發(fā)布時間：2018-07-04 07:11

  本文選題：小腦 + 間位核　； 參考：《蘇州大學》2012年博士論文

【摘要】：目的： 我們實驗室以往的研究表明小腦間位核可調(diào)節(jié)免疫功能。由于小腦與免疫系統(tǒng)之間沒有直接的結(jié)構(gòu)上的聯(lián)系，因此闡明小腦調(diào)節(jié)免疫功能的信息傳遞途徑對于更好地理解小腦的免疫調(diào)節(jié)功能具有重要作用。本研究以小腦—下丘腦的神經(jīng)投射為切入點來揭示其在傳遞小腦免疫調(diào)節(jié)信息中的作用，為小腦神經(jīng)免疫調(diào)節(jié)提供新的資料，有助于拓寬小腦功能的知識，促進臨床上對小腦神經(jīng)免疫調(diào)節(jié)異常疾病的思考和研究。 方法： 1.在一側(cè)小腦間位核內(nèi)微量注射順行神經(jīng)束路追蹤劑葡聚糖-德克薩斯紅（dextran-texas red, dextran-TR），觀察小腦間位核至下丘腦神經(jīng)投射的路徑及終止部位。 2.在一側(cè)下丘腦外側(cè)區(qū)（順行追蹤顯示下丘腦內(nèi)終止纖維最豐富的區(qū)域）微量注射逆行神經(jīng)束路追蹤劑紅色熒光金（fluoro-ruby, FR），觀察小腦間位核內(nèi)FR逆行標記的神經(jīng)元，同時，結(jié)合谷氨酸熒光免疫組織化學法探討小腦間位核投射至下丘腦的神經(jīng)元中是否存在谷氨酸能神經(jīng)元。 3.在雙側(cè)小腦間位核內(nèi)微量注射谷氨酰胺酶抑制劑6-重氮-5-氧代-L-正亮氨酸（6-diazo-5-oxo-L-norleucine, DON），以抑制間位核內(nèi)谷氨酸能神經(jīng)元合成谷氨酸，并以雙側(cè)小腦間位核內(nèi)注射等量生理鹽水及未經(jīng)任何處理的動物作為對照。在上述三組（未處理對照組、生理鹽水對照組、DON組）大鼠的單側(cè)下丘腦外側(cè)區(qū)微量注射逆行神經(jīng)束路追蹤劑FR，5天后，在雙側(cè)小腦間位核內(nèi)注射DON或生理鹽水，3天后，同一層面的間位核腦片應(yīng)用谷氨酸熒光免疫組織化學技術(shù)，觀察小腦間位核—下丘腦外側(cè)區(qū)谷氨酸能神經(jīng)投射的數(shù)量變化。 4.在雙側(cè)小腦間位核內(nèi)注射DON后第3天，用高效液相色譜法（high performanceliquid chromatography, HPLC）測定下丘腦內(nèi)谷氨酸含量的變化。 5.在雙側(cè)小腦間位核內(nèi)注射DON后第3天，用血細胞計數(shù)法檢測動物外周血白細胞中淋巴細胞百分比的變化；用羧基二乙酸熒光素琥珀酰亞胺酯（carboxyfluorescein diacetate succinimidyl ester, CFSE）和結(jié)合PE的抗大鼠CD3抗體雙標腸系膜淋巴結(jié)細胞，流式細胞儀檢測T淋巴細胞對刀豆蛋白A（concanavalinA,Con A）刺激的增殖能力的變化；用酶聯(lián)免疫吸附測定法（enzyme-linked immunosorbentassay, ELISA）檢測血清中抗綿羊紅細胞IgM抗體水平的變化。 結(jié)果： 1.小腦間位核內(nèi)微量注射dextran-TR后示蹤可見間位核神經(jīng)元發(fā)出離核纖維，行走于同側(cè)小腦上腳中，經(jīng)小腦上腳交叉到達對側(cè)，然后繼續(xù)走行于小腦上腳中，進入下丘腦后較多纖維終止于下丘腦外側(cè)區(qū)，少數(shù)纖維終止于下丘腦室旁核和下丘腦后區(qū)。說明小腦間位核神經(jīng)元發(fā)出纖維直接投射至下丘腦（主要是外側(cè)區(qū)）。 2.下丘腦外側(cè)區(qū)內(nèi)微量注射FR后可逆行追蹤到對側(cè)小腦間位核具有FR標記的神經(jīng)元，這進一步證明了小腦間位核與下丘腦之間存在直接的神經(jīng)投射；利用谷氨酸熒光免疫組織化學法染色小腦間位核，發(fā)現(xiàn)這些FR標記的神經(jīng)元中有許多谷氨酸陽性神經(jīng)元。