小脑间位核调节免疫功能的作用途径研究

发布时间：2018-07-04 07:11

  本文选题：小脑 + 间位核　； 参考：《苏州大学》2012年博士论文

【摘要】：目的： 我们实验室以往的研究表明小脑间位核可调节免疫功能。由于小脑与免疫系统之间没有直接的结构上的联系，因此阐明小脑调节免疫功能的信息传递途径对于更好地理解小脑的免疫调节功能具有重要作用。本研究以小脑—下丘脑的神经投射为切入点来揭示其在传递小脑免疫调节信息中的作用，为小脑神经免疫调节提供新的资料，有助于拓宽小脑功能的知识，促进临床上对小脑神经免疫调节异常疾病的思考和研究。 方法： 1.在一侧小脑间位核内微量注射顺行神经束路追踪剂葡聚糖-德克萨斯红（dextran-texas red, dextran-TR），观察小脑间位核至下丘脑神经投射的路径及终止部位。 2.在一侧下丘脑外侧区（顺行追踪显示下丘脑内终止纤维最丰富的区域）微量注射逆行神经束路追踪剂红色荧光金（fluoro-ruby, FR），观察小脑间位核内FR逆行标记的神经元，同时，结合谷氨酸荧光免疫组织化学法探讨小脑间位核投射至下丘脑的神经元中是否存在谷氨酸能神经元。 3.在双侧小脑间位核内微量注射谷氨酰胺酶抑制剂6-重氮-5-氧代-L-正亮氨酸（6-diazo-5-oxo-L-norleucine, DON），以抑制间位核内谷氨酸能神经元合成谷氨酸，并以双侧小脑间位核内注射等量生理盐水及未经任何处理的动物作为对照。在上述三组（未处理对照组、生理盐水对照组、DON组）大鼠的单侧下丘脑外侧区微量注射逆行神经束路追踪剂FR，5天后，在双侧小脑间位核内注射DON或生理盐水，3天后，同一层面的间位核脑片应用谷氨酸荧光免疫组织化学技术，观察小脑间位核—下丘脑外侧区谷氨酸能神经投射的数量变化。 4.在双侧小脑间位核内注射DON后第3天，用高效液相色谱法（high performanceliquid chromatography, HPLC）测定下丘脑内谷氨酸含量的变化。 5.在双侧小脑间位核内注射DON后第3天，用血细胞计数法检测动物外周血白细胞中淋巴细胞百分比的变化；用羧基二乙酸荧光素琥珀酰亚胺酯（carboxyfluorescein diacetate succinimidyl ester, CFSE）和结合PE的抗大鼠CD3抗体双标肠系膜淋巴结细胞，流式细胞仪检测T淋巴细胞对刀豆蛋白A（concanavalinA,Con A）刺激的增殖能力的变化；用酶联免疫吸附测定法（enzyme-linked immunosorbentassay, ELISA）检测血清中抗绵羊红细胞IgM抗体水平的变化。 结果： 1.小脑间位核内微量注射dextran-TR后示踪可见间位核神经元发出离核纤维，行走于同侧小脑上脚中，经小脑上脚交叉到达对侧，然后继续走行于小脑上脚中，进入下丘脑后较多纤维终止于下丘脑外侧区，少数纤维终止于下丘脑室旁核和下丘脑后区。说明小脑间位核神经元发出纤维直接投射至下丘脑（主要是外侧区）。 2.下丘脑外侧区内微量注射FR后可逆行追踪到对侧小脑间位核具有FR标记的神经元，这进一步证明了小脑间位核与下丘脑之间存在直接的神经投射；利用谷氨酸荧光免疫组织化学法染色小脑间位核，发现这些FR标记的神经元中有许多谷氨酸阳性神经元。表明小脑间位核投射至下丘脑外侧区的神经纤维中有谷氨酸能神经。 3.双侧小脑间位核内注射DON（50mM）后第3天，间位核内FR和谷氨酸双标神经元的数目明显少于生理盐水对照组和未处理对照组，而两组对照之间无明显差异，说明小脑间位核DON注射减少了小脑间位核—下丘脑外侧区的谷氨酸能神经投射。 4.双侧小脑间位核内注射DON（50mM）后第3天，下丘脑内谷氨酸含量显著低于生理盐水对照组和未处理对照组，而两组对照之间无显著性差异。双侧小脑间位核内注射低剂量DON（5mM）后，下丘脑内谷氨酸含量没有明显改变。此结果进一步证明双侧小脑间位核内注射DON（50mM）减少了小脑—下丘脑的谷氨酸能神经投射。 5.在小脑间位核—下丘脑谷氨酸能神经投射减少的同时，即在双侧小脑间位核内注射DON（50mM）后的第3天，动物外周血中淋巴细胞的百分比、淋巴结T细胞对Con A的增殖能力和血清中抗绵羊红细胞的IgM抗体水平均显著降低，而两组对照（生理盐水对照和未处理对照）之间无显著性差异。然而，低剂量的DON（5mM）注射没有明显改变上述免疫功能。说明减少小脑间位核—下丘脑的谷氨酸能神经投射可导致免疫功能减弱。 6.