CN1334921A - 前列腺素转运蛋白和凋亡 - Google Patents
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- CN1334921A CN1334921A CN99816032A CN99816032A CN1334921A CN 1334921 A CN1334921 A CN 1334921A CN 99816032 A CN99816032 A CN 99816032A CN 99816032 A CN99816032 A CN 99816032A CN 1334921 A CN1334921 A CN 1334921A
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Abstract
本发明提供凋亡调控物质的筛选方法,其筛选通过作用于PGT的候选物质。本发明的细胞保护剂,其有效成分是通过前列腺素转运蛋白(PGT)介导而被摄入细胞内的物质;包括测定通过PGT介导而摄取到细胞内的筛选方法。本发明的凋亡诱导剂,其活性成分为具有PGT抑制作用的物质;包括测定诱导PGT表达细胞凋亡能力的筛选方法。本发明的细胞保护剂由于具有细胞凋亡抑制作用,可用作神经细胞凋亡抑制剂、神经细胞保护剂等,并能用于预防或治疗神经疾病等。本发明的凋亡诱导剂可用于预防和/或治疗肿瘤等细胞增生性疾病。
Description
技术领域
本发明涉及凋亡调控物质、细胞保护剂、凋亡诱导剂及其筛选方法和应用。
背景技术
前列腺素转运蛋白(以下称作PGT),在生物体内具有以载体介导(carrier-mediated)方式输送前列腺素类物质的性质。前列腺素借助PGT透过生物膜,在细胞内发挥其生理活性,或被酶等分体代谢。现已知PGT存在于生物体的肺、肾脏、脑等器官中,编码PGT的基因序列也已明确(Science,268卷,866-869页,1995年)。
穿过PGT的物质中的一种是前列腺素(以下称作PG)。据报道,PG存在有多种类型,尤其是在肺PGT的透过实验中,高速透过(Km)的有前列腺素E1(PGE1)、前列腺素E2(PGE2)(参照上述文献)。
PG类作为自体有效物质,在生物体内发挥多种生理活性。其中有改善神经障碍、抑制神经细胞凋亡等对神经细胞保护作用的报道(特开平8-277222号公报)。但是,有关PG类对细胞、尤其是神经细胞的保护作用的机理尚不明确。
近年,有关细胞组织死亡,以凋亡(apoptosis)倍受人们的关注。凋亡是指细胞自身灭亡或自然死亡的意思。这种凋亡与病理学上的细胞死亡即坏死不同,它是整合了遗传信息的细胞自身的自动死亡。也就是说,任何外部的或内部的重要因素活化诱导凋亡的信号,细胞自身就自动地崩溃、直到死亡。有关PGT与凋亡的关系未见报道。
发明公开
本发明的目的是提供利用PGT与凋亡的关系的各种技术,包括通过PGT的抑制或导凋亡、应用PGT进行凋亡调控物质的筛选及其筛选物质的应用,应用抑制凋亡物质的PGT表达细胞的培养技术。
本发明者们考虑到上述情况,进行了研究,结果首次证实了PGT与凋亡的关系。即成功地首次阐明通过PGT产生凋亡作用。具体而言,本发明发现使用对PGT有阳性作用的物质,能抑制凋亡保护细胞,相反,使用对PGT有阴性作用的物质能诱导凋亡。
也就是说,本发明的凋亡调控物质的筛选方法是以通过对PGT的作用筛选侯选物质为特征的。为此,对PGT的作用可以是测定通过介助PGT的细胞内的摄取量,也可以是测定通过介助PGT的细胞内的摄取速度。
本发明的凋亡调控物质的筛选方法,还可以是通过能阻碍PGT功能或PGT表达来测定对PGT作用的方法。
本发明的凋亡调控物质的筛选方法可以是选择对PGT的有作用,且基本上无降压作用的物质为特征的方法。
本发明的凋亡调控物质是用本发明的凋亡调控物质的筛选方法筛选出的物质。
本发明的细胞保护剂是以具有通过PGT而被细胞内吞的机能、且具有抑制凋亡作用功效的凋亡调控物质为有效成分。其中,通过PGT而被细胞内吞的机能是至少以70f mol/mg蛋白/10分钟的摄取量的细胞保护剂。另外,通过PGT而被细胞内吞的机能是用大约100nM(Km)以下的透过速度表示的有PGT亲和性的细胞保护剂。再者,本发明的细胞保护剂,其作用对脑细胞、神经细胞或肾细胞的作用。另外,本发明的细胞保护剂的特征是基本上设有降压作用。
本发明的凋亡诱导剂是以具有PGT功能或对PGT表达有阻抑作用且能诱导凋亡功效的凋亡调控物质为有效成分。其中,凋亡调控物质可以是抗PGT抗体或PGT反义物质。
本发明的细胞保护剂,是从凋亡调控物质PGK1、PGK2或二环PGE2中选择的细胞保护剂。
