丁基苯酞对慢性脑缺血老龄大鼠海马APP表达的影响
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摘要
背景和目的：慢性脑缺血是神经系统的一种常见的病理状态，伴发于血管性痴呆(Vascular dementia，VD)、阿尔茨海默病(Alzheimer's disease，AD)及Binswanger病等多种疾病的病理过程中，早期以认知功能损害为主要表现，最终导致持久或进展性认知与神经功能障碍，因此研究其发病机制对慢性脑缺血的临床防治具有普遍意义。慢性脑缺血的病理基础之一是大脑皮层及其它脑区出现大量β淀粉样蛋白质(β-amyloidprotein，Aβ)沉淀，Aβ是一种难溶性的蛋白质，一般由42(39～43)个氨基酸残基组成，分子量约为42kDa，由β-淀粉样前体蛋白(β-amyloid precursor protein，APP)水解产生，APP是一个很大的蛋白质分子，分子量为78kDa，APP基因约有170kb，含有18个外显子。丁基苯酞是从芹菜籽中提取出来的左旋体，后经人工合成为消旋体，经中国医学科学院药物研究所的科研人员20余年的研究证明，丁基苯酞对动物急性缺血性卒中具有多种机制的治疗作用，但是该药用于慢性脑缺血是否有效有待研究。海马是脑缺血最为敏感的部位，且位置特殊，与认知等有密切关系，海马CA1区与海马内部及海马外结构有广泛的纤维联络，是长时程增强(long-term potentiation，LTP)形成及学习、记忆的重要部位，因此选择了大鼠海马区作为研究对象。已有免疫组织化学研究发现，丁基苯酞可抑制皮层和海马的神经元变性、坏死，同时可减轻星形胶质细胞增生、活化，以及抑制少突胶质细胞减少，并能改善慢性脑缺血引起的认知功能障碍。有关丁基苯酞治疗慢性脑缺血对Aβ表达影响的研究目前国内外尚未见报道。本文旨在运用免疫组织化学法、RT-PCR法及免疫斑点法观测应用该药物干预后慢性脑缺血老龄大鼠海马区APP mRNA及蛋白水平的表达变化，进一步研究该药对慢性脑缺血干预作用的治疗机制，探讨该药在细胞学、分子生物学及蛋白水平的作用机制，可以让临床医生更准确地理解和把握该药的作用机制和临床适应症，为该药治疗慢性脑缺血提供理论依据。
材料和方法：
1．动物分组与模型制备：
试验动物选用健康Wistar大鼠150只(郑州大学实验动物中心提供)，雌雄不拘，12-14月龄，体重450-550克。随机分为模型组(A)、假手术组(B)、预防组(C)、低剂量组(D)和高剂量组(E)，共5组，每组30只。假手术组仅暴露双侧颈总动脉而不结扎，余各组采用双侧颈总动脉结扎(2VO)法制备动物模型。C组先给予丁基苯酞80mg.Kg~(-1).d~(-1)，1月后停药，行2VO术；B组正常饲养2月后仅给予安慰剂；A、D、E组行2VO术，正常饲养2月后，A组给予安慰剂，D组给予丁基苯酞60mg.Kg~(-1).d~(-1)，E组给予丁基苯酞120mg.Kg~(-1).d~(-1)，连续灌胃1个月；共3月后处死动物。
2．标本采集：
①免疫组织化学法标本采集：麻醉动物，37℃生理盐水灌洗后断头迅速取出脑组织，放入4％多聚甲醛中后4℃冰箱内固定24小时，经梯度酒精脱水，二甲苯透明，石蜡包埋，制作脑部冠状石蜡切片(厚度3um)，以备免疫组化染色。
②RT-PCR及免疫斑点法标本采集：麻醉动物，断头取血后迅速冰上分离脑组织，装入1.5ml EP管中，-80℃保存，以备RT-PCR及免疫斑点实验使用。
3．免疫组织化学法
①抗体：一抗兔抗APP单克隆抗体
二抗HRP标记山羊抗兔多克隆抗体
②免疫组织化学染色步骤：烘片、脱蜡至水、抗原修复、血清封闭、加一抗、加二抗、DAB显色、苏木素复染、分化、封片、照相；
③图像分析
4．RT-PCR：
①组织总RNA提取：超低温冰箱取标本，迅速冰上分离，取出其中一侧海马，用Trizol法提取组织总RNA，测定样品中260nm、280nm吸收峰的OD值，鉴定RNA纯度和含量。
②逆转录-聚合酶链反应：
*引物β-Actin：5’GGGAAATCGTGCGTGACAT 3’
5’TCAGGAGGAGCAATGATCTTG 3’
APP：5’GGGTCTGGGTTGACAAACATC 3’
5’CATTCTGCTGCATCTTGGAGA 3’
* 25ul体系，50℃，15min，cDNA合成；
94℃，2min，预变性；
94℃，30s，变性；
56℃，30s，退火；(经过反复试验确定最适退火温度为56℃)
72℃，1.5min，延伸；
4℃，forever。
*琼脂糖凝胶电泳配制2％琼脂糖凝胶，10xBuffer上样，EB染色。
③图象分析
5．免疫斑点实验：
①组织总蛋白提取：取另一半海马用单去污裂解液法提取组织总蛋白。
②免疫斑点实验：
*抗体一抗：小鼠抗大鼠IgG1 APP单克隆抗体
二抗：HRP标记羊抗小鼠IgG多克隆抗体
*步骤组织总蛋白液(即抗原液)按1：20稀释；
在醋酸纤维素(nitrocellulose，NC)膜上点样，每点10μl，37℃烤干；
脱脂奶粉室温下封闭1小时；
TBS液洗膜滤纸吸干后加入一抗，室温下摇床混匀4小时；
TBS液洗膜滤纸吸干后加入二抗，室温下摇床混匀4小时；
TBS液洗膜滤纸吸干后加入DAB显色液显色，室温下1-3分钟；
③图象分析
6．统计学处理
应用SPSS13.0统计软件进行数据处理。实验数据用均数±标准差((?)±s)表示。多样本均数间比较采用单因素方差分析及Q-检验法处理。以α=0.05作为显著性检验水准。
结果：
1．实验各阶段对大鼠进行一般行为学评价显示：造模成功后大鼠均出现精神萎靡、反应迟钝、少食、少饮；术后1天，假手术组精神恢复，余组改善不明显，部分大鼠出现共济失调，无明显肢体瘫痪；3-5天后逐渐恢复，1周后基本正常，体重恢复，伤口逐渐愈合；灌服药物干预后，预防组、低剂量组、高剂量组大鼠行为学均较模型组明显改善，高剂量组改善最明显。
2．免疫组织化学染色见大鼠海马CA1区假手术组细胞排列正常，细胞胞膜及胞内APP染色较浅，模型组细胞排列紊乱，锥体细胞形态异常，APP染色沉着明显；预防组细胞排列及细胞形态较模型组明显改善，APP染色沉着减少；低剂量组细胞排列紊乱情况介于模型组和预防组之间，APP染色沉着减少；高剂量组细胞排列紊乱情况与预防组无明显差异，APP染色沉着明显减少。
3．免疫组织化学各实验组APP表达阳性区平均灰度值反映了各实验组海马CA1区细胞内APP免疫组织化学蛋白水平表达量，分别为：A组91.612±4.526，B组69.543±1.659，C组84.499±2.005，D组82.098±2.118，E组76.593±2.342；单因素方差分析显示F=92.899，P=0.000，组内差异有显著性；Q-检验结果显示：A组与其余四组比较P＜0.05，B组与C组、D组、E组比较P＜0.05，C组与D组比较P＞0.05，C组与E组比较P＜0.05，D组与E组比较P＜0.05。除C组与D组比较差异无显著性外，其余各组间比较差异均有显著性。
4．RT-PCR实验各实验组APP相对β-actin平均光密度值反映了各实验组APP mRNA表达量，分别为：A组105.851±6.372，B组72.350±16.368，C组94.650±2.728，D组93.684±3.982，E组85.182±5.275；单因素方差分析显示F=20.570，P=0.000，组内差异有显著性；Q-检验结果显示：A组与其余四组比较P＜0.05，B组与C组、D组、E组比较P＜0.05，C组与D组比较P＞0.05，C组与E组比较P＜0.05，D组与E组比较P＜0.05。除C组与D组比较差异无显著性外，其余各组间比较差异均有显著性，与结果(3)一致。各实验组β-actin平均光密度值反映了各实验组β-actin mRNA表达量，分别为：A组166.333±11.487，B组161.138±13.974，C组161.631±11.184，D组165.509±11.368，E组164.267±16.551；单因素方差分析显示F=0.312，P=0.869，组内差异无显著性。
