CN1189482C - 一种新的血管发生抑制剂 - Google Patents
一种新的血管发生抑制剂 Download PDFInfo
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- CN1189482C CN1189482C CNB998169072A CN99816907A CN1189482C CN 1189482 C CN1189482 C CN 1189482C CN B998169072 A CNB998169072 A CN B998169072A CN 99816907 A CN99816907 A CN 99816907A CN 1189482 C CN1189482 C CN 1189482C
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Abstract
本发明提供了一种新的血管发生抑制剂,LK68,其氨基酸序列与人载脂蛋白(a)kringle结构域IV36,IV37和V38的一致,一种编码LK68的cDNA序列,一种含有所述cDNA的重组表达载体,一种用所述重组表达载体转化的重组微生物,以及LK68作为抗癌剂的新用途和治疗血管发生介导的疾病的方法。LK68,LK6,LK7和LK8对培养的内皮细胞增殖和内皮细胞迁移表现抑制活性。LK68和其单一kringle还抑制鸡胚绒毛尿囊膜(CAM)中毛细血管的正常发育。还显示LK68的系统给药抑制原发性肿瘤生长,其与诱导肿瘤的血管发生的抑制有关。因此,LK68蛋白,其单一kringle或它们的功能等同物可以用于开发有效的抗癌剂,该抗癌剂对血管发生介导的疾病,包括癌症、类风湿性关节炎、牛皮癣、眼血管形成性疾病等高度有效。
Description
发明背景
发明领域
本发明涉及一种新的血管发生抑制剂,LK68,其氨基酸序列与人载脂蛋白(a)kringle结构域IV36、IV37和V38相同,更具体地说,本发明涉及一种LK68的氨基酸序列,编码LK68的cDNA序列,包含该cDNA的重组表达载体,用该重组表达载体转化的重组微生物,以及LK68作为抗癌剂的新用途,和治疗血管发生介导的疾病的方法。
现有技术描述
血管发生是生成新血管进而形成组织或器官的生物过程。在正常生理条件下,人和动物仅在非常特殊的限制条件下才进行血管发生。例如,正常情况下在伤口愈合、胎儿和胚胎发育以及黄体、子宫内膜和胎盘形成时观察到血管发生。据报道,一些血管发生调节剂严格控制新血管生长(参见:Folkman,J.,Nature Med.,1:27-31,1995a),血管发生表型的开关依赖于血管生成刺激物的上调作用与血管生成抑制物的下调作用之间的净平衡。
据报道血管生成过程的不平衡导致病理性疾病例如糖尿病性视网膜病、类风湿性关节炎和牛皮癣(参见:Folkman,J.,Nature Med.,1:27-31,1995a)。尤其是原发性和转移性肿瘤都需要血管生成的血管恢复生长(参见:Folkman,J.,New Engl.J.Med.,285:1182-1186,1971;Folkman,J.,J.Biol.Chem.,267:10931-10934,1992)。如果这种血管发生活性能够被抑制或消除,那么肿瘤即使存在,也将停止生长。一些报道表明,抑制肿瘤的血管发生可以提供一种长期控制疾病的可操作方法。阻断血管发生的阳性调控剂或应用阴性调控剂抑制血管发生导致实验肿瘤的延迟或抑制。如果这种血管发生活性能够被抑制或消除,那么肿瘤即使存在,也将停止生长。而且,在疾病状态,防止血管发生能够有效转移由于微血管系统的侵害造成的损害。因此,旨在控制血管发生过程的治疗会导致这类疾病的消除或缓解。
因此,需要一种新的血管发生抑制剂,其能够抑制血管的不期望的生长,特别是生长成为肿瘤。含有血管发生抑制剂的抗癌剂应该能够抑制前转移性肿瘤中的内源性生长因子的活性,并防止肿瘤中毛细血管的形成,由此抑制肿瘤的生长。这种抗癌剂还应该能够调节其它血管发生过程,例如伤口愈合和生殖过程的毛细血管的形成。最后,这种抗癌剂和抑制血管发生的方法应该优选为非毒性的并产生较少副作用。
迄今为止,本领域已经鉴定了至少10中内源性血管发生抑制物(参见:O′Reilly,M.S.等,Cell,88:277-285,1997)。这类分子中的一种是制管张素,其由血纤维蛋白溶酶原kringle I到IV构成(参见:O′Reilly,M.S.等,Cell,79:315-328,1994)。当系统应用时,制管张素有效抑制原发性肿瘤的生长和转移,且无毒性,同时也抑制由bFGF诱导的血管发生(参见:O′Reilly,M.S.等,Nature Med.,2:689-692,1996)。这些抗肿瘤作用伴随肿瘤块中微血管密度的显著降低,表明血管发生的抑制与肿瘤生长的抑制有关。
