【作者】 秦嘉；

【导师】 冯甲棣；

【作者基本信息】 中国医科大学 ， 生理学， 2003， 硕士

【摘要】 前言 众所周知，非甾体类抗炎药物（non-steroidal anti-inflammatory drug，NSAID）是指一大类具有抗炎、止痛和解热作用的非固醇类药物。药理学上NSAID_s发挥作用的靶位点是环氧化酶（COX）。前列腺素PG_s，特别是PGE₂是公认的炎症和免疫调节的介导物质。作为（PG_s）合成的关键酶，COX有由不同基因编码的两种同工酶：COX-1和COX-2。COX-1在各种组织中连续性表达；COX-2只在某些细胞由炎症刺激，细胞因子等介导生成。两种COX由于对NSAID_s的反应不同而在药理上存在显著差异，这一发现重新激起药物学家设计高选择性COX-2抑制剂的热情。然而，医药界在NSAID_s对影响COX mRNA和蛋白表达方面仍存在很大分歧。有文献报道，在IL-1刺激下的人的成骨细胞，吲哚美辛（indomethacin，INDO）、吡罗昔康（piroxicam）和布洛芬（flurbiprofen）在一定程度上抑制免疫血清引起的COX-2 mRNA的表达；然而，在兔肺泡巨噬细胞中，阿司匹林（aspirin，ASA）和奈普生（naproxen）对LPS引起COX-2转录和表达没有作用。另据报道，Cao等人发现i．p．或i．v．注射LPS可引起大鼠体温升高和脑内血管壁细胞COX-2mRNA的表达，Kanato指出预先给予COX-2抑制剂可抑制LPS所致大鼠的发热。那么，选择性COX-2抑制剂美洛昔康能否影响LPS所致大鼠脑内COX-2蛋白的表达呢?此方面在国内外文献还未见报道。本实验从上述问题着手，观察预先p．o．美洛昔康后再i．v．LPS对大鼠体温以及脑内COX-2蛋白的影响，从而进一步探讨COX-2抑制剂的具体作用机制，为今后新型药物更好的开发和应用于临床提供实验依据和理论基础。 材料与方法 材料：选用中国医科大学实验动物部提供的健康雄性Wister大鼠，体重180－2209。体温测定在以下给药的大鼠组中进行（l只／组X门）对照组与Lp组：LPS组：静注LPS厂 才kg)对照组：静注生理盐水（与ifs等容积人八2）对照组与抑制剂组：抑制剂组：预先灌胃美洛昔康门．sin4kg)3Inl，3h后再静注u扫门皿pg／kg八对照组：预先灌胃蒸馏水（与美洛昔康等容积）再静注LPS门 人方法：O)测温：将数字温度计的探头插人大鼠直肠6cm。在给药前0．sh到给药后8 ／J’时每隔30nha测一次体温。p)免疫组化在以下三组中进行卜只／组入生理盐水对照组；蒸馏水＋LPS组；美洛昔康＋LPS组。步骤：①组织取材及固定：大鼠在静脉注射LPS后2．5小时用乌拉坦麻醉，再经左心室灌注0．9％，37℃含1％肝素的生理盐水20分钟，继而从同一部位灌人4％，4℃，PH6．5的含8％蔗糖的多聚甲醛溶液250nd。取出大鼠脑组织，保存在含8％蔗糖的二％多聚甲醛溶液中24小时直至免疫组化开始。预先灌胃美洛昔康再给m组和预先灌胃蒸馏水再给Lp组在给LppZ．5小时后按上述相同方法取脑固定备用。②免疫组化及显色反应：S－P法。冰冻切片室温放置30分钟后，人 4℃丙酮固定 10分钟；脑组织切片用 0．IMPBS漂洗 5分钟＊3；3％过氧化氢室温下孵育15ndn，消除内源性过氧化物酶的活性；蒸馏水冲洗，PBS浸泡5分钟；微波法抗原修复：将片子放人装有抗原修复液的容器中（PH6．0的拘椽酸缓冲液入置微波炉加热至 95t以上，持续 10－15分钟，然后在室温下放置 20－30分钟，自然晾凉，使蛋白复性卢％正常羊血清（PBS稀释）封闭，室温孵育15Inin．倾去血清，不洗；滴加兔来源抗大鼠的COX－2一抗＊：100PBS缓冲液稀释X孵育12h，4t冰箱过夜；次日，将组织切片上液体甩去，不洗，经 PBS漂洗，室温下羊抗兔一过氧化物酶 ·2·O：100PBS稀释乃7℃孵育 30min；PBS连续漂洗 5分钟＊3次；将新鲜配制的DAB显色剂置于标本上，显色20分钟；充分水洗后，用苏木素复染；最后，将切片放人75％－100％梯度乙醇中脱水，”二甲苯透明，中性树胶封片。③显微图象分析技术：使用显微图象分析技术，测定脑组织COX－2阳性反应产物的平均灰度值和整合光密度值。④统计学处理：用SPSS软件对实验数据进行统计学分析，所有实验数据均用均值土标准差表示，StUdent t检验。 结 果 1．LPS，生理盐水，COX上抑制剂对大鼠体温的影响 115组在给LPS后体温．显著升高，于160ndn达到顶峰，随后逐渐下降并保持在正常水平；静注LPS于预先给美洛昔康组则抑制LPS对体温升高的影响。抑制剂的抑制LPS致热的效应在静注1152．sh最为明显。 2．特定条件下大鼠脑组织COX－2蛋白的表达 1）光镜下对照组组织切片上的COX－2的定位表达 在冰冻切片经免疫组化，DAB显色后，可在光镜下观察到对照组COX－二蛋白阳性产物呈棕黄色颗粒，主要位于海马处，其余呈极少量全脑散在分布。 n预先 P。蒸馏水 3h后再 i．y．LPS组大鼠脑组织切片上的COX－2蛋白的定位表达 光镜下观察到，该组染色切片上 COX－二蛋白阳性产物呈棕黄色表达位于脑内包括第三脑室脉络丛在内的血管壁细胞及管周细胞的胞质中，呈全脑分布。神经元细胞的COX－2蛋白分布同对照组。 3)COX－更多还原

