發(fā)布時(shí)間：2018-05-31 05:57

  本文選題：癲癇發(fā)生過程 + 小膠質(zhì)細(xì)胞　； 參考：《重慶醫(yī)科大學(xué)》2017年博士論文

【摘要】：第一部分癲癇發(fā)生過程中腦內(nèi)小膠質(zhì)細(xì)胞表型的動(dòng)態(tài)變化目的:探索小鼠在癲癇發(fā)生過程中的不同時(shí)期腦內(nèi)小膠質(zhì)細(xì)胞表型以及表型相關(guān)細(xì)胞因子的動(dòng)態(tài)變化。方法:選用7-8周齡C57BL/6J小鼠,分為正常對(duì)照組和實(shí)驗(yàn)組。實(shí)驗(yàn)組采用匹魯卡品腹腔注射誘導(dǎo)驚厥持續(xù)狀態(tài)(Status epilepticus,SE),同時(shí)進(jìn)行腦電監(jiān)測(cè)對(duì)模型進(jìn)行驗(yàn)證。采用FCM對(duì)SE后不同時(shí)期小膠質(zhì)細(xì)胞表型的變化進(jìn)行動(dòng)態(tài)監(jiān)測(cè);酶聯(lián)免疫標(biāo)記分析(Enzyme-linked immunosorbent assay,ELISA)監(jiān)測(cè)SE后不同表型小膠質(zhì)細(xì)胞所分泌的相關(guān)細(xì)胞因子白介素-1β(Interleukin-1β,IL-1β)、白介素-4(Interleukin-4,IL-4)、白介素-10(Interleukin-10,IL-10)的變化情況。結(jié)果:(1)匹魯卡品能夠誘發(fā)C57BL/6J小鼠出現(xiàn)驚厥表現(xiàn),并且SE后腦電圖改變符合癲癇發(fā)生過程中的各時(shí)期腦電圖變化。(2)小鼠腦內(nèi)小膠質(zhì)細(xì)胞表型比例在SE后癲癇發(fā)生的過程中動(dòng)態(tài)變化,M1型小膠質(zhì)細(xì)胞比例在SE后迅速升高,并維持在較高水平,直至SE后20天左右開始逐漸下降,隨后降至接近對(duì)照水平;M2型小膠質(zhì)細(xì)胞比例在SE后迅速下降,隨后逐漸升高,在SE后第20天左右達(dá)到高峰,并于SE后第28天將至接近對(duì)照水平。(3)腦內(nèi)M1型小膠質(zhì)細(xì)胞分泌的典型細(xì)胞因子IL-1β的水平的波動(dòng)趨勢(shì)與M1型小膠質(zhì)細(xì)胞比例的變化趨勢(shì)基本一致;M2型小膠質(zhì)細(xì)胞分泌的典型細(xì)胞因子IL-4、IL-10的水平與M2型小膠質(zhì)細(xì)胞比例的變化趨勢(shì)基本一致。結(jié)論:SE后癲癇發(fā)生過程中小膠質(zhì)細(xì)胞的不同表型所占比例動(dòng)態(tài)變化,表型相關(guān)細(xì)胞因子的水平與表型變化趨勢(shì)基本一致,說明小膠質(zhì)細(xì)胞的表型變化參與了癲癇發(fā)生過程。第二部分腹腔注射IFN-γ/IL-4調(diào)節(jié)腦內(nèi)小膠質(zhì)細(xì)胞表型及相應(yīng)細(xì)胞因子目的:探索能否通過腹腔注射Th1細(xì)胞因子(IFN-γ)或Th2細(xì)胞因子(IL-4)調(diào)控腦內(nèi)小膠質(zhì)細(xì)胞的表型方法:根據(jù)第一部分結(jié)果,在M1比例顯著升高的早期分別給予IFN-γ和IL-4以達(dá)到進(jìn)一步促進(jìn)M1比例升高和抑制M1比例升高的作用,在M2比例顯著升高的中期分別給予IFN-γ和IL-4以抑制M2的產(chǎn)生和進(jìn)一步促進(jìn)M2比例升高。選用7-8周齡C57BL/6J小鼠,采用匹魯卡品腹腔注射誘導(dǎo)驚厥持續(xù)狀態(tài),隨機(jī)分成6組:早期生理鹽水干預(yù)組、早期IFN-γ干預(yù)組、早期IL-4干預(yù)組、中期生理鹽水干預(yù)組、中期IFN-γ干預(yù)組、中期IL-4干預(yù)組。應(yīng)用FCM檢測(cè)干預(yù)后小膠質(zhì)細(xì)胞的表型變化,進(jìn)一步應(yīng)用ELISA檢測(cè)細(xì)胞因子IL-1β、IL-4、IL-10的水平,了解干預(yù)后不同小膠質(zhì)細(xì)胞表型是否發(fā)生了變化,及表型相關(guān)細(xì)胞因子水平的變化是否與表型一致。結(jié)果:(1)腹腔注射細(xì)胞因子能夠達(dá)到增加外周血及腦組織細(xì)胞因子濃度的作用。(2)早期補(bǔ)充IFN-γ不能進(jìn)一步增加SE后M1的比例。中期補(bǔ)充IL-4也無法進(jìn)一步增加M2的比例。SE后表型相關(guān)細(xì)胞因子在SE后第15天達(dá)到峰值,而早期IFN-γ組、中期IL-4組腦內(nèi)IL-1β、IL-4、IL-10的峰值水平與生理鹽水干預(yù)組無顯著性差異。(3)早期給予IL-4可以顯著抑制SE后早期M1的持續(xù)升高。同樣的,中期給予IFN-γ抑制了M2的升高。在SE后第15天,早期IL-4組及中期IFN-γ組腦內(nèi)IL-1β、IL-4、IL-10的峰值水平較生理鹽水干預(yù)組明顯降低。(4)抑制表型發(fā)生偏移并不是通過抑制小膠質(zhì)細(xì)胞活化實(shí)現(xiàn)的。結(jié)論:進(jìn)一步增加SE后小膠質(zhì)細(xì)胞表型偏移程度的干預(yù)措施不能到達(dá)預(yù)期效果,而抑制SE后小膠質(zhì)細(xì)胞表型偏移的干預(yù)措施均成功抑制了表型的偏移。第三部分抑制SE后小膠質(zhì)細(xì)胞表型偏移對(duì)癲癇發(fā)生及認(rèn)知功能損傷的作用目的:探索在SE后通過抑制小膠質(zhì)細(xì)胞的表型的偏移能否影響癲癇的發(fā)生及腦認(rèn)知功能損傷。方法:在SE后第50天對(duì)實(shí)驗(yàn)組(生理鹽水干預(yù)組、早期IL-4干預(yù)組、中期IFN-γ干預(yù)組)及對(duì)照組(正常對(duì)照組、IFN-γ對(duì)照組、IL-4對(duì)照組)的小鼠植入顱內(nèi)電極,利用腦電連續(xù)檢測(cè)14天,觀察自發(fā)性癲癇發(fā)作(Spontaneous recurrent seizures,SRS)情況。應(yīng)用Morris水迷宮檢測(cè)干預(yù)后小鼠的空間學(xué)習(xí)記憶能力來反應(yīng)認(rèn)知功能的變化。結(jié)果:在腦電檢測(cè)過程中,對(duì)照組(正常對(duì)照組、IFN-γ對(duì)照組、IL-4對(duì)照組)未出現(xiàn)驚厥發(fā)作。早期IL-4干預(yù)組、中期IFN-γ干預(yù)組的SRS發(fā)生率雖然無統(tǒng)計(jì)學(xué)差異,但是SRS的持續(xù)時(shí)間、發(fā)作頻率、嚴(yán)重程度均較生理鹽水干預(yù)組有所改善。早期IL-4干預(yù)組、中期IFN-γ干預(yù)組的Morris水迷宮表現(xiàn)也優(yōu)于生理鹽水干預(yù)組,但仍不及正常對(duì)照組。結(jié)論:根據(jù)小膠質(zhì)細(xì)胞表型在SE后的變化趨勢(shì),精準(zhǔn)化的抑制表型的偏移,能夠改善SE的預(yù)后,降低SRS的持續(xù)時(shí)間、發(fā)作頻率及嚴(yán)重程度,并且減輕SE后認(rèn)知功能損傷。