5．免疫斑点实验各实验组APP平均光密度值反映了各实验组APP酶联免疫斑点蛋白水平表达量，分别为：A组39.757±7.928，B组14.016±2.888，C组31.149±7.300，D组28.107±6.586，E组20.657±3.541；单因素方差分析显示F=29.545，P=0.000，组内差异有显著性；Q-检验结果显示：A组与其余四组比较P＜0.05，B组与C组、D组、E组比较P＜0.05，C组与D组比较P＞0.05，C组与E组比较P＜0.05，D组与E组比较P＜0.05。除C组与D组比较差异无显著性外，其余各组间比较差异均有显著性，与结果(3)和结果(4)一致。
6．三组实验结果均显示APP mRNA及蛋白表达量A组最高，B组最低，丁基苯酞干预后，C组、D组和E组APP mRNA及蛋白表达量均较A组明显降低，且三组间比较E组＜D组＜C组。
结论：
1．从细胞学水平、分子生物学水平和蛋白水平进一步证实APP过量表达与慢性脑缺血关系密切。
2．丁基苯酞明显改善慢性脑缺血大鼠的一般行为学评价及组织学变化，减轻细胞结构紊乱，降低APP在海马CA1区锥体细胞内的沉积，抑制海马APP mRNA及蛋白的表达，对慢性脑缺血治疗有效，有明显的脑保护作用。
3．丁基苯酞降低APP在慢性脑缺血老龄大鼠海马的表达，高剂量组海马细胞内APP沉积、mRNA及蛋白表达量明显低于低剂量组及预防组。
4．对老龄大鼠预防性给予预防剂量丁基苯酞可能对慢性脑缺血起到预防作用。预防组与低剂量组APP mRNA及蛋白表达量差异无显著性，但预防组海马CA1区细胞排列情况较低剂量组改善明显，提示预防用药效果可能优于慢性脑缺血后低剂量用药。
5．丁基苯酞可能通过降低海马CA1区细胞内APP沉积、抑制海马APP mRNA及蛋白的表达对慢性脑缺血老龄大鼠发挥脑保护作用。
Background and objective:
Chronic cerebral ischemia is a frequent pathologic state of nervous system (NS). It always concomitant with many diseases such as vascular dementia(VD) Alzheimer disease(AD) Binswanger's disease and so on. Harmfulness of the cognition ability is the main manifestation in the earlier period, and lead to the persistent or advancing dysfunction of cognition and nerves at last. For this reason, the study of pathogenesis and drugs has the widespread significance for the clinical prevention and cure to chronic cerebral ischemia.
One of the pathologic foundations of chronic cerebral ischemia is that devils of p-amyloid protein (Aβ) precipitate in the cerebral cortex and other encephalic region. Aβis a difficult solvable protein, composed of 42 amino acid residues generally. The molecular weight is about 42kDa. It is hydrolyzed fromβ-amyloid precursor protein (APP).
The hippocampal formation is a bilateral structure sandwiched between the cerebral cortex and the thalamus. It is very sensitive to the ischemia.
Butylphthalide is an 1-isomer extracted from celery seed. Then it was made racemic by artificially synthesize. After years of investigation, scholars found that dl3 normal-butylphthalide is effective to acute ischemic stroke. But there is lots of study and research have to do to proof whether it is effective or not to chronic ischemic stroke.
In our study, we use three methods to survey the variance of the APP expression in different drug intervention group. The methods include immunohistochemistry reverse transcription- polymerase chain reaction(RT-PCR) immunodotting. Our study will provide whether it's effective to chronic ischemic stroke.
Materials and methods:
1. Subgroup of animal and preparation of models:
150 healthy Wistar rats, female or male, 12-14 months old, weight 450-550g. The rats were randomly divided into 5 groups: model group(A) control group(B) obviate group(C) low dose group(D) high dose group(E). 30 rats in every group. Control group (B) only exposed double common carotid artery, no ligation. Else groups ligated double common carotid artery. Obviate group(C) was given butylphthalide 80mg.Kg~(-1).d~(-1) one month before ligating double common carotid artery, model group(A) low dose group(D) high dose group(E) were ligated two months before giving drug, after two months, group A was given placebo, group D was given butylphthalide 60mg.Kg~(-1).d~(-1) , group E was given butylphthalide 120mg.Kg~(-1) .d~(-1) , one month continue. Then 3 months later we executed the rats.