本发明的PGT表达细胞的培养方法,其特征在于,应用添加了以具有抑制凋亡作用的凋亡调控物质为有效成分的细胞保护剂的培养基。其中,所添加的细胞保护剂的特征在于,它具有通过PGT而被细胞内吞的作用。还有一个特征是添加的细胞保护剂为PGE1。
本发明的凋亡调控方法,其特征在于,施用上述本发明的细胞保护剂或凋亡诱导剂的有效量。
本发明的凋亡诱导剂的使用,其特征在于,以制备凋亡调控药剂为目的,使用上述本发明的细胞保护剂或凋亡诱导剂。
本发明的最佳实施方案
以下对本发明进行具体说明。
(A)凋亡调控物质的筛选方法
本发明的凋亡调控物质的筛选方法的特征是,以侯补物质对PGT的作用为指标来筛选所期望的物质。所谓凋亡调控物质是指具有抑制凋亡作用的物质,或者具有诱导凋亡作用的物质。本发明的凋亡调控物质是具有抑制或诱导PGT功能或PGT表达作用的物质。
①具有抑制凋亡作用的物质的筛选方法
本发明的具有抑制凋亡作用物质的筛选方法是选择具有通过PGT而被细胞内吞性质的物质,更优选的是选择具有PGT亲和性、且具有通过PGT而被细胞内吞性质的物质。具体而言“具有PGT亲和性”是指应用强制表达人PGT的细胞,如Hela细胞,测定细胞内吞时,Km应达到100nM以下的程度。“通过PGT被细胞内吞”是指,例如应用分化的PC12细胞,与氚标记物37℃孵育10分钟后测定细胞中的放射活性时,应达到10fmol/mg蛋白/10分钟以上的程度。选择满足这些条件的物质。具体的方法虽然有如上面说的,以PGT表达细胞中的透过速度或摄取量或凋亡抑制能力为指标进行选择的方法,但并不限定于这些方法。另外更优选选择基本上无降压作用的物质。
②具有诱导凋亡作用的物质的筛选方法
本发明的具有诱导凋亡作用的物质的选择方法,是通过选择具有PGT阻碍作用的物质及具有抑制PGT自身表达作用的物质实现。具体而言,例如应用制表达人PGT的Hela细胞等PGT表达细胞来筛选诱导细胞凋亡的物质。另外,可用放射线同住来标记已知通过PGT而被细胞内吞物质的,例如PGE1等,通过选择PGT表达细胞中使其透过速度或摄取量减少或消失的物质而得到具有凋亡诱导作用的物质。本发明的具有凋亡诱导作用质的筛选方法,可以是筛选出具有PGT阻抑作用的物质及具有抑制PGT自身表达作用物质的方法即可,不限于上述例子。另外,优选选择基本上无降压作用的物质。(B)细胞保护剂
本发明的细胞保护剂,是其有效成分是具有通过PGT而被细胞内吞性质的物质。最是以具有PGT亲和性且通过PGT而被细胞内吞性质的物质为有效成分。其中所讲的“具有PGT亲和性”具体是指,应用强制表达人PGT的Hela细胞测定细胞内吞时,Km应在100nM以下。另外,“通过PGT而被细胞内吞”具体是指,应用分化的PC12细胞,与氚标记物37℃孵育10分钟后测定细胞中放射活性时,应达到70fmol/mg蛋白/10分钟以上的程度。
本发明的细胞保护剂包括用上述(A)中(1)具有凋亡抑制作用的物质的筛选方法选择出的物质,该物质具有抑制凋亡作用。这些物质可由其它途径合成、分离提取、或基因工程学方法制备,常规的制剂技术制备,作为医药品供给。
这样的物质包括PGE1、PGE2、PGF2α、PGD2、PGK1、PGK2、二环PGE2等,或其活性衍生物或其前体,例如烷基酯(特开昭59-216820号)、烷氧基羰基烷基、烷基羰基氧基烷基酯体(特开昭59-206344号、60-13779号)、硫代(特开昭58-110562号)、9-酰基氧基(特开昭58-39660号、特开平3-204853号、5-213862号),或者有与这些物质同等程度的PGT亲和性及通过PGT而被细胞内吞性质的物质。优选具有通过PGT而被细胞内吞性质的新型物质,或者还未发现其有通过PGT而被细胞内吞性质的已知物质。
特别是PGK1、PGK2和二环PGE2显示了淀粉样β肽引起的大鼠大脑皮层神经元凋亡的抑制作用。本作用是通过PGT抑制凋亡。PGK1、PGK2和二环PGE2虽然是已知的物质,但是目前为止尚没有关于通过PGT的抑制凋亡作用的报道。再者已知PGE1具有降压作用,作为药物使用时存在副作用的问题。但是,实验表明适当剂量的PGK1、PGK2和二环PGE2基本上不引起降压作用,但可充分抑制凋亡,所以其应用性优于PGE1。
该物质具有细胞保护作用,特别是对神经细胞的保护作用,具体而言,对神经细胞的变性产生作用,具有神经细胞的凋亡诱导抑制作用时,可作为神经细胞保护剂应用。可用于相关的疾病有神经疾病、伴随神经病变的疾患、阿尔茨海默病、帕金森氏病、亨亨顿氏舞蹈病、肌肉萎缩性侧索硬化症、脊椎管狭窄症等疾病的预防、治疗、改善等。
另外,该物质也可作为肾细胞的保护剂应用,可用于预防、治疗肾炎、肾功能不全、肾小球性肾炎、肾病综合征等肾脏疾病。尤其是通过抑制因NO(一氧化氮)造成的细胞损伤而用于预防、治疗急性肾功能不全、药物性肾功能不全、慢性肾功能不全等与自由基和凋亡相关的肾脏疾病。
