CN102755636A - bFGF在制备治疗人类缺血性脑中风药物中的应用 - Google Patents
bFGF在制备治疗人类缺血性脑中风药物中的应用 Download PDFInfo
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Abstract
本发明涉及医药技术领域，特别涉及碱性成纤维细胞生长因子(bFGF)在制备治疗人类脑中风药物中的应用。bFGF在制备治疗人类缺血性脑中风药物中的应用，药物的形式可以是粉针剂、水剂、脂质体等。bFGF对缺血性脑中风有治疗作用，小剂量的bFGF可以抑制内质网应激，减少内质网引起的神经元凋亡，从而对缺血性脑中风产生治疗作用。
Description
技术领域：
本发明属医药技术领域，具体涉及碱性成纤维细胞生长因子(bFGF)在制备治疗缺血性脑中风药物中的应用。
背景技术：
脑中风又称脑卒中或脑血管意外，是一组以脑部缺血或出血性损伤症状为主要临床表现的疾病，主要分为出血性脑中风(脑缺血及蛛网膜下腔出血)和缺血性脑中风(脑梗塞及脑血栓形成)两大类，其中以脑缺血性脑中风最为常见。该疾病发病急，病死率高，是世界上最为主要的致死性疾病之一。据统计，我国每年新发完全性脑中风120～150万人，死亡80～100万人，存活者中约75％致残，5年复发率高达41％；而美国每年亦有50万人发病，其中15万人死亡。脑中风的死亡率和年龄呈正相关，目前我国已进入老年化社会，其发病率及对国人的影响将有进一步增加的趋势，因此研究与开发治疗脑中风尤其是缺血性脑中风的有效药物是医药界的一个重要课题。
目前对缺血性脑中风尚缺乏有效治疗措施，临床上常见的治疗方法有药物治疗、导管擦入、颈动脉内膜切除等，其中药物治疗是最常见的治疗方法，作用方式包括溶栓、扩血管或抗血小板聚集等。上述治疗药物的共同特点是：针对性强、作用环节明确、但疗效单一，难以克服缺血性脑中风引起神经细胞损伤的多种病理学效应，且有不同程度的毒副作用。如溶栓药物很难通过血脑屏障进入受损脑组织发挥作用，因而疗效不肯定，且有引起再灌注损伤及出血的副作用；扩血管药物可使正常闹部位血管扩张，造成病变区血液流向正常脑组织，产生所谓“窃血”现象。因此寻找具有新的作用机理，且疗效明确、毒副作用 小的有效药物是一项亟待探索的工作。
碱性成纤维细胞生长因子(Basic fibroblast growth factor，bFGF)是由155个氨基酸组成的18kD多肽，其化学结构如图1所示，主要分布于垂体、脑和神经组织及视网膜、肾上腺、胎盘等组织，可促进来源于中胚层和神经外胚层的多种类型细胞增殖、分化，具有广泛的生物学效应。它的主要生物学作用是促进细胞生长、增殖和分化。小分子量的bFGF定位于细胞质，大分子量的bFGF定位于细胞核和核糖体，并通过自分泌或旁分泌的方式运输到作用部位发挥生理作用。bFGF是重要的肽类神经营养因子，对中枢神经组织和周围神经神经组织具有修复和保护作用。25mg/ml的bFGF可刺激有髓神经纤维的生长而促进大鼠面神经的生长。氧化应激与中枢神经疾病病理生理过程联系密切，许多中枢神经系统疾病如缺血再灌注损伤、帕金森病和衰老等都涉及到氧化损伤，病灶周围都产生大量的活性氧自由基。H2O2是一种活性氧成分，它参与了许多神经系统疾病的发病机制，常用作神经细胞氧化损伤的诱导剂。PC12细胞从可移植的大鼠嗜铬细胞瘤克隆而来，具有交感神经元的生理、生化特征。该细胞株具有相当高的稳定性和同质性，分化程度高，被广泛应用于神经元相关的研究。FGF-2是一种很强的丝裂原，具有促进各种软组织损伤修复的作用，能动促进组织损伤的修复。FGF-2保护神经的作用是多方面的：(1)保护神经元；(2)促进轴突再生；(3)促进SC增殖；(4)促进血管发生，改善血供微环等。研究表明，中枢神经受伤后，和周围神经一样可以再生，但再生能比周围神经差，导致这种差异的原因不在于神经元本身的差异，主要和合适的组织环境即神经再生的微环境有关。
发明内容：
本发明的目的在于提供bFGF的新用途，即在制药中得新用途。
为达到上述目的，本发明涉及bFGF在制备治疗人类缺血性脑中风药物中得应用。