2. Collection of the specimen:
(1)For immunohistochemistry experiment: after the rats were anesthetized with 10% Chloral Hydrate and lavaged with Sodium Chloride, we took out of the rat brain quickly. Then fixed with paraformaldehyde, dehydration with gradient alcohol, made it transparent with Xylene, embeded with paraffin wax, cut sheet and reserve.
(2)For RT-PCR and immunodotting experiment: after the rats were anesthetized with 10% Chloral Hydrate, we took out of the rat brain and reciped blood quickly, then separated the brain on ice, built in 1.5 mL EP tube, maintained in - 80℃and reserve.
3. Immunohistochemistry experiment:
(1)Antibody: First antibody: rabbit anti-APP monoclonal antibodySecond antibody: HRP marked goat anti-rabbit polyclonal antibody
(2)Procedure: parch, deparaffinage, antigen reparation, blood serum blocking, addin first antibody, addin second antibody, colouration by DAB, hematoxylin counterstain, differentiation, mounting.
(3)Photograph and analyze.
4. RT-PCR:
(1)Extraction of tot RNA: Trizol liquid were used.
(2)Reverse transcription- polymerase chain reaction:* Primer:β-actin: 5' GGGAAATCGTGCGTGACAT 3'5' TCAGGAGGAGCAATGATCTTG 3' APP: 5' GGGTCTGGGTTGACAAACATC 3'5' CATTCTGCTGCATCTTGGAGA 3'* 25ul system, 50℃, 15min, cDNA synthesis;94℃, 2min, force-degeneration; 94℃, 30s, degeneration; 56℃, 30s, annealing; 72℃, 1.5min, extension ; 4℃, forever.* Agarose gel electrophoresis.
(3)Photograph and analyze.
5. Immunodotting experiment:
(1)Extraction of tot protein: single decontaminant lysate were used.
(2)Immunodotting experiment:* Antibody: First antibody: mouse anti-rat IgG_1 APP monoclonal antibodySecond antibody: HRP marked goat anti-mouse polyclonal antibody* Procedure: dilution of antigen (1:20), spotting, blocking, addin first antibody, addin second antibody, colouration by DAB.
(3)Photograph and analyze.
6. Statistical treatment:
All date were entered into a computer and analyzed using software SPSS 13.0 for windows. The date were presented as means±SD( x±s).one way analysis of variance was used to determine significant differences among five groups, and Q-test was used to determine significant differences between each groups. The significant testing standard wasα=0.05.
Results:
1. Ethology appreciation of different stage dislapyed that: after the operations, all the animals behaved depressed, responded slowly; One day after the operation, group B recovered, some rats of the else groups showed up ataxia; One week later, they began to recover; after dosage, group C D E improve gradually, Group E is the most obviously.
2. From immunohistochemistry experiment we can find that there is no obviously ab -normality in group B. The shape and alignment of pyramidal cell in hippocampus is abnormality in group A and the staining is the deepest. Group C is better than group A and D, but can not compare with group B. There is no abviously differenc -e between group C and E.
3. The immunohistochemistry experiment showed the average dates of the APP positive area which reflect the APP expression of protein level in hippocampus CA1 were: group A 91.612±4.562, group B 69.543±1.659, group C 84.499±2.005, group D 82.098±2.118, group E 76.593±2.342; the one-way analyse of variance show that F=92.899, P=0.000, there was significant difference between groups; Q-test displayed that group A compare with else four group P<0.05, group B compared with group C. D E, P<0.05, group C compared with group D, P>0.05, group C compared with group E, P<0.05, group D compared with group E, P<0.05, the comparation between groups all have significance except C and D.
4. The RT-PCR experiment showed the average dates of the corr. APP optical density value(OD value) which reflect the APP mRNA expression in hippocampus were: group A 105.851±6.372, group B 72.350±16.368, group C 94.650±2.728, group D 93.684±3.982, group E 85.182±5.275; the one-way analyse of variance show that F=92.899, P=0.000, there was significant difference between groups; Q-test displayed that group A compare with else four group PO.05, group B compared with group C D E, P<0.05, group C compared with group D, P>0.05, group C compared with group E, P<0.05, group D compared with group E, P<0.05, the comparation between groups all have significance except C and D, it was identical with result 3. The average dates of theβ-actin optical density value(OD value) which reflect theβ-actin mRNA expression in hippocampus were: group A 166.333±11.487, group B 161.138±13.974, group C 161.631±11.184, group D 165.509±11.368, group E 164.267±16.551; the one-way analyse of variance show that F=0.312, P=0.869, there was no significant difference between groups.
5. The immunodotting experiment showed the average dates of the APP optical density value(OD value) which reflect the APP expression of enzyme-linked immunospot protein level in hippocampus were: group A 39.757±7.928, group B 14.016±2.888, group C 31.149±7.300, group D 28.107±6.586, group E 20.657±3.541; the one-way analyse of variance show that F=29.545, P=0.000, there was significant difference between groups; Q-test displayed that group A compare with else four group P<0.05, group B compared with group C, D E, P<0.05, group C compared with group D, P>0.05, group C compared with group E, P<0.05, group D compared with group E, P<0.05, the comparation between groups all have significance except C and D, it was identical with result 3 and result 4.
6. The results of three experiments are coherent. The mean value of APP mRNA and protein expression in group A was the biggest, the group B was the least, after dosage the mean value of APP mRNA and protein expression in group C groupD and groupE descented obviously, and groupE Conclusion:
1. Our study confirmed that the over expression of APP has some thing to do with chronic cerebral ischemia again from cytology molecular biology and proteomics.
2. Butylphthalide obviously improve the ethology score and histology changes. Lighten the confused architecture, lower the APP deposition on CA1 of hippocampus, and inhibit the expression of APP mRNA and protein in hippocampus. It is effective to chronic cerebral ischemia, the brain protection is obviously.
3. Butylphthalide can lower the expression of APP in hippocampus. The deposition of APP on CA1 of hippocampus pyramidal cell and the expression of APP mRNA and protein in hippocampus of high dose group are obviously lower than obviate group and low dose group.