再者,由于该物质及PGE1等具有细胞保护作用,所以可通过向PGT表达细胞的培养体系中添加这些物质而有效地维持PGT表达细胞的生存。尤其是使脑细胞和神经细胞等培养困难的细胞的长期培养成为可能。
该物质可以制成乙醇溶液、脂质体、脂肪乳剂、环糊精包接化,使活体投给成为可能。必要时,也可以应用常规的制剂化技术。
乙醇溶液化时,可将该物质溶解于乙醇。应用该乙醇溶液时,可用生理盐水或葡萄糖液等进行稀释。
脂质体化可这样进行,如将磷脂溶解到有机溶剂(氯仿)中,向该溶液中加入已溶解于媒溶剂(乙醇)中的该物质的溶液,然后馏去溶剂,向其中加入磷酸缓冲液,振荡,超声波处理及离心后,过滤回收上清液。
脂肪乳化可这样进行,如将该物质与油成分(大豆油、蓖麻油、橄榄油等植物油、MCT等)、乳化剂(磷脂等)等混合,加热后,向溶液中加入必要量的水,用乳化机(匀浆器,例如高压喷射型、超声波型等)进行乳化、匀浆处理。另外,亦可将之冷冻干燥。进行脂肪乳化时可添加助乳化剂。助乳化剂有甘油、糖类(例如:葡萄、糖醇、果糖等)。
环糊精包接化可这样进行,例如将该物质溶解于溶剂(乙醇等)中,向该溶液加入加热溶解于水中的环糊精溶液,然后,冷却,滤过析出的沉淀,灭菌干燥。此时使用的环糊精时,依照该物质的大小,宜选择空隙直径不同的环糊精(α、β、γ型)。
该物质的投给量应根据患者的病情、性别、年龄、体重适当选择。投给途径包括经口、非经口均可。最好是注射制剂应静脉注射。其用量,每日1-1000μg左右。(C)凋亡诱导剂
本发明的凋亡诱导剂,其有效成分是有PGT阻抑作用的物质,包含对PGT功能或PGT表达有抑制作用的物质。例如,PGT反义物质、PGT抗体等。
另外,本发明的凋亡诱导剂是用上述(A)中具有凋亡诱导作用物质的筛选方法筛选的物质,该物质具有抑制PGT功能或PGT表达的作用。这些物质可由其它途径合成、分离提取、或基因工程学方法制造,可通过常规的制剂化技术作为医药品提供。①GPT反义物质
反义PGT是指与编码PGT的基因具有互补关系的基因序列,并由于在细胞核内的DNA链上与编码PGT的基因发生置变,结果能阻抑PGT。具体而言,如具有5’-GGCTTGAGCAGGAGCCCCAT-3’等DNA序列的寡核苷酸。可应用常规的DNA合成方法进行制备。另外可以对这些物质进行巯基末端化修饰。
PGT反义物质最好是制成与脂质转化体等的混合物、脂质体、脂肪乳剂、环糊精包裹等制剂,可以活体投给。另外,为了提高与癌细胞的特异性结合和/或聚集,可将这些合成物与血管生成因子(例如:血管生物素(ァンギォポェチン)生物素、氨肽酶A等)结合使用。必要时,可旋用常规的制剂化技术。
PGT抗体没有特殊限定,只要识别作为抗原的PGT即可。PGT抗体可以是多克隆抗体,也可是单克隆抗体,可按照常规方法进行制备。例如:多克隆抗体的制备是用PGT免疫动物,回收提取免疫动物的抗血清。另外,单克隆抗体的制备是将PGT免疫动物的脾细胞与骨髓瘤细胞样的增殖细胞融合,制备杂交瘤,或者用EB病毒转化该脾细胞,培养得到的转化体,由之产生(临床研究杂志,98卷,5号,1142-1149页,1996年;美国专利5792851等)。
另外,PGT抗体也可是嵌合抗体或人源化抗体等。嵌合抗体的制备是将上述非人源的单抗的可变区与人抗体的恒定区域进行组合。人源化抗体是将上述嵌合体中可变区的氨基酸置换成人相容的(不产生新的抗原性),再应用基因工程学方法进行制备。
含有PGT抗体的制剂可按照常规的制剂化方法进行制备。
本发明的具有PGT阻抑作用的物质的投给量按照患者的病情、性别、年龄、体重等进行适当选择。例如:成人患者为0.001-1000mg/天左右。其投给路径为,经口或非经口均可。
本发明的PGT阻抑作用的物质具有凋亡诱导作用,能用于预防和/或治疗由肿瘤或病原细胞而产生的疾病。例如:作为癌化疗药、抗癌药、抗肿瘤药作用,或者用于预防和/或治疗肿瘤以外的增生性疾病、各种病毒感染性疾病、HIV病毒感染,特别是获得性免疫缺陷综合症(AIDS)。[实施例]
为了详细说明本发明,列举部分实施例及实验例,但本发明并不限于此。[实施例1](环糊精包接化)
将PGE1 17mg溶解于0.2ml乙醇中,向该溶液中加入将257mg β-环糊精加温溶解于6ml水中的溶液,45℃混和后,放置室温,析出沉淀。将之0℃放置一夜后,过滤,用50%乙醇水溶液洗净后,无菌干燥,得到环糊精包裹剂。[实施例2](脂质体化)