Kringle是由大约80个氨基酸和三个分子内二硫键构成的蛋白质结构域。Kringle结构在一些蛋白质中发现,例如凝血酶原(参见:Walz,D.A.等,Proc.Natl.Acad.Sci.,U.S.A.,74:1969-1973,1977)、血纤维蛋白溶酶原(参见:Ponting,C.p.,Blood Coagul.& Fibrinolysis,3:605-614,1992)、尿激酶(参见:Pennica,D.等,Nature,301:579-582 1983)、肝细胞生长因子(参见:Lukker,N.A.等,Protein Eng.,7:895903,1994)和载脂蛋白(″apo″)(a)(参见:McLean,J.W.等,Nature,330:132-137,1987)。这些结构域看起来是一种独立折叠单元,但它们的功能作用尚不清楚。以前的报道指出kringle结构能够作为血管发生过程中内皮细胞迁移和增殖的抑制剂。特别是凝血素的kringle 2和血纤维蛋白溶酶原的kringle 1-4、和5已经表明抗血管发生(参见:Ji,W.R.等,FASEB J.,15:1731-1738,1998a;Ji,W.R.等,Biochem.Biophys.Res.Commun.,247:414-419,1998b;Cao,Y.等,J.Biol.Chem.,271:29461-29467,1996;Cao,Y.等,J.Biol.Chem.,272:22924-22928,1997;Barendsz-Janson,A.F.,J.Vasc.Res.,35:109-114,1998;Lee,T.H.等,J.Biol.Chem.,273:28805-28812,1998)。
载脂蛋白(a),一种具有kringle结构的蛋白质,是一种新的血管发生抑制剂的候选物。Apo(a)与apoB-100共价结合,是形成脂蛋白(a)的低密度脂蛋白(LDL)的主要蛋白成分(参见:Fless,G.M.,J.Biol.Chem.,261:8712-8718,1986)。脂蛋白(a)浓度的升高代表动脉粥样硬化的一种主要独立风险因素(参见:Armstrong,V.W.等,Artherosclerosis,62:249-257,1986;Assmann,G.,Am.J.Cardiol.,77:1179-1184,1996;Bostom,A.G.等,JAMA,276:544-548,1996)。虽然已经报道了apo(a)的几种病原性活性,但其生理作用还未曾确定(参见:Lawn,R.M.等,J.Biol.Chem.,271:31367-31371,1996;Scanu,A.M.和Fless,G.M.,J.Clin.Invest.,85:1709-1715,1990;Utermann,G.,Science,246:0904-910,1989)。
Apo(a)含有两种类型的kringle结构域和一种无活性的蛋白酶样结构域:前37个kringle结构域与血纤维蛋白溶酶原kringle IV有~75%等同性,最后kringle结构域与血纤维蛋白溶酶原kringle V有90%的等同性。有趣的是,kringle IV样结构域在不同的人的apo(a)基因的等位基因中存在15-40拷贝。从这一点上说,应用Apo(a)kringle结构研制一种肿瘤血管发生和生长的抑制剂是可行的。
发明概述
按照本发明,本发明人克隆和表达了一种作为重组蛋白LK68的含有IV36,IV37和V38的人apo(a)kringles并发现:LK68蛋白和其单一kringle,LK6,LK7和LK8能够在体外抑制内源性生长因子例如bFGF的血管发生活性;并且它们可以用作抗癌制剂的活性成分。
因此,本发明的第一个目的在于提供一种新的LK68蛋白,其由人apo(a)kringle结构域IV36,IV37和V38构成,以及编码LK68蛋白的cDNA。
本发明的第二个目的在于提供一种新的重组载体,其含有编码人apo(a)kringle结构域IV36,IV37和V38的cDNA。
本发明的第三个目的在于提供一种抗癌剂,其含有LK68蛋白或其单一kringle,LK6,LK7和LK8作为活性成分。
本发明的第四个目的在于提供一种通过应用LK68蛋白治疗由血管发生介导的疾病的方法。
附图简述
通过下面结合附图的描述,本发明的上述和其它目的和特征将变得更加清楚,其中:
图1是分析在大肠杆菌中表达的重组LK68蛋白的SDS-聚丙烯酰胺凝胶电泳的照片。
图2是显示LK68对鸡绒毛尿囊膜(CAM)血管发生的抑制作用的照片。
图3(A)是显示CAM中血管生长的抑制作用随LK68变化的图。