脑缺血与神经细胞凋亡有关，细胞凋亡又称细胞程序性死亡，是指细胞在一定的生理或病理条件下，遵循自身的程序，自己结束其生命的过程。其受控于一系列基因顺序表达而发现的主动的耗能的自杀性死亡，是维持机体细胞种类和数量动态平衡的重要机制，与多种疾病的发生发展密切相关。目前已知的三条主要信号转导途径来控制细胞凋亡：1、线粒体通路；2、死亡受体通路；3、内质网通路。内质网通路机制是近年来发现的一条新的凋亡途径，且很多动物实验已被初步证明内质网通路参与了脊髓损伤的继发性病理机制。内质网是真核细胞中重要的细胞器，参与蛋白质合成、折叠和寡聚化，还参与脂类代谢、类固醇代谢的合成以及Ca2+调节等。内质网非常敏感，葡萄糖/营养素的缺乏、蛋白质糖基化的抑制、二硫键形成的障碍、蛋白质运转异常、Ca2+耗竭等刺激均可导致内质网功能失调，即内质网应激。内质网应激时内质网内蛋白质不能正确折叠而被大量滞留，导致未折叠蛋白反应，激发内质网和高尔基体、细胞核之间的信号转导，抑制蛋白质合成并启动内质网相关基因转录，一定程度的内质网应激可增强内质网的应激能力，促进内质网功能恢复。但是内质网应激持续存在或过强时将诱导内质网相关性蛋白细胞凋亡，造成组织损伤。脊髓损伤时，发生的缺血缺氧、酸中毒、ATP耗竭、钙超载以及大量自由基生成等均可诱导内质网应激，从而使其在脑缺血发生发展中具有重要意义。我们利用过氧化氢和脑缺血/再灌注模型鼠，分别在体外和体内水平上复制的氧化应激诱导神经细胞损伤中，能够检测到持续的内质网应激变化及相关的细胞凋亡过程，而bFGF给药则能够在一定程度上抑制这一过程，这也是首次证实了bFGF的神经 保护作用并不是仅仅局限于神经营养和神经再生功能之中，也与内质网应激有着密切的联系。
对于治疗人类缺血性脑中风来说，bFGF的给药方式可以是肌肉注射给药，剂量为4000U/d，10d为一疗程，连用20d。按照脑卒中病人临床神经功能缺损评分标准，bFGF治疗组为11.1±6.02，对照组为14.4±8.61，bFGF组明显优于对照组，总有效率达到92％，在临床上表现为神经系统症状逐渐消失，感觉和运动功能逐渐恢复。
总之，bFGF能显著的减少脑缺血缺氧造成的神经元损伤，其机制可能与减少内质网应激诱导的脑神经元凋亡有关，bFGF是一种对于人体相对安全的蛋白生物药，未发现药物不良反应。因此，本研究结果可望在将来为临床治疗缺血性脑中风提供一种新的药物。
具体实施方式
下面结合附图及实施例对本发明作进一步描述：
bFGF在制备治疗人类缺血性脑中风药物中的应用，药物的形式可以是粉针剂、水剂、脂质体等。bFGF对缺血性脑中风有治疗作用，小剂量的bFGF可以抑制内质网应激，减少内质网引起的神经元凋亡，从而对缺血性脑中风产生治疗作用。
实施例一：
1、动物实验：
取健康雄性B6小鼠，体重22-25g，SPF级。该小鼠由温州医学院实验中心提供，将小鼠随即分成三组，分别为正常对照组、模型组、bFGF治疗组。治疗组小鼠按bFGF 20μg/只剂量鼻腔给药1h后进行双侧颈动脉结扎性脑缺血模 型(BCCA)，缺血24h和72h后分别进行自发性行为学分析，HE染色，免疫组化及Western blot分析。并且与模型组进行对比，从而得到结果。
2.小鼠鼻腔注射给药：
将三组小鼠分别用4％浓度的水合氯醛，以0.01ml/g剂量腹腔麻醉，然后将其背部朝下固定在木板上，将细导管由小鼠嘴部插入到气管，然后将通过计算将20μg/只的bFGF分别分次用微型注射器加到小鼠鼻孔中。而气管导管能起到防止药物直接进入肺部的作用。对照组按照同样的方法给予相同量的PBS。
3.双侧颈动脉结扎全脑缺血模型(BCCA)的建立：
将小鼠背部固定，颈正中切口，分离两侧颈总动脉，用动脉夹同时结扎两侧颈总动脉持续20min，去动脉夹，缝合皮肤。手术过程中保持小鼠体温37℃，直到小鼠从麻醉中苏醒。
4.自发行为学分析：
将各组小鼠分别在脑缺血模型后1天和3天进行自发行为学分析，将小鼠放入一个(40×40×40cm)的箱子中，摄像机自动记录小鼠在一个小时内的运动轨迹，行为。并自动计算出其在一个小时内的运动总路程，平均速度等指标。通过比较给药组和无给药组总路程的变化，来阐明bFGF对小鼠行为能力的改善情况。
5.HE及免疫组化分析：
6.Western blot分析海马组织相应蛋白变化：