将60mg卵磷脂及11mg油胺溶解于5ml氯仿中,然后加入将30μgPGE1溶解于100μl乙醇中的溶液,放入到茄型烧瓶中,用旋转蒸发仪馏去溶剂。向其中加入0.1M等的磷酸缓冲溶液(pH5)1ml,振荡,超声波处理,离心后,用0.2μm的膜滤器将上清过滤,得到胶质体制剂。[实施例3](乙醇溶液)
将500μg PGE1溶解于1ml乙醇中,得到乙醇溶液。应用时,用生理盐水或葡萄糖溶液稀释。[实施例4](脂肪乳剂化)
向30g精制大豆油中加入5.4g高度精制的卵磷脂、1.5mg PGE1和0.72g油酸,40-75℃加热溶解后,加入200ml蒸馏水,接着加入7.5g日本药局方甘油,用20-40℃的蒸馏水补足总量到300ml,用匀浆器进行粗乳化。再用マントン一ガゥリン型器进行高压乳化,得到均质的微细的脂肪乳剂。该乳剂的平均粒径为0.15-0.4μm,不含有1μm以上的粒子。[实施例5](脂肪乳化)
除用PGE2代替PGE1以外,按照实施例4,同样制备脂肪乳剂。[实施例6]
用DNA合成仪合成PGT特异的反义寡核苷酸-5’-GGCTTGAGCAGGAGCCCCAT-3’构成的DNA序列。然后进行硫酯化修饰。再与脂质转化体(Gibco公司)等量混合。[实施例7]
将PGT或其片段与等量的佐剂一起,每隔2-3天给兔子多次投给。其后回收抗血清。用常规方法纯化抗血清,制备PGT抗体。[实施例8]
用PGK1、PGK2或二环PGE2替代PGE1,除此之后,按照实施例3制备乙醇溶液。[实施例9]
用大鼠PGT的N末端片段(相当于N末端第1-23。氨基酸序列为MGLLLKPGAR QGSGTSSVPD RRC)与KLH(匙孔血蓝蛋白)的复合体作免疫原,制备抗PGT抗体。
将免疫原与等量的佛氏完全佐剂(或不完全佐剂)一起,每次1mg,免疫注射国产兔。以后每间隔2周追加免疫,共追加3次。2个月后采全血获得抗血清。
用0.02M等渗磷酸缓冲液(pH7,以下称PBS)将抗血清稀释2倍,加饱和硫酸铵溶液达到40%饱和度。静置后离心,回收沉淀部分,溶解于PBS中。加入40%饱和硫酸铵,静置后离心分离,回收沉淀物,再溶于PBS。加入透析袋在水中透析,除去硫酸铵。
用将上述PGT片段固化剂琼脂糖的固化载体进行亲和层析,再纯化。抗体部分溶在PBS中,上柱。用PBS及含有1M氯化钠的PBS洗涤,用4M氯化镁溶液进行洗脱,回收抗体成分。用PBS透析,纯化抗体。用伯乐(BioRad)公司的蛋白质测定试剂盒测定抗体浓度,所得纯化抗体的浓度为大约8μg/ml。[实验例1]1)神经细胞的凋亡抑制①用含10ml/L热灭活马血清和5ml/L热灭活胎牛血清的RPMI1640使大鼠肾上腺髓质的褐色细胞PC12细胞(引自ATCC,大日本制药社引入)增殖后,用含有小鼠BNGF(100ng/ml)、N2补充液及TIP(转铁蛋白5μg/ml、胰岛素5μg/ml、孕酮10ng/ml)的RPMI 1640培养,使之向神经样细胞分化。②用Neurobasal培养基洗涤分化的PC12细胞,用不含BNGF、N2补充液和TIP的同一培养基交换后,加入小鼠βNGF抗体(终浓度50ng/ml),培养24h,诱导凋亡。③在交换该培养基时,将PGE1乙醇溶液添加到新鲜培养基(终浓度0.01-1μM)中,同时间培养。阴性对照组只添加乙醇,也相同时间培养。凋亡的检测是:用1%戊二醛固定细胞后(室温30分钟),用Hoechst33342染色(1mM室温反应2分钟)。荧光显微镜下任意观察5个视野,计数总细胞数及凋亡的阳性细胞数,计算出凋亡阳性率。④所得值以平均值±标准差表示。统计学分析是:2组间用Student’st-test(以下称t检验),多组间是一元分配分散分析(One-WayANOVA)后,用Punnet法称,机率在5%以下的均为显著差异。结果如表1示。由表1可以判断PGE1为1μM时间可抑制细胞凋亡。[表1]
*表示与阴性对照相比,有显著性差异。2)凋亡与PGT表达
PGE1浓度 | 凋亡发生率(%) | 检验(危险率) |
实验前 | 4.6±1.0 | |
无(阴性对照) | 26.3±1.8 | |
0.01μM | 21.3±1.5 | |
0.1μM | 19.1±8.1 | |
1μM | 17.1±0.6 | * |
用トリゾ一ル试剂(Gibco公司)从分化的PC12细胞中分离提取总RNA。参照大鼠PGT的cDNA序列(Kanai N等,Science,Vol.268,868-869,1995)设定RT-PUR(逆转录酶链式反应)中应用的PGT的引物。所用的PGT引物如下所示。
正义(开始碱基的序号为264):
5’-GAGCAGTCTCACCACAATCG-3’
反义(开始的碱基序号为670):
5’-GGCTCGGCAAAGTCATCCAC-3’