图3(B)是显示CAM中血管生长的抑制作用随单一kringles,LK6,LK7,LK8和对照变化的图。
图4(A)是显示重组LK68和制管张素对BCE细胞增殖的抑制作用的图。
图4(B)是显示重组LK6,LK7和LK8对BCE细胞增殖的抑制作用的图。
图4(C)是显示重组LK68和LK8对HUVEC细胞增殖的抑制作用的图。
图5(A)是显示在存在重组LK68和LK8条件下,LLC细胞的BrdU标记指数的图。
图5(B)是显示在存在重组LK68和LK8条件下,Y1细胞的BrdU标记指数的图。
图5(C)是显示在存在重组LK68和LK8条件下,TIB74细胞的BrdU标记指数的图。
图5(D)是显示在存在重组LK68和LK8条件下,CHO细胞的BrdU标记指数的图。
图5(E)是显示在存在重组LK68和LK8条件下,MSF细胞的BrdU标记指数的图。
图5(F)是显示在存在重组LK68和LK8条件下,NIH3T3细胞的BrdU标记指数的图。
图6(A)是显示重组LK68,LK8和PK5对HUVEC细胞迁移的抑制作用的图。
图6(B)是显示重组LK68,LK6,LK7和LK8对HUVEC细胞迁移的抑制作用的图。
图7是显示制管张素,重组LK68,LK6,LK7,和LK8,以及单一kringle的组合对BCE细胞迁移的抑制作用的图。
图8以总体积作为时间的函数,显示施用LK68对已移植Lewis肺癌细胞的小鼠的影响。
图9(A)到9(C)是显示通过苏木精和曙红(H/E)染色的Lewis肺癌细胞组织学分析照片。
图10以总体积作为时间的函数,显示给已移植人肺癌A549细胞的裸鼠施用LK68的作用。
发明详述
本发明提供了一种新的蛋白质LK68,其可以从人载脂蛋白(″apo″)(a)kringle作为重组蛋白克隆和表达。LK68蛋白由人载脂蛋白(a)kringle结构域IV36(氨基酸8到80),IV37(氨基酸122到194)和V38(氨基酸226到300)的氨基酸序列以连续的方式构成(参见:SEQ ID NO:2)。LK68的前两个kringle结构域(即IV36和IV37)与人血纤维蛋白溶酶原kringle IV同源,第三个kringle结构域V38与人血纤维蛋白溶酶原kringle V同源。本发明还提供了编码LK68蛋白的cDNA(参见:SEQ ID NO:1)以及含有所述cDNA和表达载体例如pET载体系列的重组载体。
在描述本发明的kringle结构域时,人载脂蛋白(a)kringle IV36,IV37和V38分别简称为KIV36,KIV37和KV38;应用LK68意味着应用含有所述三种kringle结构域的重组蛋白;应用LK6,LK7和LK8分别意味着应用KIV36,KIV37和KV38重组蛋白。
由于载脂蛋白(a)含有血纤维蛋白溶酶原-IV和V型kringle结构域,设想载脂蛋白(a)可能具有抗血管发生活性。有一个实验证据表明载脂蛋白(a)可能具有作为肿瘤血管发生和生长抑制剂的生物活性(参见:Trieu,V.N.和Uckun,F.M.,Biochem.Biophys.Res.Commun.,257:714,1999)。据报道LL/2(Lewis肺癌)肿瘤生长在apo(a)转基因小鼠中延迟,apo(a)转基因小鼠的LL/2肿瘤的微血管密度比作为对照的野生型小鼠的低。
在这种情况下,本发明人设想LK68蛋白,其单一kringle或它们的功能等同物可能具有抗-血管发生活性。为了证明所述抗-血管发生活性,研究重组LK68和其单一kringle(即,LK6,LK7和LK8)是否在体外以及在体内是有效的抗血管发生因子。结果发现,LK68,LK6,LK7和LK8对培养的内皮细胞的增殖和对内皮细胞迁移表现抑制活性。LK68和其单一kringle还抑制鸡胚绒毛尿囊膜(CAM)中毛细血管的正常发育。并且显示LK68的系统给药抑制原发性肿瘤生长,其与诱导肿瘤的血管发生的抑制有关。既然每个单一kringle蛋白,LK6,LK7和LK8均表现抗血管发生活性,料想也抑制原发性肿瘤生长或转移。
因此,LK68蛋白、其单一kringle或它们的功能等同物可以应用于开发强效抗癌剂,其对于由血管发生介导的疾病,包括类风湿性关节炎、牛皮癣、或眼血管发生性疾病等具有高度有效性。
另外,LK68蛋白,其单一kringle或它们的功能等同物还可以与其它组合物和方法联合应用于治疗疾病。例如,可以常规地用手术、放疗或化疗结合LK68、其单一kringle或它们的功能等同物治疗肿瘤,接着可以给患者施用LK68、其单一kringle或它们的功能等同物,以延长微转移的休眠期,同时稳定和抑制任何残存原发性肿瘤的生长。
下面进一步以实施例说明本发明,它们不应当用来限制本发明的范围。
实施例1:重组LK68的克隆和表达
