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  本文關(guān)鍵詞： 實驗性自身免疫性腦脊髓炎 LCN2 CXCL10 多發(fā)性硬化 氟西汀　出處：《山西醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景 多發(fā)性硬化（multiple sclerosis,MS）是中樞神經(jīng)系統(tǒng)（central nervous system,CNS）炎性脫髓鞘疾病，病因和發(fā)病機制尚不明確。近年來其發(fā)病呈現(xiàn)明顯的增高趨勢，是導(dǎo)致青年人神經(jīng)功能障礙的主要疾病,嚴(yán)重威脅著青年人的健康和生存質(zhì)量。載脂蛋白2（Lipocalin2,LCN2）作為新發(fā)現(xiàn)存在于腦脊液和腦實質(zhì)的蛋白質(zhì)，在炎癥、免疫反應(yīng)中起到重要作用。在MS患者腦脊液可以檢測到增高的LCN2水平，提示載脂蛋白2水平可能與MS有關(guān)聯(lián)，包括成為MS腦脊液中的標(biāo)記物。隨著科技日新月異,涉及中樞神經(jīng)系統(tǒng)脫髓鞘疾病的診斷方法越來越精細，從而能夠更好的服務(wù)臨床。但是，當(dāng)前更多的MS患者通過用藥僅能緩解病情，所以，MS的病理生理機制需要進行進一步深入研究，從而為多發(fā)性硬化的治療提供更多理論及實驗依據(jù)。 目的 通過髓鞘少突膠質(zhì)細胞糖蛋白（myelin oligodendrocyteglycoprotein35-55,MOG35-55)免疫小鼠構(gòu)建經(jīng)典的實驗性自身免疫性腦脊髓炎（experimental autoimmune encephalomyelitis，EAE）模型，測定小鼠腦組織中LCN2表達情況及其血清中CXCL10水平，以及使用氟西汀后的變化情況，探討氟西汀在MS疾病中的作用機制。 方法 1.實驗分組和干預(yù)方法：選取雌性C57BL/6小鼠總共80只，8-10周齡大小，體重在18～20g，被隨機分為四組，每組20只。用生理鹽水加完全弗氏佐劑（Complete Freund'sAdjuvant，CFA）免疫動物對照組小鼠。以MOG35-55及含有卡介苗的CFA免疫動物EAE組小鼠，即建立EAE模型。干預(yù)組建模前經(jīng)紫外線（280～320nm）照射，以MOG35-55及含有卡介苗的CFA免疫動物，建立EAE模型。氟西汀組建模前經(jīng)紫外線（280～320nm）照射，以MOG35-55及含有卡介苗的CFA免疫動物，建立EAE模型，免疫當(dāng)天開始給予氟西�。�10mg/kg）灌胃，直至處死。對照組、干預(yù)組和EAE組小鼠均每天給予等量生理鹽水灌胃處理，并持續(xù)至處死。 2.構(gòu)建動物模型方法：制作誘導(dǎo)乳劑是通過向MOG35-55中加入生理鹽水進行稀釋，稀釋濃度為3mg/ml，再將此液體與等體積的完全弗氏佐劑混合，使用玻璃注射器最終抽吸成油包水狀。按0.2ml/只給予模型組及干預(yù)組C57BL/6小鼠背部中央偏頭側(cè)皮下4點注射誘導(dǎo)乳劑，于免疫后第0天、第2天分別進行腹腔注射百日咳毒素400ng，建立EAE動物模型。 3.神經(jīng)功能評分：免疫結(jié)束后，每天觀察小鼠精神狀態(tài)，進食以及活動情況等有無變化。同時評估小鼠神經(jīng)功能，采用國際認(rèn)可的Kono評分標(biāo)準(zhǔn)。 4.檢測指標(biāo)：取額葉及腦室周圍腦組織做石蠟切塊，行HE染色，用免疫組化化學(xué)法測定腦組織中LCN2的表達。ELISA試劑盒檢測血清CXCL10含量。 結(jié)果 1.C57BL/6小鼠發(fā)病情況：小鼠經(jīng)抗原免疫后臨床癥狀逐漸加重，可表現(xiàn)為精神萎靡，食欲差，體重下降，皮毛欠光滑等情況，并且出現(xiàn)尾部無力，繼而后肢無力，最后前肢無力甚至大小便均失禁等臨床癥狀，小鼠發(fā)病臨床表現(xiàn)呈現(xiàn)動態(tài)性改變，以后穩(wěn)步進入慢性期階段。干預(yù)組、氟西汀組兩組小鼠發(fā)病時間和病情高峰期均較EAE組出現(xiàn)后移，臨床癥狀也相對減輕。 2.HE染色結(jié)果：EAE組小鼠腦組織伴有明顯的炎性細胞浸潤現(xiàn)象，在腦組織病變主要集中在軟膜下和腦室周圍白質(zhì)，可見血管袖套現(xiàn)象和明顯的腦膜炎表現(xiàn)，以單核細胞浸潤為主；干預(yù)組、氟西汀組中炎性細胞浸潤現(xiàn)象明顯減少。 3.免疫組化染色結(jié)果：各組均有陽性細胞存在，EAE組白質(zhì)空泡形成、疏松明顯。