用引物对计算出扩增的PUR产物的分子量为444bp。RT-PCR应用RNA PCR试剂盒(Takara公司)进行。DNA的复制反应是95℃1分钟,60℃2分钟、72℃3分钟的循环进行25个循环,用DNA热循环仪(PJ2000,PE公司)进行。反应终了后,取名PT-PCR产物的样本1/5量进行2%琼脂糖电泳,EB染色后紫外照射下进行分析。
PC12细胞mRNA扩增出1条基因片段。由此可确定是PGT特异的持录。[实验例2]
关于PGE1的神经细胞的凋亡抑制作用,除应用TUNEL染色进行凋亡检测以外,按照实验例1的方法得行。结果如表2所示。表2表明PGE10.1μM和1μM时对细胞凋亡有抑制作用。[表2]
**表示与阴性对照相比有显著性差异。[实验例3]1)将分化的PC12细胞与氚标记的PGE1(添加浓度0.1μM)37℃孵育10分钟。其后,细胞依次用加有5g/L牛血清白蛋白的预冷的等渗磷酸缓冲液①,预冷的等渗磷酸缓冲液洗涤,再测定放射活性。计算出摄入PC12细胞内的PGE1量。2)在添加PGE1时,同时向培养基中加入PGT阻抑剂溴化甲醇绿(Bromcresol green,以下称BrCG)。其后与上述1)同样地处理。结果如表3所示。[表3]
数值表示均值±标准误。*表示用t检验时,与PGE1添加组相比P<5%,有显著性差异。3)PGT cDNA特异的反义寡核苷酸的设计如下:5’-GGCTTGAGCAGGAGCCCCAT-3’
PGE1浓度 | TUNEL阳性细胞率(%) | 检定(危险率) |
实验前 | 7.00±1.27 | |
无(阴性对照) | 30.93±2.31 | |
0.1μM | 13.80±0.75 | **P=0.0006 |
1μM | 8.10±1.57 | **P=0.0001 |
BrCG添加量 | PGE1摄取量(fmol/mg蛋白/10分钟) |
未添加 | 78±11 |
3μM | 60±15 |
10uM | 20±8* |
对该反义寡核苷酸进行硫酯化修饰,用Lipofectin(GiBco),在与PGE1培养剂的2天,转化PC12。其后与上述1)进行同样处理。结果如表4所示。[表4]
数值表示均值±标准误。*表示用t检验时,与PGE1添加组相比P<5%,有显著性差异。4)通过SAPK/JNK的细胞内信号转导
反义添加量 | PGE1摄取量(fmol/mg蛋白/10分钟) |
未添加 | 78±11 |
0.5μM | 56±17 |
1.5μM | 14±14* |
SAPK/JNK(Stress Activated Protein Kinase/Jun IV-terminalKinase的略写)与凋亡诱导相关。这里从对SAPK/JNK活性变化的效果来探讨SAPK/JNK介导的细胞内信。
本实验用Western blotting进行分析。按上述实验例那样向PC12细胞的培养基中添加各种添加物。除去NGF后培养1-4小时。用缓冲液融解PC12细胞,离心,提取细胞上清液。将该上清液与JNK1多克隆抗体(サンタクルズ公司)4℃下孵育1小时后,添加Protein-A Sepharose,再孵育1小jf,供给以下的免疫沉淀用。用SOS-PAGE电泳法分离100mg细胞融解物,转到尼龙膜(Pharmacia公司)上。将该膜与磷酸化(活化型)JNK抗体(サンタクルズ公司)一起印迹,按说明书检测条带。细胞内信号通过化学发光/自发放射活性(ECL公司,Pharmacia)被增强。结果如表5所示。[表5]
发光度:+++表示极强;+表示可看到发光;±表示有若干发光。
添加物 | 发光度 |
未添加 | +++ |
PGE1(1μM) | ± |
PGE1(0.1μM) | + |
PGE1(0.1μM)+BrCG(10μM) | +++ |
PGE1(0.1μM)+PGT反义物质 | +++ |
SAPK/JNK在除去NGF1小时后开始活化。在添加PGE1的PC12细胞中未见到这种活性变化,即抑制了活化。PGT阻抑剂BrCG或反义PGT能阻抑了PGE1对SAPK/JNK活化的抑制作用。
从上述结果可以看出具有通过PGT能被细胞内吞性质的物质与细胞凋亡有关系。
存在于PGT近旁的光换质一旦被摄取到细胞内,就能抑制细胞凋亡。此时,当PGT阻抑剂共存,阻抑了该物质向细胞内的摄取时,不能抑制凋亡。
