本文選題：18 + ku轉(zhuǎn)位蛋白(TSPO)�。� 參考：《河北北方學(xué)院》2017年碩士論文

【摘要】：18 ku轉(zhuǎn)位蛋白(Translocator protein,TSPO),曾被稱為外周型苯二氮卓類受體,其在腦內(nèi)主要位于膠質(zhì)細(xì)胞線粒體外膜上,近年來被認(rèn)為是焦慮癥和抑郁癥治療的潛在新靶標(biāo)。該蛋白的主要功能是介導(dǎo)膽固醇進(jìn)入線粒體內(nèi)膜,經(jīng)過酶促反應(yīng)合成神經(jīng)類固醇(如四氫孕酮),進(jìn)而通過作用于GABAA受體調(diào)節(jié)情緒行為。AC-5216是國際公認(rèn)的TSPO高選擇性配體,但具有不溶于水、生物利用度低等缺陷。YL-IPA08是軍事醫(yī)學(xué)科學(xué)院毒物藥物研究所對AC-5216進(jìn)行結(jié)構(gòu)優(yōu)化合成的新型配體化合物,具有TSPO親和力和選擇性更高、易溶于水,生物利用度高等優(yōu)點(diǎn)。前期已證實(shí)YL-IPA08在嚙齒動物實(shí)驗(yàn)上具有顯著的抗焦慮、抗抑郁、抗PTSD作用,且腦內(nèi)TSPO及其介導(dǎo)的四氫孕酮合成釋放介導(dǎo)了上述行為活性。本研究利用斑馬魚行為模型和皮質(zhì)酮(corticosterone,CORT)誘導(dǎo)BV-2膠質(zhì)細(xì)胞系凋亡模型,探討YL-IPA08抗焦慮、抗抑郁作用及其抗細(xì)胞凋亡機(jī)制,對解析TSPO的靶標(biāo)價(jià)值及其配體藥物作用機(jī)制具有重要意義。采用新魚缸實(shí)驗(yàn)、黑白偏好實(shí)驗(yàn)、鏡像實(shí)驗(yàn)這三個經(jīng)典的斑馬魚行為模型,連續(xù)進(jìn)行YL-IPA08藥物處理一周于第八天進(jìn)行行為學(xué)檢測,探討該藥在斑馬魚模型上的行為學(xué)效應(yīng)。采用高濃度CORT誘導(dǎo)BV-2細(xì)胞凋亡模型,以YL-IPA08和(或)TSPO拮抗劑PK11195預(yù)處理細(xì)胞2 h,而后與CORT共處理24 h,采用流式細(xì)胞技術(shù)檢測細(xì)胞凋亡率,CCK-8法檢測細(xì)胞活性,Western蛋白印跡法檢測TSPO的表達(dá),酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測細(xì)胞上清中四氫孕酮的水平。本研究結(jié)果如下:(1)與正常對照組相比,新魚缸實(shí)驗(yàn)中,YL-IPA08 0.012mg·L~(-1)在不影響游動距離的情況下能夠增加實(shí)驗(yàn)魚開始6 min內(nèi)在魚箱中的上下穿梭次數(shù)和在魚箱上半部分的停留時(shí)間(P0.05);黑白偏好實(shí)驗(yàn)中,YL-IPA08 0.012 mg·L~(-1)能夠降低實(shí)驗(yàn)魚開始6 min內(nèi)在暗區(qū)的停留時(shí)間百分比(P0.05),同時(shí)增加其在明區(qū)的停留時(shí)間百分比(P0.05);鏡像實(shí)驗(yàn)中,YL-IPA08 0.012 mg·L~(-1)能夠減少實(shí)驗(yàn)魚首6 min在魚箱中央?yún)^(qū)的游動距離百分比和停留時(shí)間百分比(P0.05),但不減少進(jìn)入中央?yún)^(qū)的次數(shù);(2)采用流氏細(xì)胞儀進(jìn)行細(xì)胞凋亡檢測發(fā)現(xiàn):與溶劑對照組相比,CORT誘導(dǎo)細(xì)胞凋亡實(shí)驗(yàn)中,100、200μmol·L~(-1)的CORT能夠有效誘導(dǎo)BV-2細(xì)胞凋亡(P0.05,P0.01),且200μmol·L~(-1)時(shí)作用更為顯著;YL-IPA08 1~100 nmol·L~(-1)與陽性化合物AC-5216一樣能夠顯著降低CORT處理的BV-2細(xì)胞的凋亡率(P0.01),且具劑量依賴關(guān)系,該作用能夠被TSPO拮抗劑PK11195 100 nmol·L~(-1)所拮抗(P0.05);CCK-8細(xì)胞活性檢測顯示,YL-IPA08 100 nmol·L~(-1)能夠顯著升高細(xì)胞活性(P0.01),PK11195 100 nmol·L~(-1)處理會拮抗該作用(P0.01);Western蛋白印跡法結(jié)果顯示YL-IPA08 100 nmol·L~(-1)能夠顯著增加CORT處理的BV-2細(xì)胞TSPO蛋白的表達(dá)(P0.05),同時(shí)ELISA法檢測顯示YL-IPA08 100nmol·L~(-1)顯著升高細(xì)胞培養(yǎng)液中四氫孕酮的水平(P0.01),PK11195 100nmol·L~(-1)顯著逆轉(zhuǎn)該現(xiàn)象,降低四氫孕酮的水平(P0.05)。以上結(jié)果提示,TSPO選擇性配體YL-IPA08在斑馬魚行為模型上具有抗焦慮、抗抑郁行為作用,并且顯著對抗高濃度皮質(zhì)酮誘導(dǎo)的BV-2細(xì)胞凋亡,增加細(xì)胞活性,YL-IPA08的行為作用可能與其升高四氫孕酮水平并產(chǎn)生膠質(zhì)細(xì)胞保護(hù)作用有關(guān)。
[Abstract]:18 Ku transposition protein (Translocator protein, TSPO), once known as the peripheral type of benzene two azeptors, is mainly located on the outer membrane of the mitochondria of the glial cells. In recent years, it has been considered as a potential new target for the treatment of anxiety and depression. The main function of this protein is to mediate cholesterol into the mitochondrial membrane and synthesize by enzymatic reaction. Neurosteroid (such as four hydro progesterone), and then through the action of the GABAA receptor to regulate emotional behavior.AC-5216 is an internationally recognized TSPO high selective ligand, but it is insoluble in water and low bioavailability.YL-IPA08 is a new ligand compound for the structure optimization of AC-5216 by the Toxicological Research Institute of Military Medical Science Academy of the PLA. TSPO has the advantages of high affinity and selectivity, easy to dissolve in water and high bioavailability. Earlier, it has been proved that YL-IPA08 has significant anti anxiety, antidepressant and anti PTSD effects in rodent experiments, and