为了验证人apo(a)kringle的抗血管发生活性,本发明克隆和表达了含有IV36,IV37和V38的最后三个kringle,作为一种重组蛋白LK68。从人肝cDNA PCR扩增了apo(a)从核苷酸12052到12975的DNA片段(参见:McLean J.W.等,Nature,330:132,1987),并将得到的924-bp NdeI-BamHI片段连接到大肠杆菌表达载体pETlla(Novagen,USA)中。对于PCR扩增,应用标准PCR方案,使用寡核苷酸引物A(SEQ ID NO:9)和F(SEQID NO:14)(参见:表1)。该克隆命名为″pETlla/LK68″,其编码包括人apo(a)kringle结构域,IV36,IV37和V38在内的308个氨基酸(参见:SEQ NOID:2)。该克隆的前两个kringle结构域,IV36和IV37与人血纤维蛋白溶酶原kringle IV同源,第三个kringle结构域V38与人血纤维蛋白溶酶原kringle V同源。
该克隆的核苷酸序列在两个方向上都得到验证。当将该克隆的核苷酸序列与人apo(a)的相同区域(参见:McLean J.W.等,Nature,330:132,1987)比较时,它们的核苷酸序列除了核苷酸554位上发生一个碱基改变外其余都相同。我们的克隆在该位置含有一个胞嘧啶,而McLean等(参见:McLean J.W.等,Nature,330:132,1987)报道的序列中为胸腺嘧啶(thymidine),导致从Met到Thr的一个氨基酸改变。其它研究组也曾报道过该取代(参见:Van der-Hoek,Y.Y.等,Hum.Mol.Genet.,2:361-366,1993;LoGrasso,P.V.等,J.Biol.Chem.,269:21820-21827,1994),并且看起来是apo(a)的主要等位基因。
用一种表达质粒pETlla/LK68转化大肠杆菌BL21(DE3),在下面的条件下表达重组LK68蛋白。用10ml过夜培养的携带pETlla/LK68质粒的大肠杆菌BL21(DE3)接种1升含氨苄青霉素的Luria-Bertani肉汤培养基,在37℃振摇培养。当培养物的OD600达到0.4-0.6时,加入终浓度为1mM的异丙基硫代-β-D-半乳糖苷(IPTG)。细胞经诱导后再生长4小时。在4℃,8000xg离心30分钟收获细胞。将这些细胞沉淀超声,通过SDS-PAGE分析过表达的蛋白质(参见:图1)。在图1中,Mr代表分子量标记(Boehringer Mannheim,Germany),1道,无IPTG诱导的重组LK68蛋白的表达;2道,有IPTG诱导的重组LK68蛋白的表达。分子量为37kDa的重组LK68蛋白在大肠杆菌中很好地表达,通过扫描凝胶的图象分析表明,该蛋白累积到总蛋白的约20-30%。由此制备的转化体被命名为′大肠杆菌BL21/LK6-8′,并于1999年6月9日保藏在国际保藏机构,韩国典型培养物保藏中心,#52 Oun-dong,Yusong-ku,Taejon 305-333,韩国,保藏号为No.KCTC0633BP。
各单一kringle结构域,IV36,IV37和V38分别如上述克隆到表达载体pET15b中。用于克隆的寡核苷酸引物在表1中列出:即用于克隆KIV36的是A(SEQ ID NO:9)和D(SEQ ID NO:12);用于克隆KIV37的是B(SEQID NO:10)和E(SEQ ID NO:13);用于克隆KV38的是C(SEQ ID NO:11)和F(SEQ ID NO:14)。这三对寡核苷酸引物用于以标准PCR方案进行PCR扩增,得到的克隆命名为″pET15b/LK6″、″pET15b/LK7″和″pET15b/LK8″,它们各自分别包括单一人apo(a)kringle结构域IV36,IV37和V38。用各表达质粒pET15b/LK6,pET15b/LK7和pET15b/LK8转化E.coli BL21(DE3)感受态细胞。如此由质粒pET15b/LK6制备的转化体命名为′大肠杆菌BL21(DE3)/LK6′,于1999年9月3日保藏在国际保藏机构,韩国典型培养物保藏中心,#52 Oun-dong,Yusong-ku,Taejon 305-333,韩国,保藏号为No.KCTC0655BP。如此用质粒pET15b/LK7制备的转化体命名为′大肠杆菌BL21(DE3)/LK7′,于1999年9月3日保藏在国际保藏机构,韩国典型培养物保藏中心,与上述相同地址,保藏号为No.KCTC0656BP。如此用质粒pET15b/LK8制备的转化体命名为′大肠杆菌BL21/LK8′,于1999年6月9日保藏在国际保藏机构,韩国典型培养物保藏中心,与上述相同地址,保藏号为No.KCTC0634BP。