从以上结果可以认为具有通过PGT介导被细胞内吞性质的物质,在通过PGT被摄取到细胞内时,诱导某种细胞内信号,其诱导的信号直接/间接地抑制细胞凋亡,尤其是神经细胞凋亡。[实验例4]1)用含有小鼠BNGF(100ng/ml)、N2补充物(1%)及TIP(转铁蛋白5μg/ml、胰岛素5μg/ml、孕酮10ng/ml)的Neurobasal培养基培养来源于大鼠肾上腺髓质的褐色细胞PC12细胞(引自ATCC,大日本制药引入),分化成神经样细胞。2)添加实施例6中制备的反义寡核苷酸溶液(终浓度600nM),培养2天。用同样地添加脂质转化体的作阴性对照,同时间培养。用10%中性福尔马林缓冲液固定细胞后(室温30分),通过TUNEL染色进行凋亡的检测,在光学显微镜下随意观察6个视野,统计总细胞数和TUNEL(凋亡)阳性细胞数,算出阳性细胞的比率。结果如表6(均值±标准差)所示。
从表6可以判明,添加了反义寡核苷酸的,促进凋亡。[表6]
[实验例5]淀粉样β肽对凋亡的诱导1)大鼠大脑皮质神经元的培养
反义 | TUNEL阳性细胞率(%) |
未添加 | 7.4±0.9 |
添加 | 12.9±1.1 |
从胎龄17或18日龄的大鼠胚胎大脑中,冰浴下摘出皮质部位,剪碎后用神经细胞分散液(SUMILON)分散细胞。其后,将细胞以1.5×105个/cm2的密度接种到预先用多聚乙烯亚胺包被的培养瓶中,培养4天后供给以下试验。培养液使用添加有B27补充液(1/50体积)、2-巯基乙醇(27.5μM)、L-谷氨酸(25μM)及谷氨酰胺(0.5mM)的Nourobasal培养基(Gibco)。2)淀粉样β肽对凋亡的诱导
将淀粉样β肽25-35(Aβ25-35)溶解于蒸馏水中,浓度达1mM,37℃孵育1周,制备Aged-Aβ25-35。换成含10μM Aged-Aβ25-35的上述培养液(除去L-谷氨酸),诱导神经细胞的凋亡。3)凋亡诱导24小时后,确认该细胞有无PGT表达。根据Western blot法,用针对PGT N末端的抗体检测全细胞蛋白提取液中的PGT。结果,在分子量约40kd的部位检测出单一的条带。也就是说,脑型PGT与已知的肺型PGT(分子量70kd)有不同的分子量。4)确证PGE1对淀粉样β肽诱导的凋亡的抑制效果。将PGE1溶解于乙醇后,加入到含有上述2)中的Aged-Aβ25-35的培养液中培养。其终浓度为1μM。5)凋亡的检测
凋亡诱导24小时后,先用PBS(-)洗涤细胞,用1%戊二醛(PBS溶液)室温固定30分钟。然后用PBS(-)洗涤2次后,在1mM Hoechst33342溶液(PBS溶液)中反应2分钟。其后在荧光显微镜下,随意观察4个视野的核染色质的形态,统计正常细胞数和凋亡阳性细胞数(看到染色质片段化或凝集的细胞),它们的比值算作凋亡的发生率。例数为3例。结果如表7所示。6)确认PGT阻抑剂与淀粉样β肽诱导凋亡之间的关系。除同时添加60μM终浓度的PGT阻抑剂BrCG与PGE1之外,其余按上述1)-5)的方法进行实验。结果如表7所示。[表7]
数值表示均值±标准差。**表示用Tunnet法进行检测时与载体溶剂(仅用淀粉样β肽处理)间的概率小于1%,#表示用t检验时,与添加PGE1组间的概率小于5%,有显著的差异。
药剂 | 凋亡发生率(%) |
载体溶剂 | 30±2 |
PGE1 | 23±2** |
PGE1+BrCG | 28±1# |
PGE1抑制由淀粉样β肽诱导的凋亡。而PGT阻抑剂BrCG阻抑了PGE1的这种作用(抑制淀粉样β肽诱导的凋亡)。结果提示PGE1的这种作用是通过PGT实现的。[实验例6]
应用反义PGT,观察PGE1对淀粉样β肽诱导凋亡的抑制作用的影响。反义PGT仍使用实验例3中的寡核苷酸。其添加逍度为1.5μM。按实验例5进行凋讯检测实验。例数为3。结果如表8所示。[表8]
药剂 | 凋亡发生率(%) |
载体溶液 | 33±2 |
PGE1 | 23±2** |
PGE1+反义PGT | 32±2# |
数值以均值±标准差表示。**表示用Tunnet法进行检测时,与载体溶液(只用溶粉样β肽处理)组之间的概率<1%,#表示用t检验时,与PGE1添加组间的概率<5%,有显著性差异。[实验例7]1)检测具有抑制淀粉样β肽诱导细胞凋亡效果的药物。将被检药物(PGE1、PGK1、PGK2、二环PGE2)溶解于乙醇后,除了加入同时添加有Aged-Aβ25-35的培养液中外,按照实验例5进行实验。由正常细胞数和凋亡阳性细胞数的比率,算出凋亡诱导的抑制率。结果如表9所示。2)检测被验药物的降压作用。腹腔内注射戊巴比妥钠将正常大鼠(雄性,体重约300g)麻醉后,背位固定。接着,颈动脉插管,通过压力输送仪(日本光电)测定血压,同时连接多种波动描记器(日本光电)进行记录。将被验物溶解于乙醇后,用生理盐水稀释(×200),尾静脉注射,用量100μm/kg体重,测定平均体血压(MBP)。根据用药前后的测定值算出血压的降低度。各组例数均为3。结果如表9所示。[表9]
药剂 | 凋亡诱导抑制率(%) | 血压下降程度(%) |
PGK1 | 45.4 | -5 |
PGK2 | 36.2 | +2 |