【Abstract】 INTRODUCTIONNonsteroidal anti - inflammatory drugs ( NSAIDS ) , a group of structurally unrelated compounds, are commonly prescribed to relieve inflammation, pain and fever. The pharmacologic target of NSAIDS is cyclooxygenase (COX) ,a critical enzyme in the biosynthetic pathway of prostaglandins( PGS) . PGS, particularly PGE2, are considered to be mediators of inflammation and immunomodulators. COX exists as two isoforms encoded by two different genes: COX- 1 which is constitu-tively and ubiquitously expressed, and COX - 2 which can be induced only in certain cells by inflammatory stimulation and cytokines etc. The COX isoforms are pharmacologically distinct according to their sensitivity to inhibition by NSAIDS. This finding has renewed interest in designing drugs that highly selective for inhibition of COX - 2 activity and to date several new compounds are under study. However,there is a controversy regarding the effect of NSAIDS on expression of COX mRNA and protein. In human osteoblastic cells stimulated with inter-leukin -1 (IL - 1) it has been showed that indomethacin( INDO) , pi-roxicam and flurbiprofen can partially inhibit the expression of COX mRNA induced by serum. On the contrary, in rabbit alveolar macro-phage aspirin ( ASA) and naproxen has no effect with the expression of COX - 2 induced by LPS at the levels of transcription and translation.We observed if the Meloxicam, a selective COX - 2 inhibitor would effect the COX -2 protein expressin caused by LPS in rats train and further discussed the frondose mechanism of specific COX - 2 inhibitor.MATERIALS AND METHODSMaterials: Male Wister rats, 180 -220g,were used in this study. They were assigned into two big groups of ten animals each at random and then every big ones were divided into two small groups. The time course of rectal temperature ( T.J, ) change was studied in 10 LPS - injected rats(100g/kg) , 10 saline - injected rats ( with the same volume of LPS) , 10 Meloxicam (0. 5mg/kg)3h taken orally pretreated and LPS - injectioned rats, 10 distilled water(with the same volume of Meloxicam) taken orally pretreated and LPS - injected rats by insec-ting the sensory of the digital thermometer into the rectum for 6cm. Methods: (1) Insecting the sensory of the digital thermometer into the rectum for 6cm. and the Tab of each was measured every 30 min from 1h before and 8h after the injection. (2 ) Iirnninuhistochemistry: (1)Ani-mal perfusion and histology: The rats were anesthetized with chloral hydrate at 2.5h after the LPS injection and perfused via the left ventricle with 0.9% saline for 20 minutes,followed by 250ml 4% paraform-aldehyde solution(PH6.5) containing 8% sucrose at 4 C. The brains were removed, stored in the 2% parafonnaldehyde fixative with 8% sucrose for 24h until immunohistochemistry staining was intitiated. Meloxicam - pretreated + LI’S - injected rats, distilled water - pretreated + LPS - injected rats were killed at 2. 5h after the injection. For each time point,5 rat brains were studied. (2)Immnuohistochemis-try:The tissue was stained using S - P. Tissue sections were rinsedwith 0.1M PBS (initially and between incubation steps) and then sequentially incubated in 3% hydrogen peroxide in PBS for 30 minutes at room temperature and 3% normal goat serum . Sections were then in-curated in rabbit COX -2 primary antiserum( 1:100 in PBS diluent) for 12h at room temperature. Sections were washed in PBS and incubated in peroxidase - conjugated goat anti - rabbits IgG( 1:100 in PBS diluent)for 30 minutes at room temperature. After rinsing,the sections were incubated in 0. 01% hydrogen peroxide dissolved in PBS. The section was terminated after 6 - 10 minutes with two successive rinses in PBS. Then we used the DAB as the staining material. (3)The sections were analyzed by using microscopic image quantitative analysis technique. (4)The statistic analysis used the Student t test.REDULTS1. The Tab of the UPS - injected rats, but not that of the saline -injected rats, started to rise again between 70 min and 90 m更多还原