[Abstract]:The first part is the dynamic change of microglia phenotype in the process of epilepsy: To explore the dynamic changes of microglia phenotypes and phenotypic cytokines in the brain of mice during the different periods of epilepsy. Methods: 7-8 weeks old C57BL/6J mice were selected to be divided into normal control group and experimental group. Status epilepticus (SE) was induced by intraperitoneal injection, and the model was verified by electroencephalogram monitoring. FCM was used to monitor the changes of microglia phenotype in different periods after SE; enzyme linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA) was used to monitor the differentiation of microglia in different phenotypes after SE. The changes of interleukin -1 beta (Interleukin-1 beta, IL-1 beta), interleukin -4 (Interleukin-4, IL-4), and interleukins -10 (Interleukin-10, IL-10). Results: (1) pilocarpine can induce convulsion in C57BL/6J mice, and the changes of EEG in SE after the onset of epilepsy are in accordance with the changes of electroencephalogram during the various periods of epilepsy. (2) small The phenotypic proportion of microglia in the rat brain changed dynamically in the process of epilepsy after SE. The proportion of M1 microglia increased rapidly after SE, and maintained at a high level until after SE 20 days, and then declined gradually and then dropped to the control level. The proportion of M2 microglia decreased rapidly after SE, and then gradually increased, after SE. The peak was reached about twentieth days and was close to the control level at twenty-eighth days after SE. (3) the trend of the fluctuation trend of the typical cytokine IL-1 beta secreted by M1 microglia in the brain was basically consistent with the change trend of the M1 microglia ratio; the level of the canonical cytokine secreted by M2 microglia, the level of IL-10 and the M2 microglia, were secreted by M2 microglia cells. The change trend of cell proportion is basically consistent. Conclusion: the proportion of different phenotypes of small glial cells in the process of epilepsy after SE changes, the level of phenotypic related cytokines is basically consistent with phenotypic changes, indicating that the phenotypic change of microglia participates in the process of epileptic onset. The second part is intraperitoneally injected with IFN- gamma /IL-4 Microglia phenotypes and corresponding cytokines in the brain: explore whether Th1 cytokines (IFN- gamma) or Th2 cytokine (IL-4) are injected into the brain to regulate the phenotypic method of microglia in the brain: according to the first part, IFN- gamma and IL-4 are given to increase the ratio of M1 to increase the proportion of M1 in the early stage of the significant increase in M1 ratio. The effect of increasing the proportion of M1 was given to IFN- gamma and IL-4 to inhibit the production of M2 and to further promote the increase of M2 ratio in the middle of the M2 ratio. The 7-8 week old C57BL/6J mice were selected to induce the persistent state of convulsion by intraperitoneal injection of pilocarpine, which were randomly divided into 6 groups: early physiological saline intervention group, early IFN- gamma intervention group, early IFN- gamma intervention group, and early IFN- gamma intervention group. Phase IL-4 intervention group, medium-term saline