7.数据统计分析：数据均以均数±标准差(Mean±SD)表示，统计分析采用单因素方差分析，P＜0.05为统计学差异有显著性。
8.结论：
图1为双侧颈动脉结扎性脑缺血小鼠注射bFGF后自发性行为的比较图。图 中BCCA模型后小鼠行为能力显著性下降，而给予bFGF后能显著改善小鼠行为能力。图2为bFGF给药后脑切片海马区域HE和CHOP，caspase-1免疫组化的比较，HE染色显示bFGF能改善脑缺血造成的神经细胞的凋亡，而免疫组化显示，bFGF能显著抑制脑缺血引起的内质网应激，降低CHOP，caspase-12的表达，增加p-AKT，p-ERK的表达。图3通过Western blot进一步验证bFGF能显著抑制脑缺血引起的内质网应激，降低CHOP，caspase-12的表达并增加p-AKT，p-ERK的表达。使用bFGF的脑缺血小鼠和未使用bFGF的脑缺血小鼠比较，bFGF能显著改善小鼠的行为能力，抑制内质网应激，减少脑缺血带来的不良影响。
9.结论：bFGF可显著改善小鼠的行为能力，抑制内质网应激，对缺血性脑中风具有一定的防治作用。
实施例二：
1.PC12细胞H2O2氧化损伤实验：
PC12细胞(大鼠肾上腺髓质嗜珞细胞瘤神经细胞株)用含10％小牛血清的DMEM液培养，接种于96孔板中，在37℃、95％O2和5％CO2条件下培养至细胞铺满单层。实验一分为5组，分别为正常组，H2O2浓度40、80、120、160μM作用12h。另设实验二分为5组，在120μM H2O2条件下依次加入0、20、40、80、160ng/ml bFGF作用12h。
2.MTT法测定活细胞数：
MTT全称为3-(4，5-二甲基噻唑-2)-2，5-二甲苯四氮唑溴盐，商品名：噻唑蓝。将上述各组细胞分别加入总浓度为0.5mg/ml的MTT反应4h，吸去上清液(注意避免吸走甲瓒结晶)，每孔加入150M DMSO，待孔内颗粒完全溶解后，用酶标仪测定570nm处的光密度。
3.流式细胞仪检测细胞凋亡率：
PC12细胞消化后，以2×105cells/ml，3ml/孔接种与6孔板，细胞贴壁后分为5组：a组为阴性对照组，不做任何处理；b组：120μM H2O2作用细胞12h；c组：40ng/ml bFGF作用细胞24h，d组：40ng/ml bFGF 2h后再加入120μMH2O2作用细胞12h，按照Annexin V-FITC凋亡检测试剂盒说明书处理细胞，流式细胞仪测定细胞凋亡率。
4Western blot检测蛋白表达：
取对数生长期的PC12细胞，饥饿同步化24h。细胞分为空白对照组、bFGF组、H2O2组、bFGF+H2O2组，处理浓度、时间和顺序分别为40ng/mL bFGF作用2h，120μMH2O2处理12h。收集细胞，按全蛋白提取试剂盒操作步骤提取细胞蛋白并用BCA法测定蛋白浓度。调整各样本蛋白浓度一致，进行10％SDS-PAGE凝胶电泳，经PVDF膜转移，5％脱脂奶粉封闭后，分别加CHOP，Caspase-12，p-AKT，AKT，ERK1/2和p-ERK1/2(1∶200)多克隆抗体过夜，PBS洗涤，再相对应得二抗IgG-HRP(1∶2000)结合，再次洗涤，加化学荧光试剂，X光曝光，凝胶图像分析仪进行光密度扫描与分析。
5.数据统计分析：
数据均以均数±标准差(Mean±SD)表示，统计分析采用单因素方差分析，P＜0.05为统计学差异有显著性。
6.实验结果和结论：
附图4为PC12细胞经H2O2处理应用bFGF后经MTT法及流式测定细胞后的对比图；从图中可知bFGF可提高PC12细胞的存活率，对细胞起到抗凋亡作用。图5为PC12为细胞经H2O2处理应用bFGF后CHOP，caspase-12，p-ERK，p-AKT等western blot检测蛋白的变化，结果显示bFGF能显著抑制脑缺血引起 的内质网应激，降低CHOP，caspase-12的表达，增加p-ERK，p-AKT的表达。而加入p-AKT和p-ERK抑制剂以后，bFGF的抑制内质网应激作用减低，说明bFGF抑制内质网应激要通过AKT和ERK介导的信号通路。数据均以均数±标准差表示，统计分析采用单因素方差分析，P＜0.05为统计学差异有显著性。在PC12细胞H2O2氧化损伤模型中，bFGF能够增加PC12细胞的存活率并抑制PC12细胞的凋亡，并且抑制内质网应激。
