模式生物斑马鱼药物依赖模型的建立以及中药的干预机制
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摘要
背景
目前,苯丙胺类中枢兴奋剂(amphetamine-type stimulants,ATS)滥用增长势头迅猛,世界五大洲21个国家的兴奋剂滥用人数已超过海洛因和可卡因滥用人数之和,呈全球蔓延之势。欧洲7个国家的一项调查表明,ATS已成为第二大类最常滥用的成瘾物质。在我国,苯丙胺类中枢兴奋剂等新型毒品滥用形势严峻,又因其严重损害中枢神经系统、心血管系统等器官功能,易发生群体淫乱、人身攻击等暴力,引起社会和公共卫生问题,严重败坏社会风气,因而研制针对其依赖及其戒断症状的治疗药物意义深远。在我国中医药戒毒已经有200多年历史,积累了丰富的经验,形成了一套特有的理论和方法。中医药戒毒以扶助正气,排解烟毒为治疗思想,突出辨证论治的特色。中药疗法具有低毒价廉、药源广泛、无成瘾性等独特的优势,注重从病证出发,辨证施治,因此戒毒中药很可能在戒毒问题上取得突破性进展。
大量研究结果表明中枢谷氨酸神经系统与药物依赖有着密切的联系,有关NMDA受体和AMPA受体与药物依赖的报道较多,而TH作为多巴胺合成的关键酶在药物依赖形成过程中也起着十分重要的作用,但相关的报道不多。用绿色荧光蛋白(Green Fluorescent Protein,GFP)标记TH是一种新的分子生物学技术,它使TH表达的变化变得更加直观。GFP是一种能够在活细胞中表达的发光蛋白,不需要外源底物或辅助因子,在蓝光的激发下能发出绿色荧光。GFP作为荧光标记分子,既具有敏感的标记检测率,又没有放射性的危害。
条件性位置偏爱(conditioned place preference,CPP)实验是一种通过测定实验动物对体验药物效应的位置是否产生偏爱,从而评价药物精神依赖性潜力的有效方法。目前关于药物精神依赖的研究的实验动物,多集中啮齿类的大鼠、小鼠等,而随着越来越多样化的毒品的出现,我们需要寻找新的可用的模式生物作为传统模式生物的补充,应用于成瘾医学领域。
斑马鱼作为一种新型模式动物,与大鼠、小鼠等哺乳动物相比,由于其交配行为受光周期控制、产卵量多、受精卵在体外受精、繁殖周期快、易于饲养；而且前期胚胎整体透明,易于活体观察等优点,斑马鱼在行为学中的应用前景逐渐被研究者所认识。
实验目的
1.拟建立成年斑马鱼的甲基苯丙胺CPP动物模型,观察成年斑马鱼CPP效应,从行为学方面来评估成年斑马鱼的依赖效果,同时用中药有效成分进行干预,观察形成依赖后成年斑马鱼的行为变化。
2.采用蛋白免疫印迹(Western blotting)方法,观察甲基苯丙胺条件性位置偏爱成年斑马鱼脑内NR2B受体、GluR2受体和TH受体表达的改变及中药活性成分钩藤总碱对其干预后是否发生变化。
3.用GFP来标记多巴胺合成的关键酶—TH,然后利用转基因技术培育出TH-GFP转基因斑马鱼,通过观察转基因斑马鱼幼鱼头部荧光的强弱来判断其甲基苯丙胺依赖程度,同时初步摸索对于斑马鱼幼鱼大叶钩藤水提物的有效浓度。
4.证实斑马鱼是一个研究苯丙胺类兴奋剂依赖的理想模型和进一步研究斑马鱼的甲基苯丙胺依赖的作用机理以及中药对其的干预机制提供实验证据。
实验方法