二环PGE2 | 40.6 | 0 |
PGE1 | 38.2 | -55 |
本表结果表明用PGK1、PGK2、二环PGE2在基本上不引起血压下降的投给量时可充分抑制凋亡。[实验例8]1)在肾细胞中由NO(一氧化氮)诱导凋亡。将大鼠肾小管上皮细胞株(主要来自远曲小管)的NRK52E细胞在用I型胶原包被的小室-玻片中培养24小时。其后将培养基变为不含血清的培养基,添加NO供体NOC12(0.4mM)或S-亚硝基-L-谷胱甘肽(GSNO;1mM),诱导凋亡。2)确认诱导凋亡的肾细胞中有无PGT表达。根据Wostern印迹的方法,应用针对PGT N末端的抗体检测肾脏总蛋白提取液中的PGT。结果,在肾脏中也检测出与脑型PGT一样、分子量为40kd的单一条带。3)确认诱导凋亡的肾细胞中有无GPT的局部存在。通过组织免疫染色,用PGT N末端抗体识别肾脏组织切片的PGT。可以看到运曲小管及集合管有强反应。4)确认肾细胞中PGE1的凋亡抑制效果。在添加NO供体的同时添加终浓度达1或10μm的PGE1。24小时后用1%戊二醛固定细胞,用Hoechst33342染色。每一张切片任选3个视野,算出视野中(400倍)全部细胞的核片段化及发生凝集的细胞数占总细胞数的比率。例数为5。结果如表10及表11所示。[表10](添加NOC12的场合)
PGE1添加量 | 凋亡比例(%) |
无添加(载体溶液) | 16±1 |
1μM | 11±2* |
10μM | 6±1** |
数值表示的是均值±标准差。*表示用Dunnet法进行检测时,与载体溶液间的概率<5%,有显著的差异。**表示概率<1%,有显著性差异。[表11](添加GSNO的情况)
PEG1添加量 | 凋亡比率(%) |
无添加(载体溶液) | 25±4 |
1μM | 10±2** |
10μM | 11±3* |
数值表示的是均值±标准差。*表示用Turnet法进行检测时,与载体溶剂间的概率<5%,有显著性差异。**表示相同情况下概率<1%,有显著性差异。
PGE1抑制肾细胞凋亡。具体而言,它能抑制大鼠肾小管上细胞株(NRK52E细胞)中GSNO诱导的凋亡。5)证明GSNO诱导的肾细胞亡与PGT阻抑剂的关系。PGT阻抑剂BrCG的终浓度为60μM,除了同时添加GSNO及PGE1外,按上述4)中的方法进行实验。PGE1的逍度为1μM。例数为3。结果如表12所示。[表12]
药剂 | 凋亡比例(%) |
未添加(载体溶液,只有GSNO) | 19±1 |
PGE1 | 8±2 |
PGE1+BrCG | 13±1* |
数值表示的是均值±标准差。*表示用t检验时,与PGE1添加组间的概率<5%,有显著性差异。
BrCG阻碍PGE1诱导的凋亡抑制作用。该结果证明PGE1在大鼠肾小管上皮细胞株(NRK52E细胞)中对GSNO抑制诱导凋亡的作用是通过PGT介导的。[实验例9]1)检测对供体诱导的肾细胞凋亡有抑制效果的药物。除用PGK1、二环PGE2作为被验药物外,按实验例8进行实验。被检药物的浓度为1μM。例数为5。结果如表13所示。[表13]
药物 | 凋亡比例(%) |
载体溶液(只有GSNO) | 13±1 |
PGK1 | 8±1** |
载体溶液PGE2 | 6±1** |
数值表示均值±标准差。**表示用载体溶液法检验时,与载体溶液组间的概率<1%,有显著性差异。
PGK1、二环PGE2对GSNO诱导的肾细胞凋亡有抑制作用。2)确认PGT阻抑剂对PGK1、载体溶液PGE2抑制肾细胞凋亡作用的影响。PGT阻抑2剂BrCG的终浓度为60μM,除了同时添加GSNO及被验药物外,按实验1)的方法进行实验。例数为4。结果如表14及表15所示。[表14]
药剂 | 凋亡比例(%) |
未添加(载体溶液,只有GSNO) | 22±2 |
PGE1 | 8±1 |
PGE1+BrCG | 12±1* |
数值表示均值±标准差。**表示t检验时,与添加PGK1组间的概率<10%,有显著性差异。[表15]
药剂 | 凋亡比例(%) |
未添加(载体溶液,只有GSNO) | 22±2 |
PGE1 | 9±2 |
PGE1+BrCG | 16±3* |
数值表示均值±标准差。*表示t检验时,与二环PGE2添加组间的P值<5%,有显著性差异。
BrCG能部分阻抑PGK1、二环PGE2对凋亡的抑制作用。
产业上利用的可能性
本发明由于应用具有通过PGT介导被细胞内吞性质的物质来抑制细胞凋亡,所以该物质可用作细胞保护剂。尤其是用作神经细胞凋亡的抑制剂、神经细胞保护剂、肾细胞凋亡的抑制剂及肾细胞保护剂。有望用于预防和/或治疗神经疾病、伴随神经病变的疾病、阿尔茨海默病、帕金森氏病、享亨顿氏舞蹈病、肌肉萎缩性侧索硬化症、脊椎管狭窄症等疾病。