4. Butylphthalide probably has the prophylactic effect to chronic cerebral ischemia. There is no significant difference of APP mRNA and protein expression between obviate group and low dose group. But the cell condition of obviate group is better than low dose group, it hint that prophylactic administration may be better than low dose administration after chronic cerebral ischemia.
5. Perhaps butylphthalide get the brain protection effect by lower the APP deposition on CA1 of hippocampus and inhibit the expression of APP mRNA and protein in hippocampus.
材料和方法：
1．动物分组与模型制备：
试验动物选用健康Wistar大鼠150只(郑州大学实验动物中心提供)，雌雄不拘，12-14月龄，体重450-550克。随机分为模型组(A)、假手术组(B)、预防组(C)、低剂量组(D)和高剂量组(E)，共5组，每组30只。假手术组仅暴露双侧颈总动脉而不结扎，余各组采用双侧颈总动脉结扎(2VO)法制备动物模型。C组先给予丁基苯酞80mg.Kg~(-1).d~(-1)，1月后停药，行2VO术；B组正常饲养2月后仅给予安慰剂；A、D、E组行2VO术，正常饲养2月后，A组给予安慰剂，D组给予丁基苯酞60mg.Kg~(-1).d~(-1)，E组给予丁基苯酞120mg.Kg~(-1).d~(-1)，连续灌胃1个月；共3月后处死动物。
2．标本采集：
①免疫组织化学法标本采集：麻醉动物，37℃生理盐水灌洗后断头迅速取出脑组织，放入4％多聚甲醛中后4℃冰箱内固定24小时，经梯度酒精脱水，二甲苯透明，石蜡包埋，制作脑部冠状石蜡切片(厚度3um)，以备免疫组化染色。
②RT-PCR及免疫斑点法标本采集：麻醉动物，断头取血后迅速冰上分离脑组织，装入1.5ml EP管中，-80℃保存，以备RT-PCR及免疫斑点实验使用。
3．免疫组织化学法
①抗体：一抗兔抗APP单克隆抗体
二抗HRP标记山羊抗兔多克隆抗体
②免疫组织化学染色步骤：烘片、脱蜡至水、抗原修复、血清封闭、加一抗、加二抗、DAB显色、苏木素复染、分化、封片、照相；
③图像分析
4．RT-PCR：
①组织总RNA提取：超低温冰箱取标本，迅速冰上分离，取出其中一侧海马，用Trizol法提取组织总RNA，测定样品中260nm、280nm吸收峰的OD值，鉴定RNA纯度和含量。
②逆转录-聚合酶链反应：
*引物β-Actin：5’GGGAAATCGTGCGTGACAT 3’
5’TCAGGAGGAGCAATGATCTTG 3’
APP：5’GGGTCTGGGTTGACAAACATC 3’
5’CATTCTGCTGCATCTTGGAGA 3’
* 25ul体系，50℃，15min，cDNA合成；
94℃，2min，预变性；
94℃，30s，变性；
56℃，30s，退火；(经过反复试验确定最适退火温度为56℃)
72℃，1.5min，延伸；
4℃，forever。
*琼脂糖凝胶电泳配制2％琼脂糖凝胶，10xBuffer上样，EB染色。
③图象分析
5．免疫斑点实验：
①组织总蛋白提取：取另一半海马用单去污裂解液法提取组织总蛋白。
②免疫斑点实验：
*抗体一抗：小鼠抗大鼠IgG1 APP单克隆抗体
二抗：HRP标记羊抗小鼠IgG多克隆抗体
*步骤组织总蛋白液(即抗原液)按1：20稀释；
在醋酸纤维素(nitrocellulose，NC)膜上点样，每点10μl，37℃烤干；
脱脂奶粉室温下封闭1小时；
TBS液洗膜滤纸吸干后加入一抗，室温下摇床混匀4小时；
TBS液洗膜滤纸吸干后加入二抗，室温下摇床混匀4小时；
TBS液洗膜滤纸吸干后加入DAB显色液显色，室温下1-3分钟；
③图象分析
6．统计学处理
应用SPSS13.0统计软件进行数据处理。实验数据用均数±标准差((?)±s)表示。多样本均数间比较采用单因素方差分析及Q-检验法处理。以α=0.05作为显著性检验水准。
结果：
1．实验各阶段对大鼠进行一般行为学评价显示：造模成功后大鼠均出现精神萎靡、反应迟钝、少食、少饮；术后1天，假手术组精神恢复，余组改善不明显，部分大鼠出现共济失调，无明显肢体瘫痪；3-5天后逐渐恢复，1周后基本正常，体重恢复，伤口逐渐愈合；灌服药物干预后，预防组、低剂量组、高剂量组大鼠行为学均较模型组明显改善，高剂量组改善最明显。
2．免疫组织化学染色见大鼠海马CA1区假手术组细胞排列正常，细胞胞膜及胞内APP染色较浅，模型组细胞排列紊乱，锥体细胞形态异常，APP染色沉着明显；预防组细胞排列及细胞形态较模型组明显改善，APP染色沉着减少；低剂量组细胞排列紊乱情况介于模型组和预防组之间，APP染色沉着减少；高剂量组细胞排列紊乱情况与预防组无明显差异，APP染色沉着明显减少。
3．免疫组织化学各实验组APP表达阳性区平均灰度值反映了各实验组海马CA1区细胞内APP免疫组织化学蛋白水平表达量，分别为：A组91.612±4.526，B组69.543±1.659，C组84.499±2.005，D组82.098±2.118，E组76.593±2.342；单因素方差分析显示F=92.899，P=0.000，组内差异有显著性；Q-检验结果显示：A组与其余四组比较P＜0.05，B组与C组、D组、E组比较P＜0.05，C组与D组比较P＞0.05，C组与E组比较P＜0.05，D组与E组比较P＜0.05。除C组与D组比较差异无显著性外，其余各组间比较差异均有显著性。
4．RT-PCR实验各实验组APP相对β-actin平均光密度值反映了各实验组APP mRNA表达量，分别为：A组105.851±6.372，B组72.350±16.368，C组94.650±2.728，D组93.684±3.982，E组85.182±5.275；单因素方差分析显示F=20.570，P=0.000，组内差异有显著性；Q-检验结果显示：A组与其余四组比较P＜0.05，B组与C组、D组、E组比较P＜0.05，C组与D组比较P＞0.05，C组与E组比较P＜0.05，D组与E组比较P＜0.05。除C组与D组比较差异无显著性外，其余各组间比较差异均有显著性，与结果(3)一致。各实验组β-actin平均光密度值反映了各实验组β-actin mRNA表达量，分别为：A组166.333±11.487，B组161.138±13.974，C组161.631±11.184，D组165.509±11.368，E组164.267±16.551；单因素方差分析显示F=0.312，P=0.869，组内差异无显著性。
5．免疫斑点实验各实验组APP平均光密度值反映了各实验组APP酶联免疫斑点蛋白水平表达量，分别为：A组39.757±7.928，B组14.016±2.888，C组31.149±7.300，D组28.107±6.586，E组20.657±3.541；单因素方差分析显示F=29.545，P=0.000，组内差异有显著性；Q-检验结果显示：A组与其余四组比较P＜0.05，B组与C组、D组、E组比较P＜0.05，C组与D组比较P＞0.05，C组与E组比较P＜0.05，D组与E组比较P＜0.05。除C组与D组比较差异无显著性外，其余各组间比较差异均有显著性，与结果(3)和结果(4)一致。
6．三组实验结果均显示APP mRNA及蛋白表达量A组最高，B组最低，丁基苯酞干预后，C组、D组和E组APP mRNA及蛋白表达量均较A组明显降低，且三组间比较E组＜D组＜C组。
结论：
1．从细胞学水平、分子生物学水平和蛋白水平进一步证实APP过量表达与慢性脑缺血关系密切。
2．丁基苯酞明显改善慢性脑缺血大鼠的一般行为学评价及组织学变化，减轻细胞结构紊乱，降低APP在海马CA1区锥体细胞内的沉积，抑制海马APP mRNA及蛋白的表达，对慢性脑缺血治疗有效，有明显的脑保护作用。
3．丁基苯酞降低APP在慢性脑缺血老龄大鼠海马的表达，高剂量组海马细胞内APP沉积、mRNA及蛋白表达量明显低于低剂量组及预防组。
4．对老龄大鼠预防性给予预防剂量丁基苯酞可能对慢性脑缺血起到预防作用。预防组与低剂量组APP mRNA及蛋白表达量差异无显著性，但预防组海马CA1区细胞排列情况较低剂量组改善明显，提示预防用药效果可能优于慢性脑缺血后低剂量用药。
5．丁基苯酞可能通过降低海马CA1区细胞内APP沉积、抑制海马APP mRNA及蛋白的表达对慢性脑缺血老龄大鼠发挥脑保护作用。
Background and objective:
Chronic cerebral ischemia is a frequent pathologic state of nervous system (NS). It always concomitant with many diseases such as vascular dementia(VD) Alzheimer disease(AD) Binswanger's disease and so on. Harmfulness of the cognition ability is the main manifestation in the earlier period, and lead to the persistent or advancing dysfunction of cognition and nerves at last. For this reason, the study of pathogenesis and drugs has the widespread significance for the clinical prevention and cure to chronic cerebral ischemia.
One of the pathologic foundations of chronic cerebral ischemia is that devils of p-amyloid protein (Aβ) precipitate in the cerebral cortex and other encephalic region. Aβis a difficult solvable protein, composed of 42 amino acid residues generally. The molecular weight is about 42kDa. It is hydrolyzed fromβ-amyloid precursor protein (APP).
The hippocampal formation is a bilateral structure sandwiched between the cerebral cortex and the thalamus. It is very sensitive to the ischemia.
Butylphthalide is an 1-isomer extracted from celery seed. Then it was made racemic by artificially synthesize. After years of investigation, scholars found that dl3 normal-butylphthalide is effective to acute ischemic stroke. But there is lots of study and research have to do to proof whether it is effective or not to chronic ischemic stroke.
In our study, we use three methods to survey the variance of the APP expression in different drug intervention group. The methods include immunohistochemistry reverse transcription- polymerase chain reaction(RT-PCR) immunodotting. Our study will provide whether it's effective to chronic ischemic stroke.
Materials and methods:
1. Subgroup of animal and preparation of models:
150 healthy Wistar rats, female or male, 12-14 months old, weight 450-550g. The rats were randomly divided into 5 groups: model group(A) control group(B) obviate group(C) low dose group(D) high dose group(E). 30 rats in every group. Control group (B) only exposed double common carotid artery, no ligation. Else groups ligated double common carotid artery. Obviate group(C) was given butylphthalide 80mg.Kg~(-1).d~(-1) one month before ligating double common carotid artery, model group(A) low dose group(D) high dose group(E) were ligated two months before giving drug, after two months, group A was given placebo, group D was given butylphthalide 60mg.Kg~(-1).d~(-1) , group E was given butylphthalide 120mg.Kg~(-1) .d~(-1) , one month continue. Then 3 months later we executed the rats.
2. Collection of the specimen:
(1)For immunohistochemistry experiment: after the rats were anesthetized with 10% Chloral Hydrate and lavaged with Sodium Chloride, we took out of the rat brain quickly. Then fixed with paraformaldehyde, dehydration with gradient alcohol, made it transparent with Xylene, embeded with paraffin wax, cut sheet and reserve.
(2)For RT-PCR and immunodotting experiment: after the rats were anesthetized with 10% Chloral Hydrate, we took out of the rat brain and reciped blood quickly, then separated the brain on ice, built in 1.5 mL EP tube, maintained in - 80℃and reserve.
3. Immunohistochemistry experiment:
(1)Antibody: First antibody: rabbit anti-APP monoclonal antibodySecond antibody: HRP marked goat anti-rabbit polyclonal antibody
(2)Procedure: parch, deparaffinage, antigen reparation, blood serum blocking, addin first antibody, addin second antibody, colouration by DAB, hematoxylin counterstain, differentiation, mounting.
(3)Photograph and analyze.
4. RT-PCR:
(1)Extraction of tot RNA: Trizol liquid were used.
(2)Reverse transcription- polymerase chain reaction:* Primer:β-actin: 5' GGGAAATCGTGCGTGACAT 3'5' TCAGGAGGAGCAATGATCTTG 3' APP: 5' GGGTCTGGGTTGACAAACATC 3'5' CATTCTGCTGCATCTTGGAGA 3'* 25ul system, 50℃, 15min, cDNA synthesis;94℃, 2min, force-degeneration; 94℃, 30s, degeneration; 56℃, 30s, annealing; 72℃, 1.5min, extension ; 4℃, forever.* Agarose gel electrophoresis.