重组LK6,LK7和LK8蛋白在与含N-末端His-tag的融合蛋白相同的条件下表达。用pET His-tag系统,在生产商推荐的条件下纯化每种过表达的重组LK6,LK7和LK8蛋白。
表1.用于PCR克隆的寡核苷酸引物
核苷酸序列* | 描述 | 位置** | SEQ ID NO. |
A.TCCATATGAAAAGCCCTGTGGTCCAGGAT | K36-5′ | 12052-12072 | 9 |
B.CAGTCCATATGGTCCGCCAGTGCTACCATGGCA | K37-5′ | 12406-12427 | 10 |
C.GGAATTCCATATGGAACAGGACTGCATGTTT | K38-5′ | 12718-12735 | 11 |
D.CGGGATCCTTAACCTGATTCTGTTTC | K36-3′ | 12310-12323 | 12 |
E.CGGGATCCTTAGACCACAGTCCCTTC | K37-3′ | 12658-12671 | 13 |
F.CGGGATCCTTAAGAGGATGCACA | K38-3′ | 12964-12975 | 14 |
*限制性位点,为了克隆方便,加入NdeI和BamHI(下化线)。
**参见:McLean等,Nature,330:132,1987,对于核苷酸序列(登记号为X06290)。
实施例2:重组LK68的纯化
为了生产重组LK68,在5L Bioflow III生物反应器(New BrunswickScientifics,Edison,USA)中的下列培养基中进行高细胞密度发酵:4%(w/v)酵母提取物,4%(w/v)甘油,1%(w/v)磷酸氢二钠,0.2%(w/v)磷酸二氢钾和50μg/ml氨苄青霉素。当细胞在600nm的吸收值达到100,用1mMIPTG诱导蛋白质表达,然后用饲养培养基(29%(w/v)酵母提取物,39%(w/v)甘油和0.5%(w/v)硫酸镁)进行9小时的DO-stat饲养分批培养(fed-batch)。8000xg离心30分钟收获细胞。每个发酵过程产生约80g细胞/L(湿重)。
为了评价LK68在大肠杆菌细胞的可溶性细胞组分还是不溶性细胞组分中表达,本发明人分析了这些组分中LK68的表达。该分析表明LK68位于不溶性细胞组分中。因此,必须使LK68变性、重折叠和再氧化二硫键。通过使用去氧胆酸盐和其它变性剂,不溶性LK68蛋白以包涵体形式纯化,纯度大于95%。然后,用7M脲使包涵体溶解,通过快速稀释法和平衡透析机制将其折叠成天然构型。在折叠缓冲液中,纯化的包涵体可以方便地再折叠,且无可检测的蛋白凝聚。透析后,通过赖氨酸琼脂糖凝胶4B亲合色谱纯化蛋白质。结合到赖氨酸琼脂糖凝胶上的蛋白质用ε-ACA(ε-氨基-n-己酸)特异性洗脱。这表明定位在重折叠蛋白的KIV37 kringle中的赖氨酸结合位点是完全有功能的。用0.1Mε-ACA对LK68进行的亲合洗脱产生约3mg蛋白/g细胞(湿重)。随后用多粘菌素B小珠(Sigma Chemical Co.,USA)进行层析,消除所有内毒素,用鲎变形虫状细胞裂解物分析试剂盒(Biowhittaker Inc.,USA)确定残存的内毒素活性。通过SDS-PAGE分析纯化的蛋白质,然后将其在-20℃储藏,直到使用。LK68的计算pI值为6.13。纯化的LK68 N-末端氨基酸序列通过氨基酸测序证实。
实施例3:鸡绒毛尿囊膜分析
为了确定LK68在体内是否有抗血管发生活性,本发明人考察了它抑制绒毛尿囊膜(″CAM″)中毛细血管发育的能力(参见:Lee,T.H.等,J.Biol.Chem.,273:28805-28812,1998)。将受精三天的鸡蛋在37℃孵育,提取卵清蛋白后,开一个小口。孵育两天后,将一个含有重组LK68蛋白的Thermanox盖玻片(Nunc Inc.,USA)应用于个体胚的CAM。48小时后,将20%的脂肪乳注射到胚的绒毛尿囊中,考察Thermanox周围的血管形成(参见:图2)。在图2中,左照片显示CAM中毛细血管的正常发育;右照片显示LK68对CAM血管发生的抑制作用。
当给CAM施用剂量范围为3-5pg的LK68时,受测试的100个鸡蛋中超过60%在应用样品的周围显示无血管区,表明毛细血管的生长受到抑制。对于每种kringle结构域的重组蛋白,例如LK6,LK7或LK8,60-70%受测试蛋在1g/CAM的剂量范围时,显示抑制作用(参见:图3(A)和3(B))。该体内研究显示apo(a)kringle结构域具有抗-血管发生活性,并且LK68及单一kringle蛋白是血管发生的强效抑制剂。在所有受测试的鸡胚中,没有任何毒性的证据。
实施例4:内皮细胞增殖的抑制
