CN100569797C - 一种十一肽及其用途 - Google Patents
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Abstract
本发明提供了一种新的11肽，它由11个氨基酸残基组成，其序列为异亮氨酸-天冬氨酸-苏氨酸-赖氨酸-谷氨酸-甘氨酸-异亮氨酸-亮氨酸-谷氨酰胺-酪氨酸-半胱氨酸(IDTKEGILQYC)，本发明还公开了它具有神经营养作用和治疗神经元退行性变疾病的用途。
Description
分案申请的说明
本件分案申请是申请日为2000年4月11日，申请号为00105799.5，发明名称为“一种十一肽及其制备方法和用途”的中国专利申请的分案申请。
技术领域
本发明涉及肽化学，特别是涉及一种新的11肽，其制备方法和用途。
背景技术
90年代中期曾经发现β-淀粉样肽前体蛋白(β-Amyloidprecursor protein，APP)肽链中第319-335位的肽段(即APP17肽)，具有神经营养作用，包括：促进轴突生长、增加突触密度、能保护缺血引起的脑神经元损伤。
发明内容
本发明的目的是要寻找一种肽链较APP17肽短，合成较APP17肽容易的，具有神经营养作用的多肽。经过几年的研究我们最终发现了一种新的11肽，APP11肽，它不但具有神经营养作用，而且可能治疗神经元退行性变疾病。该APP11肽是我们首次发现的一种新的多肽，迄今尚未发现有关它肽的任何报道。
本发明的APP11肽是β-淀粉样肽前体蛋白N端第63-73位肽段，由11个氨基酸残基组成，序列为异亮氨酸-天冬氨酸-苏氨酸-赖氨酸-谷氨酸-甘氨酸-异亮氨酸-亮氨酸-谷氨酰胺-酪氨酸-半胱氨酸(IDTKEGILQYC)。我们经反复研究APP N端各种不同长度的多肽片段的生物活性，最终发现并确定APP11肽具有神经营养作用。
迄今为止我们的研究发现APP11肽具有如下功能：
1.在体外，能增加人神经母细胞瘤株SY5Y细胞胞体面积和轴突长度，促进细胞增殖和存活能力。
2.改善糖尿病小鼠的学习记忆功能和海马神经元中一些重要蛋白质的表达。
3.改善糖尿病小鼠坐骨神经传导速度和一些蛋白质的表达。
以上结果表明APP11肽具有神经营养作用，并存在改善实验性神经元退行性变的能力。
具体实施方式
本发明的APP11肽采用固相法合成，固相肽合成的主要思想是：先将所要合成肽链的羧基末端氨基酸的羧基以共价键的结构同一个不溶性的高分子化合物(树脂)相连接，然后以此结合在固相载体上的氨基酸作为氨基组份，经过脱去氨基保护基并同过量的活化羧基组份反应，接长肽链。这样的步骤可以反复地多次进行下去，最后达到所需要合成的肽链的长度。下式表示了这个合成过程。
原料：
HMP树脂(P-羟甲基苯氧甲基多聚乙烯树脂)
Fmoc-AA(9-芴基甲氧羰酰基保护的氨基酸)
NMP氮甲基吡咯烷酮
DCM二氯甲烷
甲醇
哌啶
DMAP二甲基氨基吡啶
HOBT羟基苯并三唑
DCC二环己基碳二亚胺
TFA三氟乙酸
EDT 1，2-乙二硫醇
硫代苯甲醚
结晶苯酚
乙腈
仪器：
多肽自动合成仪(美国ABI431A型)
旋转蒸发仪(日本YamatoRE50)
高效液相色谱仪(美国PE151A型)
冷冻干燥机
合成方法：
称取HMP树脂100mg，取代当量是1.0meq，即0.1mmol于美国ABI431A型多肽自动合成仪的反应腔内，由合成仪自动将特定的AA按不同的顺序连接起来，偶联率达99％。反应如下：
1.氨基酸的活化(HOBt/DCC法)
Fmoc保护的氨基酸
2.联接氨基酸到树脂上
3.脱去氨基酸的Fmoc保护基
4.氨基酸的活化(HOBt/DCC法)
5.偶联
重复3-5直至合成结束，得到APP11肽的肽树脂，即IDTKEGILQYC-树脂350mg。
将肽链从树脂上切落：用TFA(三氟乙酸)切割肽链，用EDT，硫代苯甲醚，H2O作清除剂，在室温下反应3.0小时，除去切割试剂，再用乙醚萃取，得到APP11肽的粗品为100mg，收率为85％。
APP11肽的纯化
高效液相色谱分离纯化：
条件：色谱柱 C810×100mm
色谱仪 ABI 151A型 美国
流动相 A-0.1％TFA(三氟乙酸)于H2O中
B-0.1％TFA(三氟乙酸)于60％乙腈
检测波长 214nm
流速 4ml/分钟
洗脱梯度 20-60％B，30分钟
HPLC(高效液相色谱)分析
色谱柱：C18 4.6×150mm
流动相：A-0.1％TFA(三氟乙酸)于H2O中
B-0.1％TFA(三氟乙酸)于乙腈中
检测波长：214nm
流速：1ML/min
洗脱梯度：0-60％B，30分钟
分析结果见附表A.