在小脑皮质内注射DON（50mM），没有改变下丘脑内谷氨酸的含量也不影响外周血中淋巴细胞百分比、淋巴结T细胞对Con A的增殖能力和血清中抗绵羊红细胞的IgM抗体水平。这一阴性结果进一步证明上述免疫功能的变化确实由小脑间位核—下丘脑的谷氨酸能神经投射所介导。 结论： 1.小脑间位核至下丘脑外侧区存在直接的谷氨酸能神经投射。 2.小脑间位核内注射DON减弱了间位核至下丘脑的谷氨酸能神经投射。 3.减弱小脑间位核—下丘脑的谷氨酸能神经投射导致免疫功能的抑制，提示小脑的免疫调节信息由小脑间位核—下丘脑的谷氨酸能神经投射所传递。 第二部分小脑间位核GABA能神经投射调节免疫功能的途径 目的： 我们在本文的第一部分研究中已说明小脑间位核—下丘脑的谷氨酸能神经投射可调节免疫功能。在这部分工作中，我们探讨小脑间位核的另一传出途径——GABA能神经投射对免疫功能的影响，以更深层次地理解和解释小脑间位核调节免疫功能的作用途径。 方法： 1.在一侧下丘脑外侧区微量注射逆行神经束路追踪剂FR，观察小脑间位核内FR逆行标记的神经元，同时，结合GABA荧光免疫组织化学法探讨小脑间位核投射至下丘脑的神经元中是否存在GABA能神经元。 2.双侧小脑间位核内分别微量注射GABA合成酶抑制剂3-巯基丙酸（3-mercaptopropionic acid,3-MP）、GABA转氨酶抑制剂氨己烯酸（vigabatrin, VGB）以分别减弱或增强间位核的GABA能神经投射，并以双侧小脑间位核内注射等量生理盐水及未经任何处理的动物作为对照。同时，腹腔注射小牛血清白蛋白（bovineserum albumin, BSA）免疫大鼠。术后第3天，流式细胞仪检测动物外周血单个核细胞中T和B淋巴细胞百分比的变化；用CFSE和结合PE的抗大鼠CD3抗体双标肠系膜淋巴结细胞，流式细胞仪检测T淋巴细胞对ConA刺激的增殖能力的变化；ELISA检测血清中抗BSAIgM抗体水平的变化。 3.双侧小脑间位核注射3-MP或VGB后第3天，HPLC测定下丘脑内GABA、淋巴结和脾脏中去甲肾上腺素（norepinephrine, NE）含量的变化。 4.双侧小脑间位核注射3-MP或VGB后第3天，ELISA试剂盒检测血清中促肾上腺皮质激素（adrenocorticotropic hormone, ACTH）、皮质醇（cortisol）、促甲状腺激素（thyroid stimulating hormone, TSH）、三碘甲状腺原氨酸（3,5,3’ triiodothyronine,T3）、四碘甲状腺原氨酸（3,5,3’,5’ tetraiodothyronine, T4）水平的变化。 结果： 1.下丘脑外侧区微量注射FR后逆行追踪至对侧小脑间位核，可见到FR标记的神经元；利用GABA荧光免疫组织化学法染色小脑间位核，发现这些FR标记的神经元中存在GABA阳性神经元，此结果证明小脑间位核与下丘脑外侧区之间存在直接的GABA能神经投射。 2.双侧小脑间位核内注射3-MP后第3天，外周血单个核细胞中的T和B淋巴细胞百分比、淋巴结T细胞对ConA的增殖能力、血清中抗BSA的IgM抗体水平均较两组对照显著升高；而双侧小脑间位核内注射VGB后第3天，外周血单个核细胞中的T和B淋巴细胞百分比、淋巴结T细胞对ConA的增殖能力、血清中抗BSA的IgM抗体水平均较两组对照显著降低。在两组对照之间，即双侧间位核内注射生理盐水的对照组和未作任何处理的对照组之间，上述免疫指标无显著性差异。这些结果表明减弱小脑间位核的GABA能神经投射促进了T和B细胞的功能，而增强小脑间位核的GABA能神经投射则抑制了T和B细胞的功能。 3.在免疫功能发生变化的同时，即在双侧小脑间位核内注射3-MP后第3天，下丘脑内GABA含量明显降低；而注射VGB后第3天，下丘脑内GABA含量显著升高。在两组对照之间，下丘脑内的GABA含量无显著性差异。这些结果表明间位核内注射3-MP减弱了间位核—下丘脑的GABA能神经投射，而间位核内注射VGB则增强了间位核—下丘脑的GABA能神经投射。因此提示，上述间位核内注射3-MP或VGB导致的免疫功能改变可由间位核—下丘脑的GABA能神经投射介导。 4.在免疫功能发生变化的同时，即在双侧小脑间位核内注射3-MP后的第3天，肠系膜淋巴结和脾脏中的NE含量降低，而注射VGB后的第3天，肠系膜淋巴结和脾脏中的NE含量升高，两对照组之间无显著性差异。这些结果表明，间位核内注射3-MP或VGB导致的免疫功能改变可由交感神经这一外周途径介导。 5.在免疫功能发生变化的同时，即在双侧小脑间位核内注射3-MP或VGB后的第3天，血清中的ACTH、皮质醇、TSH、T3和T4的水平与两对照组相比无显著性差异，说明腺垂体—肾上腺皮质/甲状腺途径可能不参与介导小脑间位核GABA能神经投射的免疫调节作用。 结论： 1.小脑间位核至下丘脑外侧区存在直接的GABA能神经投射。 2.小脑间位核的GABA能神经投射可调节T和B细胞的功能，，其作用是负性调节这些细胞的功能。 3.小脑间位核GABA能神经投射通过下丘脑—交感神经的途径实现其免疫调节作用。