(3)Photograph and analyze.
5. Immunodotting experiment:
(1)Extraction of tot protein: single decontaminant lysate were used.
(2)Immunodotting experiment:* Antibody: First antibody: mouse anti-rat IgG_1 APP monoclonal antibodySecond antibody: HRP marked goat anti-mouse polyclonal antibody* Procedure: dilution of antigen (1:20), spotting, blocking, addin first antibody, addin second antibody, colouration by DAB.
(3)Photograph and analyze.
6. Statistical treatment:
All date were entered into a computer and analyzed using software SPSS 13.0 for windows. The date were presented as means±SD( x±s).one way analysis of variance was used to determine significant differences among five groups, and Q-test was used to determine significant differences between each groups. The significant testing standard wasα=0.05.
Results:
1. Ethology appreciation of different stage dislapyed that: after the operations, all the animals behaved depressed, responded slowly; One day after the operation, group B recovered, some rats of the else groups showed up ataxia; One week later, they began to recover; after dosage, group C D E improve gradually, Group E is the most obviously.
2. From immunohistochemistry experiment we can find that there is no obviously ab -normality in group B. The shape and alignment of pyramidal cell in hippocampus is abnormality in group A and the staining is the deepest. Group C is better than group A and D, but can not compare with group B. There is no abviously differenc -e between group C and E.
3. The immunohistochemistry experiment showed the average dates of the APP positive area which reflect the APP expression of protein level in hippocampus CA1 were: group A 91.612±4.562, group B 69.543±1.659, group C 84.499±2.005, group D 82.098±2.118, group E 76.593±2.342; the one-way analyse of variance show that F=92.899, P=0.000, there was significant difference between groups; Q-test displayed that group A compare with else four group P<0.05, group B compared with group C. D E, P<0.05, group C compared with group D, P>0.05, group C compared with group E, P<0.05, group D compared with group E, P<0.05, the comparation between groups all have significance except C and D.
4. The RT-PCR experiment showed the average dates of the corr. APP optical density value(OD value) which reflect the APP mRNA expression in hippocampus were: group A 105.851±6.372, group B 72.350±16.368, group C 94.650±2.728, group D 93.684±3.982, group E 85.182±5.275; the one-way analyse of variance show that F=92.899, P=0.000, there was significant difference between groups; Q-test displayed that group A compare with else four group PO.05, group B compared with group C D E, P<0.05, group C compared with group D, P>0.05, group C compared with group E, P<0.05, group D compared with group E, P<0.05, the comparation between groups all have significance except C and D, it was identical with result 3. The average dates of theβ-actin optical density value(OD value) which reflect theβ-actin mRNA expression in hippocampus were: group A 166.333±11.487, group B 161.138±13.974, group C 161.631±11.184, group D 165.509±11.368, group E 164.267±16.551; the one-way analyse of variance show that F=0.312, P=0.869, there was no significant difference between groups.
5. The immunodotting experiment showed the average dates of the APP optical density value(OD value) which reflect the APP expression of enzyme-linked immunospot protein level in hippocampus were: group A 39.757±7.928, group B 14.016±2.888, group C 31.149±7.300, group D 28.107±6.586, group E 20.657±3.541; the one-way analyse of variance show that F=29.545, P=0.000, there was significant difference between groups; Q-test displayed that group A compare with else four group P<0.05, group B compared with group C, D E, P<0.05, group C compared with group D, P>0.05, group C compared with group E, P<0.05, group D compared with group E, P<0.05, the comparation between groups all have significance except C and D, it was identical with result 3 and result 4.
6. The results of three experiments are coherent. The mean value of APP mRNA and protein expression in group A was the biggest, the group B was the least, after dosage the mean value of APP mRNA and protein expression in group C groupD and groupE descented obviously, and groupE
1. Our study confirmed that the over expression of APP has some thing to do with chronic cerebral ischemia again from cytology molecular biology and proteomics.
2. Butylphthalide obviously improve the ethology score and histology changes. Lighten the confused architecture, lower the APP deposition on CA1 of hippocampus, and inhibit the expression of APP mRNA and protein in hippocampus. It is effective to chronic cerebral ischemia, the brain protection is obviously.
3. Butylphthalide can lower the expression of APP in hippocampus. The deposition of APP on CA1 of hippocampus pyramidal cell and the expression of APP mRNA and protein in hippocampus of high dose group are obviously lower than obviate group and low dose group.
4. Butylphthalide probably has the prophylactic effect to chronic cerebral ischemia. There is no significant difference of APP mRNA and protein expression between obviate group and low dose group. But the cell condition of obviate group is better than low dose group, it hint that prophylactic administration may be better than low dose administration after chronic cerebral ischemia.
5. Perhaps butylphthalide get the brain protection effect by lower the APP deposition on CA1 of hippocampus and inhibit the expression of APP mRNA and protein in hippocampus.
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[35] 陈多，吴春福．淀粉样前体蛋白(APP)分泌的神经及药物调节[J]．沈阳药科大学学报，2001，18(1)：75-78
[1] Martinez-Lage P, Hachinski V. Multi-Infarct dementia. In: Barnett H, Mohr JP, Stein B, Yatsu F, eds. Stroke: Pathophysiology, Diagnosis and Management [J]. 3rd Ed. Philadephia: Churchill Livingstone, 1998, 875-894
[2] 杨昆．老年性痴呆的防治[J]．博爱，2004，8(297)：47
[3] Rogers S L, Dody RS, Mobs Rc, et al. Done Pezil improves cognition and global fanction in Alzheimer' s disease; a 15-weekdouble-blind, placebo-controlled studyp[J]. Aroh Intern Med, 1998, 158(9): 1021
[4] Black S, Romn GC, Geldmacher DS, et al. Donepezil 307 Vascular Dementia Study Group. Efficacy and tolerability of donepezil in vascular dementia: positive results of a 24-week, multicenter, international, randomized, placebo-controlled clinical trial. Stroke [J]. 2003, 34(10): 2323-2330
[5] Wilkinson D, Doody R, Helme R, et al. Donepezil 308 Study Group. Donepezil in vascular dementia: a randomized, placebo-controlled study [J]. Neurology. 2003, 61 (4): 479-486.