另外,本发明通过应用有PGT阻抑2作用的物质,诱导凋亡,也适用于预离和/或治疗肿瘤等细胞增生的疾病。
再者,本发明阐明通过PGT而产生的信号与细胞凋亡的相关性,并证明有SAPK/JNK参与。也就是说,本发明的物质通过PGT而被摄取到细胞内,结果是对SAPK/JNK的活化呈抑制效果,或者通过诱导抑制性的胞内信号而抑制凋亡,表现出保护细胞的作用。
另外,应用本发明的筛选方法可简便地筛选出通过PGT被摄取到细胞内显示细胞保护的物质及具有诱导细胞凋亡作用的物质。
Claims (19)
1.凋亡调控物质的筛选方法,其特征在于,筛选作用于前列腺素转运蛋白(PGT)的候选物质。
2.权利要求1的凋亡调控物质的筛选方法,其中对前列腺素转运蛋白(PGT)的作用是通过测定通过PGT介导而被细胞内吞的摄取量。
3.权利要求1的凋亡调控物质的筛选方法,其中对前列腺素转运蛋白(PGT)的作用是靠测定通过PGT而进入细胞内的摄取速度。
4.权利要求1的凋亡调控物质的筛选方法,其中对前列腺素转运蛋白(PGT)的作用是靠测定对PGT功能或PGT表达的阻止能力。
5.权利要求1、2、4任一项的凋亡调控物质的筛选方法,均选择基本上无降压作用的物质。
6.用权利要求1~5任一项的方法筛选出的新的凋亡调控物质。
7.细胞保护剂是以具有通过PGT介导而能被摄取到细胞内的机能,且具有抑制凋亡作用的凋亡调控物质为有效成分。
8.权利要求7的细胞保护剂,通过PGT而摄取到细胞内的机能用摄取量表示时,至少为70fmol/mg蛋白/10分钟。
9.权利要求7的细胞保护剂,通过PGT而被摄取到细胞内的机能具有对PGT的亲合性,用透过速度表示时其为约100nM(Km)以下。
10.权利要求7-9任一项的细胞保护剂,其中的细胞是指脑细胞、神经细胞或肾细胞。
11.权利要求7-10任一项中的细胞保护剂都基本上无降低血压作用。
12.凋亡诱导剂,是以具有阻抑PGT功能或PGT表达作用,且具有诱导凋亡作用的凋亡调控物质为有效成分。
13.权利要求12中的凋亡诱导剂,其中凋亡调控物质为PGT抗体或PGT反义物质。
14.权利要求7~11任一项的细胞保护剂,其中凋亡调控剂选自PGK1、PGK2或二环PGE2。
15.PGT表达细胞的培养方法,该方法使用添加了以具有凋亡抑制作用的凋亡调控物质为有效成分的细胞保护剂的培养基。
16.权利要求15的培养方法,其中上述的细胞保护剂具有通过PGT而被摄取到细胞内的作用。
17.权利要求15或16的培养方法,其中上述的细胞保护剂为PGE1。
18.凋亡的调控方法,至少包括施用有效量的权利要求7-12、13或14任一项中的细胞保护剂或凋亡诱导剂。
19.权利要求7-12、13或14任一项中的细胞保护剂或凋亡诱导剂的应用,其目的是制造用于凋亡调控的药物。
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- 1999-12-02 CA CA002359928A patent/CA2359928A1/en not_active Abandoned
- 1999-12-02 AU AU14147/00A patent/AU1414700A/en not_active Abandoned
- 1999-12-02 KR KR1020017006956A patent/KR20010082323A/ko not_active Application Discontinuation
- 1999-12-02 WO PCT/JP1999/006766 patent/WO2000034778A1/ja not_active Application Discontinuation
-
2001
- 2001-06-04 US US09/871,721 patent/US20020127228A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
EP1146337A4 (en) | 2002-04-17 |
WO2000034778A1 (fr) | 2000-06-15 |
EP1146337A1 (en) | 2001-10-17 |
AU1414700A (en) | 2000-06-26 |
KR20010082323A (ko) | 2001-08-29 |
US20020127228A1 (en) | 2002-09-12 |
CA2359928A1 (en) | 2000-06-15 |
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