[Abstract]:Purpose :
The previous studies in our laboratory have shown that the cerebellum can regulate the immune function . Because there is no direct structural connection between the cerebellum and the immune system , the information transmission way of regulating the immune function of the cerebellum has an important role in the better understanding of the immunoregulatory function of the cerebellum .
Method :
1 . Microinjection of dextran - TX - TR was performed on one side of the cerebellum , and the pathway and termination of the nucleus - to - hypothalamus nerve projection were observed .
2 . In the lateral hypothalamic area of the hypothalamus ( follow - up follow - up of the most abundant area of termination fiber in the hypothalamus ) , a trace injection of retrograde nerve beam tracking agent red fluorescent gold ( fluoro - ruby , FR ) was used to observe the neurons labeled with FR in the nucleus of the cerebellum .
3 . 6 - diazo - 5 - oxo - L - noradrenaline ( DON ) was injected into the nucleus of bilateral cerebellar interneurons .
4 . The content of glutamic acid in the hypothalamus was measured by high performance liquid chromatography ( HPLC ) on the third day after injection of DON on the bilateral cerebellum .
5 . The changes of lymphocyte percentage in peripheral blood cells of animals were measured by blood cell counting method on the third day after injection of DON on bilateral cerebellar interneurons .
The proliferation of T lymphocytes stimulated by concanavalin A ( Con A ) was detected by flow cytometry ( FCM ) .
Changes in serum anti - sheep red blood cell IgM antibody levels were detected by enzyme - linked immunosorbentassay ( ELISA ) .