[6] HONG Xia, ZHANG Zhen-xin, WANG Lu-ning, et al. A randomized study comparing the effect and safety of galantam ine and donepezil in patients with mild to moderate Alzheimer's disease [J]. Chin J Neurol, 2006, 39(6): 379-382
[7] Kumar V, Anand R, Messina J, et al. An efficacy and safety analysis of Exelon in Alzheimer' s disease patients with concurrent Vascular risk factors[J]. Eur J Neurol, 2000, 7: 159-169.
[8] Moretti-R, Torre P, AntoneIlo RM, et al. Rivastigmine superior to aspirin plus nimodipine in subcortical vascular dementia: an open, 16-month, comparative study[J]. Int J Clin Pract, 2004, 58(4): 346-353.
[9] 国家药典委员会编．临床用药须知[M]．北京：人民卫生出版社，2005：60
[10] 陶纪宁．胆碱酯酶抑制剂治疗AD的研究进展[J]．药学进展，2001，25(2)：205
[11] Orgogozo J-M, Rigaud A-S, Steffler A, et al. Efficacy and safety of memantine in patients with mild to moderate vascular dementia: a randomized, placebo-controlled trial (MMM300) [J]. Stroke, 2002, 33(7): 1834-1839.
[12] Wilcock G, Mabius HJ, Steffler A. on behalf of the MMM 500 group. A double-blind, placebo-controlled multicentre study of memantine in mild to moderate vascular dementia (MMM500) [J]. Internal Clin Psychopharmacol, 2002, 17(6): 297-305.
[13] 张华芳，李铮，唐勇，萘呋胺脂治疗脑血管性痴呆的临床观察[J]．临床荟萃，1997，12(14)：653-654．
[14] Kittner B. Clinical trails of propentofylline in vascular dementia. European/Canadian Propentofylline Study Group [J]. Alzheimer Dis Assoc Disorder, 1999, 13(3): 166-171
[15] Lopez-Arieta BJ. Nimodipine for primary degenerative, mixed and vascular dementia [P]. Cochrane Database Syst Rev, 2001, I: CD000147.
[16] Lopez-Arrieta J M, Birks J. Nimodipine for primary degenerative, mixed and vascular dementia[P]. Cochrane Database Syst Rev, 2002, 3: CD000147.
[17] Pantoni L, Del Ser T, Soglian AG, et al. Efficacy and safety of nimodipine in subcortical vascular dementia: a randomized placebo-controlled trail [J]. Stroke, 2005, 36(3): 619-624.
[18] 冯征，张均田，丹酚酸B对β-淀粉样蛋白介导原代培养皮层神经元毒性的保护作用[J]．药学学报，2000，35(12)：881-885．
[19] 韩恩吉，胡洪涛，何秀全，海风藤选择性抑制淀粉样前体蛋白基因表达[J]．中国临床康复，2004，8(13)：2592-2593
[20] Yun CAO, Alistair Jan GUAN, Laura BENNET, et al. Insulin like growth factor-1 reduces β-amyloid precursor protein expression after ischemic white matter damage in near-term fetal sheep [J]. Chin J Contemp Pediatr, 2004, 6(6): 449-452
[21] 里克特格登化工有限公司．作为药物的反式阿扑长春英宁酸酯衍生物[P]中国专利：96119131．3，1999-01-20．
[22] 王景华，楼风昌．天然活性成份防治AD的研究进展[J]．药学进展，2001，25(2)：76-80
[23] 刘小林，程春荣，曹威，等．石杉碱甲的药理作用与临床应用研究进展[J]，医药导报，2006，25(2)：90-95
[24] 李仪奎．中药药理世纪回眸[J]．中成药，2000，22(1)：560
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[27] CUI Li-ying, LI Shun-wei, LU Chuan-zhen, et al. The multicentric randomized study of dl-3-butylphthalide in the treatment of acute moderate ischemic stroke [J], Chin J Cerebrovasc Dis, 2005, 2(3): 112-115
[28] CUI Li-ying, LIU Xiu-qin, ZHU Yi-cheng, et al. Effects of dl-3-Butylph-thalide on treatment of acute ischemic strobe with moaerate symptoms: a multi-center, randomized, double-blind, placebo-control trial [J], Chin J Neurol, 2005, 38(4): 251-254
[29] 常文广，丁基苯酞对慢性脑缺血老龄大鼠学习记忆能力和GFAP，MBP的影响[D]，郑州大学2006届硕士研究生毕业论文，2006
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[31] 周晖，赵伟康．调心方对Aβ大鼠痴呆模型空间学习记忆障碍和胆碱能系统的影响[J]．中药药理和临床，1998，14(3)：29-31．
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[33] 刘学源，赵伟康，徐品初，调心方对淀粉样β蛋白25-35杏仁核注射诱导的痴呆模型大鼠淀粉样肽前体mRNA表达的影响[J]，中国临床康复2005，9(28)：130-132
[34] 舒斌，马世平．当归芍药散治疗老年痴呆研究进展[J]．中国老年学杂志，2002，22：236-237
[35] 蔡定芳，顾喜喜，陈依萍，等．养血清脑颗粒治疗慢性脑供血不足实验研究[J]．中华老年医学杂志，2005，24(3)：223-224
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[6] CUI Li-ying, LIU Xiu-qin, ZHU Yi-cheng, et al. Effects of dl-3-Butylph-thalide on treatment of acute ischemic strobe with moaerate symptoms: a multi-center, randomized, double-blind, placebo-control trial [J], Chin J Neurol, 2005, 38(4): 251-254
[7] 常文广，丁基苯酞对慢性脑缺血老龄大鼠学习记忆能力和GFAP，MBP的影响[D]，郑州大学2006届优秀硕士研究生毕业论文，2006
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[1] Martinez-Lage P, Hachinski V. Multi-Infarct dementia. In: Barnett H, Mohr JP, Stein B, Yatsu F, eds. Stroke: Pathophysiology, Diagnosis and Management [J]. 3rd Ed. Philadephia: Churchill Livingstone, 1998, 875-894
[2] 杨昆．老年性痴呆的防治[J]．博爱，2004，8(297)：47
[3] Rogers S L, Dody RS, Mobs Rc, et al. Done Pezil improves cognition and global fanction in Alzheimer' s disease; a 15-weekdouble-blind, placebo-controlled studyp[J]. Aroh Intern Med, 1998, 158(9): 1021
[4] Black S, Romn GC, Geldmacher DS, et al. Donepezil 307 Vascular Dementia Study Group. Efficacy and tolerability of donepezil in vascular dementia: positive results of a 24-week, multicenter, international, randomized, placebo-controlled clinical trial. Stroke [J]. 2003, 34(10): 2323-2330
[5] Wilkinson D, Doody R, Helme R, et al. Donepezil 308 Study Group. Donepezil in vascular dementia: a randomized, placebo-controlled study [J]. Neurology. 2003, 61 (4): 479-486.