the synthesis and release of four hydrogen progesterone mediated by TSPO in the brain mediates the activity. This study uses the zebrafish behavior model and the cortex Corticosterone (CORT) induces the apoptosis model of BV-2 glial cell line, and discusses the anti anxiety, antidepressant and anti apoptosis mechanism of YL-IPA08. It is of great significance to the target value of TSPO and the mechanism of ligand drug action. The three classic zebrafish behavior models are used in the new fish tank experiment, black and white preference experiment and mirror image experiment. A week of YL-IPA08 drug treatment was performed on eighth days to investigate the behavioral effects of the drug on the zebrafish model. The apoptosis model of BV-2 cells was induced by high concentration of CORT. YL-IPA08 and (or) TSPO antagonist PK11195 were used to pretreat cell 2 h, and then CORT was treated with 24 h. Flow cytometry was used to detect cell apoptosis. Rate, CCK-8 method to detect cell activity, Western blot method to detect TSPO expression, enzyme linked immunosorbent assay (ELISA) to detect the level of four hydrogen progesterone in cell supernatant. The results of this study are as follows: (1) compared with normal control group, YL-IPA08 0.012mg. L ~ (-1) can increase the experimental fish without affecting the distance of swimming. At the beginning of the 6 min, the number of up and down in the fish box and the stop time (P0.05) in the upper part of the fish box (P0.05); in the black and white preference experiment, YL-IPA08 0.012 mg. L~ (-1) can reduce the percentage of residence time (P0.05) of the experimental fish in the dark area of the 6 min, and increase the percentage of the residence time in the clear area (P0.05); in the mirror experiment, YL-IPA08 0 .012 mg. L~ (-1) can reduce the percentage of swimming distance and the percentage of residence time (P0.05) in the central area of the fish box in the central area of the fish box, but it does not reduce the number of times into the central area; (2) the apoptosis detection by the flow cytometer is found: compared with the solvent control group, the CORT energy of 100200 Mu mol. L~ (-1) in the CORT induced apoptosis experiment. BV-2 cell apoptosis (P0.05, P0.01) can be effectively induced, and the effect of 200 mu mol. L~ (-1) is more significant; YL-IPA08 1~100 nmol L~ (-1) can significantly reduce the apoptosis rate of the cells treated with the positive compound AC-5216, and has a dose-dependent relationship. 5) CCK-8 cell activity detection showed that YL-IPA08 100 nmol / L~ (-1) could significantly increase cell activity (P0.01), PK11195 100 nmol. L~ (-1) could antagonize the action (P0.01). The results showed that YL-IPA08 100nmol. L~ (-1) significantly increased the level of four h progesterone in cell culture fluid (P0.01), PK11195 100nmol. L~ (-1) significantly reversed this phenomenon and reduced the level of four hydrogen progesterone (P0.05). The above results suggest that TSPO selective ligands have anti anxiety, antidepressant action and significant antagonism on the zebrafish behavior model. High concentration of corticosterone induced apoptosis of BV-2 cells and increased cell activity. The action of YL-IPA08 may be related to the increased level of four hydro progesterone and the protective effect of glial cells.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R749

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