APP11肽的鉴定
APP11肽AA组份分析结果见附表B.
序列：IDTKEGILQYC(异亮氨酸-天门冬氨酸-苏氨酸-赖氨酸-谷氨酸-甘氨酸-异亮氨酸-亮氨酸-谷氨酰胺-酪氨酸-半胱氨酸)
以Gly(甘氨酸)做标准
注：Gln(谷氨酰胺)在酸解过程中被破坏变为Glu(谷氨酸)，所以Glu(谷氨酸)为2，而Gln(谷氨酰胺)为0。
AA组份分析结果显示与目的肽的组份相符，证明合成是成功的。
表A APP11肽的高效液相色谱分析结果
表B APP11肽的氨基酸组份分析结果
0-2850 09/21/99 17:27
SAMPLS： TAG：101 CH：1 UIRL：1 PROS-NO.：1
FILE：1 CRLC-METHOD：EXT-STD TRBLE：1 CONC：AREA
NO.NAME RT HEIGHT AREA n mol n3 RATIO
1 OXSO3H 2.20 84939 1318001 0.006 0.0 0.0
3 Ass 6.82 65851 1283838 4.144 551.6 102.7
4 Thr 7.52 64327 1289162 4.009 477.4 101.8
5 Ser 8.25 1315 24460 0.076 8.0 105.6
6 Glu 9.16 145581 2685046 9.542 1403.6 100.0
7 Gly 12.24 72550 1283799 4.199 315.4 100.6
8 Ale 12.98 1004 15472 0.054 4.8 104.2
11 Ile 17.91 83288 2176202 7.790 1022.1 100.4
12 Leu 19.04 44405 1180698 4.130 541.9 102.0
13 Tyr 19.98 60254 1131260 4.200 761.1 102.6
14 s-ABA 21.82 347 14808 0.055 5.7 565.4
16 Lys 23.40 93565 1323625 4.203 614.4 101.3
17 NH3 24.72 46691 1112520 6.688 113.7 100.1
21 Ars 30.46 373 14681 0.051 8.8 143.3
TOTAL
764490 14853572 49.147 5829.2
PEAK REJ：10000
SF：1.000
SAMP-AMT：1.000
INJ-UOL：10.00
1.实验方法：采用国际公认的实验方法进行水迷宫实验、免疫组化染色和神经细胞培养。
1)水迷宫实验
动物成模后3周行水迷宫试验，每天训练2次，连续5天，第1天2个盲端，第2天3个盲端，第3、4、5天4个盲端，以头部碰到盲端为1次错误，记录每只小鼠游完全程的时间及错误反应次数。
2)海马神经原免疫组织化学研究
经固定后的鼠脑行冰冻切片，进行各种抗体的免疫组化染色。Sp免疫组化试剂盒购自Zymed Laboratories公司。按说明书进行实验。
免疫组化定量分析：(1)每组动物各3只，每只于相应海马部位连续切片，每隔2张取1张，即共取15张切片计数。(2)用奔腾Visilog5图像分析软件对进行免疫组化标记的每张切片随机选5-6个阳性细胞，测其胞浆平均灰度。
数据资料处理：计数资料用均数±标准差表示，显著性检验用方差分析法。
3)体外实验：应用人神经母细胞瘤株SY5Y，细胞培养条件为MEM培养基中加入10％胎牛血清。将SY5Y细胞分为对照组、损伤组和APP11肽保护组。
①MTT(溴化3-[4，5-二甲基噻唑-2-基]二苯基四唑，Sigma产品)代谢率测定：
将SY5Y细胞以密度为1×103/ml接种于96孔板(Costar产品)，3天后分为对照组、损伤组和APP11肽保护组，每组8孔。接种后第4天每孔加MTT(5mg/ml)20μl，37℃孵育4小时，吸出培养液，每孔加入DMSO 200μl，振摇10分钟，用酶标仪(Diagnostic Pasteur LP400)测定550nm处光密度值(OD)。
(MTT为噻唑蓝，被培养的细胞摄取后，通过线粒体代谢生成甲赞(formazane)，故线粒体活力越旺盛，甲赞生成越多，光密度值也越高。通过测定MTT代谢率，可反应细胞生存情况)
②细胞计数
③乳酸脱氢酶(LDH)漏出率测定：按照LDH测定试剂盒所示方法(比色法)，测定450nm处光密度值，同时做LDH标准曲线。
④形态学观察：应用医学影像计算机图像处理系统测定细胞轴突长度和胞体面积。
⑤统计学处理应用SPSS软件做t检验统计。
材料：由发明人按前述方法全合成的APP11肽。实验动物为小鼠和大鼠，小鼠为昆明种，体重32-37克，8周龄。大鼠为Wistar，250克左右，16周龄。
动物模型制备和防治：分为对照组(C组)、糖尿病组(DM组)和APP11肽治疗组(APP11肽+DM组)
表1.动物模型制备和给药
糖尿病小鼠模型