1.成年斑马鱼条件性位置偏爱模型的建立：根据文献以及预实验结果可知成年斑马鱼的天然偏爱箱为褐色箱,故选择透明箱作为伴药箱。给药前用动物行为学分析系统对实验成年斑马鱼进行位置偏爱测定,剔除不符合天然偏爱习惯的动物。测定前将实验箱中间隔板抽出,当成年斑马鱼处于两个箱体之间时开始计时,逐条观察15min,以成年斑马鱼头部为准,记录成年斑马鱼在白箱中的停留时间。取经过自然位置偏爱测定合格的成年斑马鱼20条,按随机分组原则分为：①正常对照组,②甲基苯丙胺模型组。d1将斑马鱼置于独立的用于训练的CPP箱,每个CPP箱的水位不低于5cm,以保证足够的水压,适应性喂养至少2d。于d3测试正常状态下,所有斑马鱼的位置偏爱箱(15min内),并且用Noldus动物行为学分析系统来跟踪其路线图(5min内)。d4、d6、d8,将甲基苯丙胺模型组斑马鱼用tricaine麻醉后,置于柔软粗糙面上,用微量注射器,迅速腹腔注射甲基苯丙胺(40μg/g),将其置于伴药箱45min,伴药箱与偏爱箱之间用一个透明挡板隔开,使鱼不能游过去,但又不会遮挡其视线。45min后,将鱼移至较大的有蓝色环境的鱼缸,并且用70%的乙醇清洁CPP箱,最后用系统水冲洗。空白组注射等体积鱼用生理盐水,其他处理与模型组一致。d5、d7,在与d4注射的同一时间,斑马鱼用相同方法注射等体积的鱼用生理盐水,按相同的步骤,对斑马鱼进行训练。最后一次注射24h后(即d9),测试所有斑马鱼的伴药箱停留时间以及在鱼缸的路线图,比较它们在伴药箱前后停留时间的差值。
2.钩藤总碱对甲基苯丙胺依赖成年斑马鱼的影响：
2.1钩藤总碱对甲基苯丙胺依赖成年斑马鱼CPP的影响：取经过自然位置偏爱测定合格的成年斑马鱼50条,按随机分组原则分为：①正常对照组,②甲基苯丙胺模型组,③模型+钩藤总碱低剂量组(50μg/g),④模型+钩藤总碱高剂量组(100μg/g),⑤模型+氯胺酮组(150μg/g)。整个实验需要进行9d,d1将斑马鱼置于独立的用于训练的CPP箱,每个CPP箱的水位不低于5cm,以保证足够的水压,适应性喂养至少2d。d3测试正常状态下,所有斑马鱼的位置偏爱箱(15min内),并且用Noldus动物行为学分析系统来跟踪其路线图(5min内)。在d4、d6、d8,将除开空白组以外的所有斑马鱼浸入200mg/L tricaine methanesulfonate溶液中麻醉,用微量注射器迅速腹腔注射甲基苯丙胺(40μg/g),然后将其置于非偏爱箱(伴药箱)45min。之后的训练步骤与建立模型的训练步骤相同。12h后,将除开空白组以外的所有斑马鱼再次浸入200mg/L tricaine methanesulfonate溶液中麻醉,用微量注射器迅速腹腔注射相应的处理药物(模型组注射等体积生理盐水,低剂量组注射50μg/g钩藤总碱溶液,高剂量组注射100μg/g钩藤总碱溶液,氯胺酮组注射150gg/g氯胺酮溶液),之后将其移至较大的有蓝色环境的鱼缸。空白组斑马鱼用200mg/L tricaine methanesulfonate溶液麻醉后注射等体积鱼用生理盐水,其他处理与模型组一致。d5和d7在与d4注射的同一时间,给予斑马鱼注射等体积的鱼用生理盐水,然后将其置于偏爱箱(非伴药箱)45min。按上述相同的步骤,对斑马鱼进行训练。最后一次注射24h后(即d9),测试所有斑马鱼的伴药箱停留时间以及在鱼缸的路线图,比较它们在伴药箱前后停留时间的差值。