表明小腦間位核投射至下丘腦外側(cè)區(qū)的神經(jīng)纖維中有谷氨酸能神經(jīng)。 3.雙側(cè)小腦間位核內(nèi)注射DON（50mM）后第3天，間位核內(nèi)FR和谷氨酸雙標神經(jīng)元的數(shù)目明顯少于生理鹽水對照組和未處理對照組，而兩組對照之間無明顯差異，說明小腦間位核DON注射減少了小腦間位核—下丘腦外側(cè)區(qū)的谷氨酸能神經(jīng)投射。 4.雙側(cè)小腦間位核內(nèi)注射DON（50mM）后第3天，下丘腦內(nèi)谷氨酸含量顯著低于生理鹽水對照組和未處理對照組，而兩組對照之間無顯著性差異。雙側(cè)小腦間位核內(nèi)注射低劑量DON（5mM）后，下丘腦內(nèi)谷氨酸含量沒有明顯改變。此結(jié)果進一步證明雙側(cè)小腦間位核內(nèi)注射DON（50mM）減少了小腦—下丘腦的谷氨酸能神經(jīng)投射。 5.在小腦間位核—下丘腦谷氨酸能神經(jīng)投射減少的同時，即在雙側(cè)小腦間位核內(nèi)注射DON（50mM）后的第3天，動物外周血中淋巴細胞的百分比、淋巴結(jié)T細胞對Con A的增殖能力和血清中抗綿羊紅細胞的IgM抗體水平均顯著降低，而兩組對照（生理鹽水對照和未處理對照）之間無顯著性差異。然而，低劑量的DON（5mM）注射沒有明顯改變上述免疫功能。說明減少小腦間位核—下丘腦的谷氨酸能神經(jīng)投射可導致免疫功能減弱。 6.在小腦皮質(zhì)內(nèi)注射DON（50mM），沒有改變下丘腦內(nèi)谷氨酸的含量也不影響外周血中淋巴細胞百分比、淋巴結(jié)T細胞對Con A的增殖能力和血清中抗綿羊紅細胞的IgM抗體水平。這一陰性結(jié)果進一步證明上述免疫功能的變化確實由小腦間位核—下丘腦的谷氨酸能神經(jīng)投射所介導。 結(jié)論： 1.小腦間位核至下丘腦外側(cè)區(qū)存在直接的谷氨酸能神經(jīng)投射。 2.小腦間位核內(nèi)注射DON減弱了間位核至下丘腦的谷氨酸能神經(jīng)投射。 3.減弱小腦間位核—下丘腦的谷氨酸能神經(jīng)投射導致免疫功能的抑制，提示小腦的免疫調(diào)節(jié)信息由小腦間位核—下丘腦的谷氨酸能神經(jīng)投射所傳遞。 第二部分小腦間位核GABA能神經(jīng)投射調(diào)節(jié)免疫功能的途徑 目的： 我們在本文的第一部分研究中已說明小腦間位核—下丘腦的谷氨酸能神經(jīng)投射可調(diào)節(jié)免疫功能。在這部分工作中，我們探討小腦間位核的另一傳出途徑——GABA能神經(jīng)投射對免疫功能的影響，以更深層次地理解和解釋小腦間位核調(diào)節(jié)免疫功能的作用途徑。 方法： 1.在一側(cè)下丘腦外側(cè)區(qū)微量注射逆行神經(jīng)束路追蹤劑FR，觀察小腦間位核內(nèi)FR逆行標記的神經(jīng)元，同時，結(jié)合GABA熒光免疫組織化學法探討小腦間位核投射至下丘腦的神經(jīng)元中是否存在GABA能神經(jīng)元。 2.雙側(cè)小腦間位核內(nèi)分別微量注射GABA合成酶抑制劑3-巰基丙酸（3-mercaptopropionic acid,3-MP）、GABA轉(zhuǎn)氨酶抑制劑氨己烯酸（vigabatrin, VGB）以分別減弱或增強間位核的GABA能神經(jīng)投射，并以雙側(cè)小腦間位核內(nèi)注射等量生理鹽水及未經(jīng)任何處理的動物作為對照。同時，腹腔注射小牛血清白蛋白（bovineserum albumin, BSA）免疫大鼠。術(shù)后第3天，流式細胞儀檢測動物外周血單個核細胞中T和B淋巴細胞百分比的變化；用CFSE和結(jié)合PE的抗大鼠CD3抗體雙標腸系膜淋巴結(jié)細胞，流式細胞儀檢測T淋巴細胞對ConA刺激的增殖能力的變化；ELISA檢測血清中抗BSAIgM抗體水平的變化。 3.