Results :
1 . The nucleus neurons from the nucleus of the nucleus of the cerebellum were injected into the medial cerebellar peduncle , and then went on to the ipsilateral cerebellar peduncle , and then went on to the opposite side of the medial cerebellar peduncle , and then went on to the lower thalamus , and the more fibers were terminated in the lateral area of the hypothalamus , and the few fibers were terminated in the paraventricular nucleus and the posterior hypothalamus .
2 . It is further proved that there is a direct nerve projection between the nucleus of the cerebellum and the hypothalamus after the micro - injection of FR in the lateral region of the hypothalamus leads to retrograde tracing of the neurons with FR - labeled neurons in the lateral cerebellum .
The neurons of these FR - labeled neurons were found to have a lot of glutamic acid - positive neurons .
3 . There was no significant difference between the two groups of control group and untreated control group on the 3rd day after intracerebroventricular injection of DON ( 50mM ) , but there was no significant difference between the two groups .
4 . The content of glutamic acid in the hypothalamus was significantly lower than that of the normal saline control group and the untreated control group on the third day after the injection of DON ( 50 mM ) in the bilateral cerebellum , while the content of glutamic acid in the hypothalamus was not significantly changed after the low dose DON ( 5 mM ) was injected into the bilateral cerebellum .
5 . At the same time , the percentage of lymphocytes in peripheral blood of animals and lymph node T cells decreased significantly at the 3rd day after injection of DON ( 50 mM ) in the nucleus - hypothalamus of the cerebellum , but there was no significant difference between the two groups ( normal saline control and untreated control ) . However , the low dose DON ( 5 mM ) injection did not significantly alter the above - mentioned immune function .
6 . Injection of DON ( 50 mM ) in the cerebellar cortex did not alter the content of glutamic acid in the hypothalamus nor did it affect the percentage of lymphocytes in the peripheral blood , the proliferative ability of the lymph node T cells to Con A and the level of IgM antibody against the anti - sheep red blood cells in the serum .
Conclusion :
1 . There is a direct glutamic acid nerve projection in the lateral nucleus of the cerebellum to the lateral region of the hypothalamus .
2 . Intra - nucleus injection of DON attenuated glutamate - gic nerve projection from the nucleus to the hypothalamus .
3 . weakening the glutamate gic nerve projection in the nucleus - hypothalamus of the cerebellum leads to the inhibition of the immune function , suggesting that the immunoregulatory information of the cerebellum is transmitted by the glutamate gic nerve projection of the nucleus - hypothalamus .
The second part of cerebellar interbrain nucleus GABAergic neuroprojection regulation of immune function
Purpose :
In the first part of this study , we have shown that the glutamate gic nerve projection of the nucleus - hypothalamus of the cerebellum can modulate the immune function . In this part , we discuss the effect of the other Outgoing Pathway _ GABA ergic Neuroprojection on the immune function of the cerebellar interneurons , so as to understand and explain the pathway of the immune function of the cerebellar interneurons in a deeper level .
Method :
1 . In the lateral hypothalamic area , a trace injection of retrograde nerve bundle tracking agent ( FR ) was used to observe the neurons of FR retrograde labeled neurons in the nucleus of the cerebellum . At the same time , the GABA - ergic neurons in the neurons of the hypothalamus were examined by the GABA fluorescence immunohistochemical method .
2 . GABA synthetase inhibitor ( 3 - MP ) , GABA transaminase inhibitor vigabatrin ( VGB ) were injected into the nucleus of bilateral cerebellum , respectively , to reduce or enhance the GABA - ergic neuroprojection of the meta - nucleus , and to inject the same amount of physiological saline and untreated animals as control . At the same time , the percentage of T and B lymphocytes in peripheral blood mononuclear cells was detected by flow cytometry on the third day after operation .
The proliferation ability of T lymphocytes to Con A stimulated by T lymphocytes was detected by flow cytometry with CFSE and PE anti - CD3 antibody .
Changes of anti - BSAIgM antibody levels in sera were detected by ELISA .
3 . The contents of norepinephrine ( NE ) in the hypothalamus , lymph nodes and spleen were determined by HPLC on the 3rd day after 3 - MP or VGB injection .
4.鍙屼晶灏忚剳闂翠綅鏍告敞灏

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