮神经干细胞分裂增殖的能力。
[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.

【学位授予单位】：昆明医学院
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R321

【引证文献】

相关期刊论文 前1条

1 李力燕;;神经营养因子前体的研究[J];昆明医科大学学报;2013年05期

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