EAE組（57.33±3.670）、氟西汀組（48.00±5.899）和干預(yù)組（49.33±5.785）陽性細胞數(shù)平均秩次明顯高于對照組（13.00±3.742），且具有統(tǒng)計學(xué)意義（P＜0.05）。氟西汀組和干預(yù)組陽性細胞數(shù)較EAE組明顯減少（P＜0.05）。氟西汀組陽性細胞數(shù)較干預(yù)組無明顯變化（P＞0.05）。 4.小鼠血清CXCL10水平ELISA結(jié)果：EAE組CXCL10含量較氟西汀組高，，氟西汀組較對照組也升高（P＜0.05）。 5.Pearson相關(guān)分析顯示：EAE組小鼠腦組織中LCN2陽性細胞數(shù)平均秩次和血清CXCL10含量之間存在正相關(guān)（P＜0.05）。 結(jié)論 1.氟西汀可以推遲EAE小鼠的發(fā)病時間，減輕疾病對神經(jīng)系統(tǒng)功能損害。 2.氟西汀能夠減輕EAE小鼠腦組織中炎性細胞浸潤程度，對EAE小鼠起到保護作用。 3.氟西汀能夠降低EAE小鼠腦組織中LCN2表達，降低EAE小鼠血清CXCL10水平，發(fā)揮抗炎作用，提示其在治療人類MS方面具有很有希望的應(yīng)用前景。
[Abstract]:Research background
Multiple sclerosis (multiple sclerosis MS) is the central nervous system (central nervous system, CNS) in inflammatory demyelinating diseases, etiology and pathogenesis is still not clear. In recent years the incidence showed a significant increasing trend, is the leading cause of disease of young people nerve dysfunction, a serious threat to the health and survival of young people 2. The quality of apolipoprotein (Lipocalin2, LCN2) as a new found in cerebrospinal fluid and brain protein in inflammation, plays an important role in the immune response. MS can be detected in cerebrospinal fluid of patients with elevated levels of LCN2, suggesting that apo 2 levels may be associated with MS, including a marker of MS in cerebrospinal fluid the diagnosis method and with the science and technology change rapidly, involving the demyelinating disease of the central nervous system more precise, so as to better service in clinic. However, the more MS patients through medication can alleviate disease Therefore, the pathophysiological mechanism of MS needs to be further studied in order to provide more theoretical and experimental basis for the treatment of multiple sclerosis.
objective
The myelin oligodendrocyte glycoprotein (myelin, oligodendrocyteglycoprotein35-55, MOG35-55) in mice immunized with classical construction of experimental autoimmune encephalomyelitis (experimental autoimmune, encephalomyelitis, EAE) model, CXCL10 level and serum LCN2 expression in mouse brain were measured, and the change after fluoxetine, explore the mechanism of action of fluoxetine in MS disease in.