2.2钩藤总碱对甲基苯丙胺依赖成年斑马鱼脑内NR2B、GluR2和TH表达的影响：完成CPP测定后,将成年斑马鱼冷冻处死,取脑。将取出的脑,放入EP管中,加入适量的Lysis Buffer,冰浴匀浆。之后于其后于4℃,14000rpm离心10min,取其上清液。每组取上清液2μ1,将其稀释20倍,从稀释液中取5¨1至96孔板,每组取3次,共15μ1。然后,在96孔板每孔中加入Protein Assay Reagent A25μl、 Protein Assay Reagent B200μl。避光,缓慢振摇30min后,测蛋白浓度,放至-80℃冻存。配制好实验所需的溶液,待用。配制凝胶：一、分离胶,取10ml塑料离心管一支,分别加入MilliQ2.85ml、1.0M Tris-HCl(PH=8.8)3ml,30%Acrylamide2.15ml、10%SDS40μl,10%APS40μl,灌胶前加入TEMED8μl,混匀,灌胶。二、浓缩胶,取10ml塑料离心管一支,分别加入MilliQ2.1ml、1.0M Tris-HC1(PH=6.8)0.375ml、30%Acrylamide0.375ml、10%SDS15μl10%APS22.5μl,待分离胶凝好后,加入TEMED4.5μl,混匀,灌胶。SDS-PAGE电泳：将电泳槽加入足够电泳液后,准备上样。每组蛋白按30μg上样,两端,分别上MagicMark和Dual Mark。前40min,以40V,130mA电泳。40min之后,Dual Mark会逐渐分开,将电压加大,以70V,130mA电泳。转膜：在电泳结束前约5min,配制好Transfer Buffer。剪一张与分离胶差不多大小的PVDF膜,剪去一角,用100%甲醇浸润。取2块Mini Trans Blot Filter paper,与浸润好的PVDF膜一起放入Transfer Buffer中。电泳结束后,将浓缩胶与分离胶小心剥离,然后将分离胶轻轻取出,放入Transfer Buffer中。从下至上,按照夹子黑色面一Filterpaper—分离胶-PVDF膜—Filter paper—夹子红色面的顺序做好“三明治”。将“三明治”放入转移槽中,加入足够的Transfer Buffer,以15V,110mA,室温,搅拌,电转过夜。一抗、二抗的孵育：取出己转好的PVDF膜,室温下,在摇床上用1×TBST洗3次,每次10min,之后再用1×TBS洗10min。洗完后,用5%的脱脂牛奶在摇床上封闭1h。将封闭好的PVDF膜正面向上,涂上稀释成适当浓度的一抗,室温下静置2h。2h后,取出PVDF膜,于室温下,在摇床上用1×TBST洗3次,每次5min,之后再用1×TBS洗5mmin。将洗好的PVDF膜正面向上,涂上稀释成适当浓度的二抗,室温下静置1h。按照洗一抗的方法,洗去二抗。显影、定影：在最后一遍洗涤时,准备好ECL发光液,将其置于保鲜膜上,将洗好的PVDF膜,慢慢地盖上(确保没有气泡),包好,放入X一光片夹。在暗室中,将1×的显影液和定影液分别倒入塑料托盘中,在红灯下取出X一光片,并剪成合适大小(比PVDF膜略大)。打开X一光片夹,将剪好的X一光片覆盖在PVDF膜上,合上X一光片夹,根据信号强弱来调整曝光时间。拿出曝光好的X一光片,浸入显影液中,直到出现明显条带后,取出,用清水漂洗后,放入定影液2-5min,取出再用清水清洗。扫描特异条带,用Metamorph软件分析每个特异条带的OD值。