雙側(cè)小腦間位核注射3-MP或VGB后第3天，HPLC測定下丘腦內(nèi)GABA、淋巴結(jié)和脾臟中去甲腎上腺素（norepinephrine, NE）含量的變化。 4.雙側(cè)小腦間位核注射3-MP或VGB后第3天，ELISA試劑盒檢測血清中促腎上腺皮質(zhì)激素（adrenocorticotropic hormone, ACTH）、皮質(zhì)醇（cortisol）、促甲狀腺激素（thyroid stimulating hormone, TSH）、三碘甲狀腺原氨酸（3,5,3’ triiodothyronine,T3）、四碘甲狀腺原氨酸（3,5,3’,5’ tetraiodothyronine, T4）水平的變化。 結(jié)果： 1.下丘腦外側(cè)區(qū)微量注射FR后逆行追蹤至對側(cè)小腦間位核，可見到FR標記的神經(jīng)元；利用GABA熒光免疫組織化學法染色小腦間位核，發(fā)現(xiàn)這些FR標記的神經(jīng)元中存在GABA陽性神經(jīng)元，此結(jié)果證明小腦間位核與下丘腦外側(cè)區(qū)之間存在直接的GABA能神經(jīng)投射。 2.雙側(cè)小腦間位核內(nèi)注射3-MP后第3天，外周血單個核細胞中的T和B淋巴細胞百分比、淋巴結(jié)T細胞對ConA的增殖能力、血清中抗BSA的IgM抗體水平均較兩組對照顯著升高；而雙側(cè)小腦間位核內(nèi)注射VGB后第3天，外周血單個核細胞中的T和B淋巴細胞百分比、淋巴結(jié)T細胞對ConA的增殖能力、血清中抗BSA的IgM抗體水平均較兩組對照顯著降低。在兩組對照之間，即雙側(cè)間位核內(nèi)注射生理鹽水的對照組和未作任何處理的對照組之間，上述免疫指標無顯著性差異。這些結(jié)果表明減弱小腦間位核的GABA能神經(jīng)投射促進了T和B細胞的功能，而增強小腦間位核的GABA能神經(jīng)投射則抑制了T和B細胞的功能。 3.在免疫功能發(fā)生變化的同時，即在雙側(cè)小腦間位核內(nèi)注射3-MP后第3天，下丘腦內(nèi)GABA含量明顯降低；而注射VGB后第3天，下丘腦內(nèi)GABA含量顯著升高。在兩組對照之間，下丘腦內(nèi)的GABA含量無顯著性差異。這些結(jié)果表明間位核內(nèi)注射3-MP減弱了間位核—下丘腦的GABA能神經(jīng)投射，而間位核內(nèi)注射VGB則增強了間位核—下丘腦的GABA能神經(jīng)投射。因此提示，上述間位核內(nèi)注射3-MP或VGB導致的免疫功能改變可由間位核—下丘腦的GABA能神經(jīng)投射介導。 4.在免疫功能發(fā)生變化的同時，即在雙側(cè)小腦間位核內(nèi)注射3-MP后的第3天，腸系膜淋巴結(jié)和脾臟中的NE含量降低，而注射VGB后的第3天，腸系膜淋巴結(jié)和脾臟中的NE含量升高，兩對照組之間無顯著性差異。這些結(jié)果表明，間位核內(nèi)注射3-MP或VGB導致的免疫功能改變可由交感神經(jīng)這一外周途徑介導。 5.在免疫功能發(fā)生變化的同時，即在雙側(cè)小腦間位核內(nèi)注射3-MP或VGB后的第3天，血清中的ACTH、皮質(zhì)醇、TSH、T3和T4的水平與兩對照組相比無顯著性差異，說明腺垂體—腎上腺皮質(zhì)/甲狀腺途徑可能不參與介導小腦間位核GABA能神經(jīng)投射的免疫調(diào)節(jié)作用。 結(jié)論： 1.小腦間位核至下丘腦外側(cè)區(qū)存在直接的GABA能神經(jīng)投射。 2.小腦間位核的GABA能神經(jīng)投射可調(diào)節(jié)T和B細胞的功能，，其作用是負性調(diào)節(jié)這些細胞的功能。 3.小腦間位核GABA能神經(jīng)投射通過下丘腦—交感神經(jīng)的途徑實現(xiàn)其免疫調(diào)節(jié)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.鍙屼晶灝忚剳闂翠綅鏍告敞灝

本文編號：2095250