Method
1. experimental groups and intervention methods: a total of 80 female C57BL/6 mice were selected, aged 8-10 weeks, weighing 18 ~ 20g, were randomly divided into four groups, 20 rats in each group. Saline with complete Freund's adjuvant (Complete, Freund'sAdjuvant, CFA) immune animal control mice. CFA EAE mice with immune animal MOG35-55 and contain BCG, namely the establishment of EAE model. The intervention group before modeling by UV irradiation (280 ~ 320nm), CFA and MOG35-55 in animal immunity containing BCG, establish the EAE model. With the UV fluoxetine group (280 ~ 320nm) irradiation, CFA and MOG35-55 in animal immunity containing BCG, the establishment of EAE model of immune day given fluoxetine (10mg/kg) orally, until death. The control group, intervention group and EAE group were given normal saline treatment, and continued until death.
2. methods: the animal model induced by emulsion is diluted by adding normal saline to MOG35-55, dilution concentration is 3mg/ml, then the mixture of complete Freund's adjuvant and the liquid volume, the use of glass syringe aspiration into the final water in oil. According to 0.2ml/ only give the model group and intervention group C57BL/6 mice central 4 migraine lateral subcutaneous injection induced emulsion, on the zeroth day after immunization, second days were given intraperitoneal injection of pertussis toxin 400ng, an animal model of EAE was established.
3. nerve function score: after the end of immunization, we observed the changes of mental state, feeding and activity of mice daily. At the same time, we evaluated the neurological function of mice, and adopted the internationally recognized Kono scoring standard.
4. test index: take frontal lobe and periventricular brain tissues as paraffin blocks, perform HE staining, detect the expression of LCN2 in brain tissue by immunohistochemical method, and detect CXCL10 content in serum by.ELISA kit.
Result
The incidence of 1.C57BL/6 in mice: mice were immunized after clinical symptoms gradually worsened, can be manifested as listlessness, poor appetite, weight loss, less smooth fur and so on, and then the tail weakness, hind limb weakness, finally forelimb weakness or even urine will incontinence and other symptoms of onset clinical presentation of dynamic change, after steadily into the chronic phase stage. The intervention group, fluoxetine group two incidence and severity of the peak time of mice were higher than those in group EAE after the shift, the clinical symptoms are relatively reduced.
Results: 2.HE staining of brain tissue of mice in EAE group with significant infiltration of inflammatory cells in the brain lesions, mainly concentrated in the surrounding subpial and periventricular white matter, visible vascular cuff phenomenon and obvious manifestations of meningitis, infiltration of monocytes; the intervention group significantly reduced the infiltration of inflammatory cells in the fluoxetine group.
3. immunohistochemical staining results: there were positive cells were found in EAE group, the white matter vacuolization, osteoporosis group.EAE was (57.33 + 3.670), fluoxetine group (48 + 5.899) and intervention group (49.33 + 5.785) positive cell number and mean rank was significantly higher than the control group (13 + 3.742), and statistically the significance (P < 0.05). Fluoxetine group and intervention group the number of positive cells was significantly reduced compared with EAE group (P < 0.05). The number of positive cells compared with fluoxetine group the intervention group had no significant change (P > 0.05).
The results of CXCL10 level ELISA in serum of 4. mice: the content of CXCL10 in group EAE was higher than that in fluoxetine group, and in fluoxetine group was also higher than that of control group (P < 0.05).
5.Pearson correlation analysis showed that there was a positive correlation between the average number of LCN2 positive cells in the brain tissue of group EAE and the content of serum CXCL10 (P < 0.05).
conclusion
1. fluoxetine can postpone the onset time of EAE mice and reduce the damage to the nervous system.
2. fluoxetine can reduce the degree of inflammatory cell infiltration in the brain tissue of EAE mice and protect EAE mice.
3. fluoxetine can reduce the expression of LCN2 in the brain tissue of EAE mice, reduce the level of CXCL10 in serum of EAE mice, and play an anti-inflammatory role, suggesting that it has a very promising application prospect in the treatment of human MS.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R744.51

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1 張宇;氟西汀對實驗性自身免疫性腦脊髓炎小鼠LCN2及CXCL10的影響[D];山西醫(yī)科大學(xué);2014年

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