在下述条件下分析重组LK68,LK6,LK7和LK8蛋白对bFGF刺激的牛毛细血管内皮(BCE)细胞增殖的抑制活性。BCE细胞在含10%小牛血清(BCS)和3ng/ml bFGF(Upstate Biotechnology,USA)的DMEM中生长。将约3000个细胞加入96-孔组织培养平板的各个孔中,在37℃,5%CO2大气下培养。培养18小时后,用含0.5%BCS的DMEM替换培养基,并将实验样品加到各孔中。30分钟温育后,加bFGF至终浓度为1ng/ml。通过[3H]胸苷掺入法测定细胞数目。实验进行三次。
如图4所示,确定了LK68,LK6,LK7和LK8以剂量依赖方式特异性抑制BCE细胞增殖。当使用制管张素作为阳性对照时,所有受测试的Apo(a)kringle蛋白在该实验条件下看起来更为有效。测定LK68的半数最大抑制浓度(ED50)约为200-250nM,LK6约为140-170nM,LK7约为10-20nM,LK8约为10-20nM(参见:图4(A)和4(B))。
在下述条件下分析重组LK68和LK8蛋白对bFGF刺激的人脐静脉内皮细胞(HUVEC)增殖的抑制活性。HUVEC(美国典型培养物保藏中心,USA)在含10%热灭活的胎牛血清(″FBS″)(Hyclone,USA),30μg/ml内皮细胞生长添加剂(ECGS)(Sigma Chemical Co.,USA),和100μg/ml肝素(Sigma Chemical Co.,USA)的F12K培养基中生长。将细胞以2000/孔的密度接种96-孔组织培养平板中。细胞在37℃,5%CO2温育18小时,用无血清的培养基清洗一次,然后加入含有0.5%FBS的F12培养基。用不同浓度的样品处理细胞,并温育30分钟。然后,分别将终浓度为30μg/ml,100μg/ml和5ng/ml的ECGS、肝素和bFGF(Upstate Biotechnology,USA)加入细胞。温育48小时后,应用5-溴-2′-脱氧尿苷(BrdU)(BoehringerMannheim,USA)通过细胞增殖ELISA测定细胞数目。实验进行三次。
如图4(C)所示,确定了LK68以及LK8以剂量依赖方式特异性抑制HUVEC细胞增殖。
在存在LK68或单一kringle蛋白例如LK6,LK7和LK8时,BCE或HUVEC细胞的形态看起来类似未处理细胞的形态。除此而外,除去LK68后,细胞增殖可以通过bFGF刺激恢复。这些结果表明LK68以及单一kringle蛋白对毛细血管内皮细胞没有细胞毒性。而且,该抑制活性看起来对内皮细胞,例如BCE和HUVEC细胞是特异性的。另外,LK68和LK8未显示抑制非内皮细胞型细胞,例如CHO细胞、小鼠皮肤成纤维细胞NIH3T3细胞、小鼠Lewis肺癌细胞、小鼠肾上腺瘤Y1细胞和小鼠胎肝/SV40转化细胞系TIB74的增殖(参见:图5(A)到5(F))。图5(A)到5(C)代表各种肿瘤细胞例如LLC、Y1、和TIB74的敏感度,图5(D)到5(F)分别代表各种正常细胞系例如CHO,MSF,和NIH3T3的敏感度。
实施例5:内皮细胞迁移的抑制作用
在8-mm孔的Transwells(Costar,USA)中进行细胞迁移分析。简单地说,将各孔涂布纤连蛋白(25μg/ml)(Sigma Chemical Co.,USA)过夜,将HUVEC以2000/孔的密度接种到上层腔中的100μl含有0.4%胎牛血清(FCS)的Dulbecco′s改进的Eagle′s培养基中。将500μl含有0.4%FCS的DMEM加到下层腔中,在37℃温育1小时。将1μM浓度的实验样品加入到上层腔中,将25ng/ml的bFGF加入到下层腔中。温育5小时后,用棉签擦拭掉上层腔中的细胞后,对穿过纤连蛋白铺板膜的细胞定量。用Diff-Quik染料(Dade Behring Inc.,USA)按照生产商的说明书设置的方法对穿过膜的细胞染色,然后在100x放大倍数下记数。实验进行两次。
用碱性FGF(25ng/ml)刺激HUVEC细胞迁移。在剂量为1μM,LK68和单一kringle蛋白例如LK6,LK7和LK8将bFGF-诱导的HUVEC细胞迁移完全抑制到未诱导的对照水平(参见:图6(A)和6(B))。在图6中,(-)CON代表未诱导的对照,(+)CON代表bFGF-诱导的阳性对照。
如上述用BCE细胞进行迁移分析。应用两种不同浓度的LK68或单一kringle蛋白,所有受测试的Apo(a)kringle蛋白显示对BCE细胞迁移具有抑制作用。另外,LK68和其单一kringle蛋白对BCE细胞迁移的抑制比制管张素(AS)更有效(参见:图7)。
实施例6:对原发性肿瘤生长的抑制
实施例6-1:Lewis肺癌