[6] HONG Xia, ZHANG Zhen-xin, WANG Lu-ning, et al. A randomized study comparing the effect and safety of galantam ine and donepezil in patients with mild to moderate Alzheimer's disease [J]. Chin J Neurol, 2006, 39(6): 379-382
[7] Kumar V, Anand R, Messina J, et al. An efficacy and safety analysis of Exelon in Alzheimer' s disease patients with concurrent Vascular risk factors[J]. Eur J Neurol, 2000, 7: 159-169.
[8] Moretti-R, Torre P, AntoneIlo RM, et al. Rivastigmine superior to aspirin plus nimodipine in subcortical vascular dementia: an open, 16-month, comparative study[J]. Int J Clin Pract, 2004, 58(4): 346-353.
[9] 国家药典委员会编．临床用药须知[M]．北京：人民卫生出版社，2005：60
[10] 陶纪宁．胆碱酯酶抑制剂治疗AD的研究进展[J]．药学进展，2001，25(2)：205
[11] Orgogozo J-M, Rigaud A-S, Steffler A, et al. Efficacy and safety of memantine in patients with mild to moderate vascular dementia: a randomized, placebo-controlled trial (MMM300) [J]. Stroke, 2002, 33(7): 1834-1839.
[12] Wilcock G, Mabius HJ, Steffler A. on behalf of the MMM 500 group. A double-blind, placebo-controlled multicentre study of memantine in mild to moderate vascular dementia (MMM500) [J]. Internal Clin Psychopharmacol, 2002, 17(6): 297-305.
[13] 张华芳，李铮，唐勇，萘呋胺脂治疗脑血管性痴呆的临床观察[J]．临床荟萃，1997，12(14)：653-654．
[14] Kittner B. Clinical trails of propentofylline in vascular dementia. European/Canadian Propentofylline Study Group [J]. Alzheimer Dis Assoc Disorder, 1999, 13(3): 166-171
[15] Lopez-Arieta BJ. Nimodipine for primary degenerative, mixed and vascular dementia [P]. Cochrane Database Syst Rev, 2001, I: CD000147.
[16] Lopez-Arrieta J M, Birks J. Nimodipine for primary degenerative, mixed and vascular dementia[P]. Cochrane Database Syst Rev, 2002, 3: CD000147.
[17] Pantoni L, Del Ser T, Soglian AG, et al. Efficacy and safety of nimodipine in subcortical vascular dementia: a randomized placebo-controlled trail [J]. Stroke, 2005, 36(3): 619-624.
[18] 冯征，张均田，丹酚酸B对β-淀粉样蛋白介导原代培养皮层神经元毒性的保护作用[J]．药学学报，2000，35(12)：881-885．
[19] 韩恩吉，胡洪涛，何秀全，海风藤选择性抑制淀粉样前体蛋白基因表达[J]．中国临床康复，2004，8(13)：2592-2593
[20] Yun CAO, Alistair Jan GUAN, Laura BENNET, et al. Insulin like growth factor-1 reduces β-amyloid precursor protein expression after ischemic white matter damage in near-term fetal sheep [J]. Chin J Contemp Pediatr, 2004, 6(6): 449-452
[21] 里克特格登化工有限公司．作为药物的反式阿扑长春英宁酸酯衍生物[P]中国专利：96119131．3，1999-01-20．
[22] 王景华，楼风昌．天然活性成份防治AD的研究进展[J]．药学进展，2001，25(2)：76-80
[23] 刘小林，程春荣，曹威，等．石杉碱甲的药理作用与临床应用研究进展[J]，医药导报，2006，25(2)：90-95
[24] 李仪奎．中药药理世纪回眸[J]．中成药，2000，22(1)：560
[25] 钱天秀，杨世林，徐丽珍，等．银杏研究现状[J]．国外医药植物学分册，1997，12(4)：162
[26] 赵策生，严宝霞，翟所迪，等．银杏叶口服制剂有效成份质量评价研究[J]．临床药物治疗杂志，2003，1(3)：54
[27] CUI Li-ying, LI Shun-wei, LU Chuan-zhen, et al. The multicentric randomized study of dl-3-butylphthalide in the treatment of acute moderate ischemic stroke [J], Chin J Cerebrovasc Dis, 2005, 2(3): 112-115
[28] CUI Li-ying, LIU Xiu-qin, ZHU Yi-cheng, et al. Effects of dl-3-Butylph-thalide on treatment of acute ischemic strobe with moaerate symptoms: a multi-center, randomized, double-blind, placebo-control trial [J], Chin J Neurol, 2005, 38(4): 251-254
[29] 常文广，丁基苯酞对慢性脑缺血老龄大鼠学习记忆能力和GFAP，MBP的影响[D]，郑州大学2006届硕士研究生毕业论文，2006
[30] Stewart WF. Risk of Alzheimer's disease and duration of NSAIDs use[J]. Neurology, 1997, 48: 626-632.
[31] 周晖，赵伟康．调心方对Aβ大鼠痴呆模型空间学习记忆障碍和胆碱能系统的影响[J]．中药药理和临床，1998，14(3)：29-31．
[32] 王健，林水森，周如倩，等．调心方治疗阿尔茨海默氏病的临床研究[J]．北京中医药大学学报，2002，25(3)：51-53．
[33] 刘学源，赵伟康，徐品初，调心方对淀粉样β蛋白25-35杏仁核注射诱导的痴呆模型大鼠淀粉样肽前体mRNA表达的影响[J]，中国临床康复2005，9(28)：130-132
[34] 舒斌，马世平．当归芍药散治疗老年痴呆研究进展[J]．中国老年学杂志，2002，22：236-237
[35] 蔡定芳，顾喜喜，陈依萍，等．养血清脑颗粒治疗慢性脑供血不足实验研究[J]．中华老年医学杂志，2005，24(3)：223-224
[36] Fischer W, Wictorin R, Bjorklund A, et al. Amelioration of cholinergic neurons atrophy and spatial memory impairment in aged rats by nerver growth factor [J]. Nature, 1987, 329(6134): 65-68.
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