intervention group, medium-term IFN- gamma intervention group and medium-term IL-4 intervention group. The phenotypic changes of microglia were detected by FCM, and the levels of IL-1 beta, IL-4, IL-10 were detected by ELISA, and the changes of the microglia phenotypes after intervention and the phenotype related cytokines were found. The results were as follows: (1) intraperitoneal injection of cytokines could increase the concentration of cytokines in peripheral blood and brain tissue. (2) early supplementation of IFN- gamma could not further increase the proportion of M1 after SE. Medium term supplementation of IL-4 could not further increase the phenotype related cytokines after.SE in fifteenth Tianda after SE. In the early IFN- gamma group, the peak level of IL-1 beta, IL-4, IL-10 in the middle IL-4 group was not significantly different from that in the saline intervention group. (3) the early administration of IL-4 could significantly inhibit the continuous increase of M1 in the early stage of SE. The peak level of IL-4, IL-10 was significantly lower than that in the saline intervention group. (4) the inhibition of phenotypic migration was not achieved by inhibiting the activation of microglia. Conclusion: intervention measures to further increase the phenotypic migration of microglia after SE could not reach the expected effect, and the intervention measures to inhibit the phenotypic migration of microglia after SE were inhibited. The third part inhibits the effect of phenotypic migration of microglia after SE on epilepsy and cognitive impairment: To explore whether the phenotype shift of microglia can affect the occurrence of epilepsy and brain cognitive impairment after SE. Methods: the experimental group (physiological saline dry) at the end of the fiftieth day of SE Pre group, early IL-4 intervention group, medium IFN- gamma intervention group and control group (normal control group, IFN- gamma control group, IL-4 control group) were implanted with intracranial electrodes in mice, and the spontaneous epileptic seizures (Spontaneous recurrent seizures, SRS) were observed for 14 days by electroencephalography. The spatial learning and memory of the mice after the intervention were detected by the Morris water maze. Results: in the process of EEG detection, the control group (normal control group, IFN- gamma control group, IL-4 control group) did not appear convulsive seizures. The incidence of SRS in the early IL-4 intervention group and the mid-term IFN- gamma intervention group was not statistically different, but the duration of SRS, the frequency of attack, and the severity were more than that of the saline dry. In the early IL-4 intervention group, the Morris water maze in the mid-term IFN- gamma intervention group was also better than the normal saline intervention group, but it was still inferior to the normal control group. Conclusion: according to the change trend of the microglia phenotype after SE, the precision of the inhibitory phenotypic migration can improve the prognosis of SE, reduce the duration of SRS, the frequency of the attack and the frequency of the attack. Severity, and reduce cognitive impairment after SE.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R742.1

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