[0040] 上述实施例只为说明本发明的技术构思特点，其目的在于让熟悉此领域的人士能够了解本发明的内容并据此实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所做的等效变化或修饰，都应涵盖在本发明的保护范围之内。
附图说明
图1为双侧颈动脉结扎性脑缺血(BCCA)小鼠注射bFGF后自发性行为变化；
图2为bFGF给药后脑切片海马区域HE和CHOP，caspase-1免疫组化的比较；
图3为bFGF给药后动物CHOP，Caspase-12，P-Erk，P-Akt等蛋白表达；
图4为bFGF处理的H2O2损伤的PC12细胞增殖率及凋亡率；
图5为bFGF处理的H2O2损伤的PC12细胞caspase-12，p-ERK，p-AKT蛋白表达。
Claims (1)
1.本发明涉及医药技术领域，特别涉及碱性成纤维细胞生长因子(bFGF)在制备治疗人类脑中风药物中的应用。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107875394A (zh) * | 2016-09-27 | 2018-04-06 | 邬芬赞 | 多聚阳离子‑bFGF复合材料及其应用和制备方法 |
| CN112546198A (zh) * | 2020-12-17 | 2021-03-26 | 温州医科大学慈溪生物医药研究院 | 一种用于治疗脑中风和急性脑梗的药物组合物 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1654663A (zh) * | 2005-03-02 | 2005-08-17 | 首都医科大学 | 重组人VEGF与bFGF真核表达载体、融合蛋白及其用途 |
-
2011
- 2011-11-18 CN CN2011103720496A patent/CN102755636A/zh active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1654663A (zh) * | 2005-03-02 | 2005-08-17 | 首都医科大学 | 重组人VEGF与bFGF真核表达载体、融合蛋白及其用途 |
Non-Patent Citations (3)
| Title |
|---|
| 何祥等: "bFGF和大脑组织液合用治疗恢复期缺血性中风", 《天津医药》, vol. 27, no. 11, 30 November 1999 (1999-11-30), pages 681 - 682 * |
| 赵海等: "bFGF对大鼠脑缺血再灌注损伤保护作用的研究进展", 《沈阳医学院学报》, vol. 13, no. 2, 30 June 2011 (2011-06-30), pages 108 - 110 * |
| 韩江全等: "bFGF对大鼠脑缺血再灌注后神经元凋亡及fractalkine表达的影响", 《中国病理生理杂志》, vol. 27, no. 6, 30 June 2011 (2011-06-30), pages 1222 - 1225 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107875394A (zh) * | 2016-09-27 | 2018-04-06 | 邬芬赞 | 多聚阳离子‑bFGF复合材料及其应用和制备方法 |
| CN112546198A (zh) * | 2020-12-17 | 2021-03-26 | 温州医科大学慈溪生物医药研究院 | 一种用于治疗脑中风和急性脑梗的药物组合物 |
| CN112546198B (zh) * | 2020-12-17 | 2022-03-25 | 温州医科大学慈溪生物医药研究院 | 一种用于治疗脑中风和急性脑梗的药物组合物 |
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