对雄性6到8-周龄的C57BL6/J小鼠移植Lewis肺癌。小鼠背部最接近中线的皮下注射在0.1ml生理盐水中的1×106细胞。当肿瘤直径达到约5mm,对携带肿瘤的小鼠在远离肿瘤的部位皮下注射PBS中的LK68(100mg/kg体重)悬浮液。小鼠对照组仅接受虚拟治疗,仅用PBS处理。在治疗期间,每天测量肿瘤大小;用公式宽2×长×0.52计算体积,确定最后一个时间点处理组对对照组肿瘤体积(T/C)的比值。治疗进行8天,8天后所有小鼠被处死,摘下肿瘤(参见:图8)。如图8所示,可以很清楚地确定LLC原发性肿瘤的生长被系统性LK68治疗强烈抑制;剂量为100mg/kg的LK68的仅7天的治疗导致肿瘤荷重显著消退。
为了比较处理小鼠和对照小鼠的肿瘤,还从血管密度和出血形成,以及形态外观方面进行了组织学分析(参见:图9(A)到9(C))。在图9(A)到9(C)中,9(A)显示PBS-处理的对照,9(B)是10mg/kg体重LK68-处理的LLC肿瘤,9(C)是100mg/kg体重LK68-处理的LLC肿瘤。通过苏木精和曙红(H/E)染色,在LK68-处理的肿瘤中观察到明显的组织学差异:即肿瘤细胞不完整,形态学上非存活;肿瘤周围检查到带状坏死。而且,LK68-处理的肿瘤中血管密度降低。在所有用重组LK68处理的小鼠中,没有出现炎症或出血的证据。
实施例6-2:人肺癌
该实验中使用的四周龄远系繁殖的雌性nu/nu裸鼠在无菌环境下养殖。笼子、草垫、食物和水都是高压灭菌的。小鼠以12-hr光/12-hr暗循环生长。人肺癌细胞(A549,购自韩国细胞系库)在添加10%热灭活的FBS和抗生素的RPMI 1640培养基中维持生长。将大约2×107个人肺癌细胞皮下注射到裸鼠背部近中线部位。当肿瘤移植7天后,肿瘤可触知时,用剂量为100mg/kg体重的LK68处理小鼠。对照组仅用PBS处理。治疗持续17天。每隔一天测量肿瘤尺寸。
LK68的处理减弱了肿瘤生长:即,LK68-处理的A549肿瘤比对照动物的肿瘤小大约57.5%(参见:图10)。在所有处理小鼠中,没有显示任何毒性。只要给药,连续治疗使肿瘤始终维持在休眠状态。这些数据充分表明LK68的抗血管发生作用可以用于靶向多种原发性恶性肿瘤。
如前面清楚地举例说明和证明的,本发明提供了一种新的血管发生抑制剂,LK68,其氨基酸序列与人apo(a)kringle结构域IV36,IV37和V38的一致,一种编码LK68的DNA序列,一种含有所述DNA的重组表达载体,一种用所述重组表达载体转化的重组微生物,LK68作为抗癌剂的用途,及治疗血管发生介导的疾病的方法。
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Claims (7)
1.具有SEQ ID NO:2序列的LK68蛋白,由人载脂蛋白(a)kringle结构域IV36,IV37和V38的氨基酸序列以连续方式组成。
2.一种具有SEQ ID NO:1序列的cDNA序列,其编码权利要求1的LK68蛋白。
3.一种重组表达载体pET11a/LK68,含有权利要求2的cDNA,其表达权利要求1的LK68蛋白。
4.用权利要求3的重组表达载体pET11a/LK68转化的大肠杆菌BL21/LK6-8,保藏号为KCTC0633BP。
5.一种用于抗癌剂的药物组合物,其含有权利要求1的LK68蛋白为活性成分。
6.一种用于治疗血管发生介导的疾病的药物组合物,其包含治疗有效量的权利要求1的LK68蛋白。
7.权利要求6的药物组合物,其中所述血管发生-介导的疾病是癌症、类风湿性关节炎、牛皮癣、或眼血管发生性疾病。
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US7812010B2 (en) * | 2003-01-02 | 2010-10-12 | Femmepharma, Inc. | Pharmaceutical preparations for treatments of diseases and disorders of the breast |
KR100595364B1 (ko) * | 2003-02-20 | 2006-07-03 | 재단법인 목암생명공학연구소 | Lk8 단백질을 유효성분으로 포함하는 항암제 |
EP1713511B1 (en) * | 2004-01-09 | 2011-11-09 | Mogam Biotechnology Research Institute | Therapeutic agent for treatment of cancer comprising human apolipoprotein (a) kringles lk68 or lk8 genes as effective ingredient, and method for treating cancer using the same |