雄性昆明小鼠，体重32-37克，随机分为三组：正常对照组(C组)、糖尿病对照组(DM组)、APP11肽防治组(11P+DM组)。对DM组小鼠禁食12小时后按200mg/kg体重剂量腹腔注射链脲佐菌素(STZ)，三天后测非禁食血糖，大于15mM/L者认为糖尿病模型复制成功。11P+DM组于糖尿病成模后2周起至4周皮下注射APP11肽，每次0.35ug/只，每天一次。
糖尿病大鼠模型复制方法同上。
2.实验结果
1)体内实验
①糖尿病小鼠水迷宫实验结果
表2.游完水迷宫全程所需时间
*与DM组相比p＜0.05 **与DM组相比p＜0.01
表3.水迷宫测试错误反应次数
*与DM组相比p＜0.05 **与DM组相比p＜0.01
水迷宫测试结果显示：DM组游完全程时间延长，错误反应次数增多，与C组相比有非常显著性差异，与DM+APP11肽组相比有显著性差异。结果表明，DM组存在学习、记忆功能障碍，APP11肽对糖尿病小鼠的学习、记忆功能障碍有改善作用。
②神经组织免疫组化染色
表4.三组小鼠海马三种PS-1抗体阳性细胞数目比较
##与C组相比P＜0.01，**与DM组相比P＜0.01
PS-1是早老素-1，也称为早老蛋白-1。
表5.三组小鼠海马NF，NGF抗体阳性细胞数目比较
**与DM组相比P＜0.01
表6.三组小鼠海马三种PS-1抗体阳性细胞灰度比例三色值比较
##与C组相比P＜0.01，**与DM组相比P＜0.01
表7.三组小鼠海马NF，NGF抗体阳性细胞灰度比例三色值比较
**与DM组相比P＜0.01
以上结果表明，APP11肽对小鼠海马神经原NF、NGF、PS-1的表达有明显影响。
③外周神经组织和血中某些成分的改变
A)APP11肽可使糖尿病大鼠坐骨神经传导速度恢复正常
表8.APP11肽皮下注射3μg/日，2月后对大鼠传导速度的影响
*与DM+11P及C组比较P＜0.05
B)APP11肽不影响血糖及血糖调节激素。
表9.正常大鼠静推APP11肽50μg/只，对血糖的影响
表10.正常大鼠静推APP11肽10μg/只，对血糖调节激素的影响
C1：静推APP11肽前，C2：静推APP11肽后。P＞0.05
C)APP11肽对肾功能的影响
表11.皮下注射APP11肽3μg/日，2月后对肾功能的影响
*与C组比较P＜0.05
#与DM组比较P＜0.05
以上结果说明，APP11肽具有改善糖尿病大鼠神经元退变的功能，而且不是通过降低血糖途径完成的。
2)体外实验
①MTT代谢率测定结果显示：APP11肽组MTT代谢率高于对照组，有显著性差异。
表12.APP11肽对SY5Y MTT代谢率影响
*与对照组相比P＜0.05
②细胞计数结果显示：APP11肽组细胞数在第1、2、3天均高于对照组，有显著性差异。
表13.细胞计数观察APP11肽对SY5Y生长的影响(x±s)
*与对照组相比：P＜0.05，**与对照组相比：P＜0.01
③LDH漏出率测定结果显示：经细胞数标化后，APP11肽组LDH与对照组相比均有高度显著性差异。
表14.APP11肽对SY5Y LDH漏出率的影响
**与对照组相比：P＜0.01
④形态学观察：APP11肽能促进神经元轴突生长和胞体增大。
轴突长度：C组42.22±22.07 APP11肽组89.67±39.38
胞体面积：C组749.69±207.65 APP11肽组1101.66±355.31
我们认为APP11肽治疗神经原退变的可能机理是：糖尿病动物模型具有神经退行性变的表现，神经营养因子NF、NGF等表达减少，提示可能由于支持神经元功能的神经存活因子减少，使多种存活因子的受体后传导信号强度低于阈值，神经元基因转录不能被激活而导致神经元退变。给予外源性APP11肽后，可通过信号传导过程中相互对话增强神经存活因子的信号传导而激活神经元功能，治疗神经元退变。因为APP11肽并不影响血糖及其调节激素，故其作用与胰岛素无关。APP11肽的神经营养作用可能通过G蛋白偶联受体，激活胰岛素受体底物-1，最终起到防治糖尿病神经元退变的作用。
APP11肽将是世界上第一个以治疗神经退行性疾病的药物。体内外研究表明APP11肽有增强神经元的存活能力和减少有害因子的作用，所以可预期APP11肽的治疗谱至少可包括：
1.早、中期阿尔兹海默氏病(AD)
2.糖尿病神经病变
3.脑卒中对神经元损害
4.绝经期综合症
5.脑和神经损伤
6.帕金森氏病
但是APP11肽还有很大潜力。最近我们发现它对糖尿病肾病有治疗作用，对实验性肺动脉高压有降压作用，因此对小血管病变的治疗效果如能重复则对理解和治疗小血管病变、提供药物作用新的靶位-改善外周神经退行性病变，这将成为防治小血管病变如高血压、糖尿病等的新概念。
本发明的APP11肽可以单独使用，也可与各种接受的载体、赋形剂制成适用的剂形。
附件1：研究方法的主要参考文献
1.Fuller SJ，Storey E，et al.Intracellular production of βA4Amyloid of Alzheimer’s Disease：Modulation by phosphoralytion andlack of coupling to the secretion of the Amyloid precursor protein.1995，34，8091-8098，Biochemistry