KR100595864B1 (ko) * | 2004-01-27 | 2006-06-30 | 재단법인 목암생명공학연구소 | 형질전환 사카로마이세스 세레비지애 균주 및 이를 이용한lk8 단백질의 생산방법 |
US7427662B2 (en) * | 2005-02-01 | 2008-09-23 | Baord Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Inhibition of angiogenesis and destruction of angiogenic vessels by apolipoprotein A-I and high density lipoprotein |
KR100888022B1 (ko) * | 2006-12-21 | 2009-03-09 | 재단법인 목암생명공학연구소 | 면역글로불린 Fc와 인간 아포리포단백질(a)크링글절편의 융합단백질 LK8Fc |
US8933031B2 (en) * | 2008-02-04 | 2015-01-13 | Shanghai First People's Hospital | Polypeptide inhibiting angiogenesis and application thereof |
WO2012067427A2 (ko) * | 2010-11-16 | 2012-05-24 | 재단법인 목암생명공학연구소 | Lk8 단백질을 유효성분으로 포함하는 당뇨망막병증 또는 노인성 황반변성의 예방 또는 치료용 약학 조성물 |
CN103232537B (zh) * | 2013-02-28 | 2015-01-28 | 浙江海洋学院 | 一种灰星鲨软骨血管生成抑制因子的制备方法及应用 |
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Publication number | Priority date | Publication date | Assignee | Title |
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US5639725A (en) | 1994-04-26 | 1997-06-17 | Children's Hospital Medical Center Corp. | Angiostatin protein |
US5945403A (en) | 1997-05-30 | 1999-08-31 | The Children's Medical Center Corporation | Angiostatin fragments and method of use |
US5801012A (en) | 1996-09-17 | 1998-09-01 | Northwestern University | Methods and compositions for generating angiostatin |
EP1214346A1 (en) * | 1999-09-15 | 2002-06-19 | Mogam Biotechnology Research Institute | A novel angiogenesis inhibitor |
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1999
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EP1214346A1 (en) | 2002-06-19 |
US20040259202A1 (en) | 2004-12-23 |
AU5762199A (en) | 2001-04-17 |
AU774759B2 (en) | 2004-07-08 |
CN1535983A (zh) | 2004-10-13 |
WO2001019868A1 (en) | 2001-03-22 |
JP3725473B2 (ja) | 2005-12-14 |
CN1243018C (zh) | 2006-02-22 |
US7118905B2 (en) | 2006-10-10 |
CN1367791A (zh) | 2002-09-04 |
CA2384929C (en) | 2006-12-05 |
JP2003509042A (ja) | 2003-03-11 |
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