细胞内产生老年性痴呆的βA4淀粉样肽：通过磷酸化调节和缺乏淀粉样肽前体蛋白分泌的偶合 生物化学杂志
2.Ostrerova N，Petrucelli L，et al.α-Synuclein shares physical andfunctional homology with 14-3-3 proteins.The Journal of Neurosci.1999，19(14)：5782-5791
α-共核蛋白与14-3-3蛋白在生理功能上有同源性 神经科学杂志
3.Faircloth GT，Stewart D，Clement JJ.A simple screeningprocedure for the quantitative measurement of cytotoxicity to restingprimary lymphocyte cultures.Journal of Tissue Culture Methods.1988，11(4)：201-205
原代淋巴细胞培养中细胞毒性定量，测定的单纯筛查步骤 组织培养法杂志
4.赵咏梅，赵志炜，姬志娟等。APP17肽对糖尿病小鼠微管结构和tau蛋白磷酸化有关酶类的影响 中华老年医学杂志18(5)：306 1999
钱玉英赵咏梅等。APP17肽对糖尿病小鼠学习、记忆功能及海马NT-3、胆碱乙酰化酶神经元表达的影响 中华老年医学杂志18(6)：1999
Claims (1)
1.序列为异亮氨酸-天冬氨酸-苏氨酸-赖氨酸-谷氨酸-甘氨酸-异亮氨酸-亮氨酸-谷氨酰胺-酪氨酸-半胱氨酸的11肽在制备具有神经元营养作用以及治疗糖尿病神经病变和早中期阿尔兹海默氏病的药物中的应用。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994009808A1 (en) * | 1992-10-23 | 1994-05-11 | The Regents Of The University Of California | Substances having the growth-promoting effect of amyloid precursor protein |
| US5958883A (en) * | 1992-09-23 | 1999-09-28 | Board Of Regents Of The University Of Washington Office Of Technology | Animal models of human amyloidoses |
| WO1999058564A1 (en) * | 1998-05-08 | 1999-11-18 | Norsk Hydro Asa | Frameshift mutants of beta-amyloid precursor protein and ubiquitin-b and their use |
-
2000
- 2000-04-11 CN CNB2007100050477A patent/CN100569797C/zh not_active Expired - Fee Related
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5958883A (en) * | 1992-09-23 | 1999-09-28 | Board Of Regents Of The University Of Washington Office Of Technology | Animal models of human amyloidoses |
| WO1994009808A1 (en) * | 1992-10-23 | 1994-05-11 | The Regents Of The University Of California | Substances having the growth-promoting effect of amyloid precursor protein |
| WO1999058564A1 (en) * | 1998-05-08 | 1999-11-18 | Norsk Hydro Asa | Frameshift mutants of beta-amyloid precursor protein and ubiquitin-b and their use |
Non-Patent Citations (2)
| Title |
|---|
| APP17肽对糖尿病小鼠学习记忆功能和海马NT-3、胆碱乙酰化酶神经元的影响. 钱玉英等.中华老年医学杂志，第18卷第6期. 1999 * |
| APP17肽对糖尿病小鼠微管结构和tau蛋白磷酸化有关酶类的影响. 赵咏梅等.中华老年医学杂志，第18卷第5期. 1999 * |
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