3.斑马鱼幼鱼药物依赖模型的建立以及给药剂量的初步探索：选取10对繁殖正常的成年斑马鱼,于繁殖前一天晚上分别放入10个繁殖缸中,雄鱼和雌鱼中间用透明隔板分开,过夜。第二天上午,做好显微注射的准备之后,抽开中间隔板,迅速向卵黄囊中注射TH—FP质粒。然后,将注射完的鱼卵,放入恒温恒湿箱。12小时后,将普通egg water换成含PTU的egg water。5天后在激光扫描共聚焦显微镜下观察是否转基因成功。显微注射后第五天,取转基因成功并且已经能自由游动的斑马鱼幼鱼12条,按随机分组原则分为：①甲基苯丙胺(60mg/L)模型组,②甲基苯丙胺(6mg/L)模型组,③甲基苯丙胺(0.6mg/L)模型组,④甲基苯丙胺(0.06mg/L)模型组。将每组斑马鱼幼鱼放入相应浓度的甲基苯丙胺溶液中自由游动,禁食。3天后,取出。用PBST漂洗5min后,再用4%PFA固定,过夜。第二天,取出固定好的斑马鱼幼鱼,用PBST漂洗3次,每次5min。然后将固定好的斑马鱼幼鱼放入液态的低熔点琼脂糖中,调整好位置,待其凝固后,用荧光体视显微镜观察其荧光变化。
浓度摸索：显微注射后第五天,取转基因成功并且已经能自由游动的斑马鱼幼鱼21条,按随机分组原则分为：①大叶钩藤水提物(1g/mL)组,②大叶钩藤水提物(0.33g/mL)组,③大叶钩藤水提物(0.033g/mL)组,④大叶钩藤水提物(0.0033g/mL)组,⑤大叶钩藤水提物(0.00033g/mL)组,⑥甲基苯丙胺(0.6mg/L)模型组,⑦空白对照组。除第⑦组外,将每组斑马鱼幼鱼放入0.6mg/L的甲基苯丙胺溶液中自由游动,禁食。3天后,将第①组一第⑤组的斑马鱼幼鱼取出,放入相应浓度的大叶钩藤水提物中,第⑥组放入清水中,自由游动。24h后,将所有斑马鱼幼鱼取出,用PBST漂洗5mmin后,再用4%PFA固定,过夜。第二天,取出固定好的斑马鱼幼鱼,用PBST漂洗3次,每次5mmin。然后将固定好的斑马鱼幼鱼放入液态的低熔点琼脂糖中,调整好位置,待其凝固后,用激光扫描共聚焦显微镜观察其荧光变化。
实验结果
1.从实验结果可以看出,空白对照组成年斑马鱼和甲基苯丙胺模型组成年斑马鱼在非偏爱箱前后停留时间的差值,通过两独立样本t检验(P=0.000),表明两组成年斑马鱼在非偏爱箱前后停留时间的差值之间有显著性差异。斑马鱼在鱼缸的路线图显示,造模后,空白组造模前后的活动路线变化不显著,而甲基苯丙胺模型组斑马鱼在偏爱箱活动的活动轨迹与造模前相比,有明显减少。
2.从实验结果可以看出：模型组训练前后在伴药箱停留时间差与空白组训练前后在伴药箱停留时间差相比,有显著性差异(P=0.000),表明成年斑马鱼条件性位置偏爱模型建立成功。与模型组训练前后在伴药箱停留时间差相比,钩藤总碱高剂量组(P=0.000)和氯胺酮组(P=0.000)训练前后在伴药箱停留时间差有显著性差异,而钩藤总碱低剂量组训练前后在伴药箱停留时间差无明显差别(P=0.949)。
以适当比例稀释抗体进行Western blotting检测,以P-actin为内标。与空白组成年斑马鱼相比,模型组成年斑马鱼脑内的TH表达有显著性差异(P=0.001),钩藤总碱高剂量组成年斑马鱼(P=0.777)和氯胺酮组成年斑马鱼(P=0.546)脑内的TH表达无显著差异,与甲基苯丙胺模型组成年斑马鱼相比,钩藤总碱高剂量组成年斑马鱼(P=0.001)和氯胺酮组成年斑马鱼(P=0.007)脑内的TH表达有显著差异,钩藤总碱低剂量组成年斑马鱼脑内的TH表达无显著性差异(P=0.113),与空白组成年斑马鱼相比,模型组成年斑马鱼(P=0.001)和钩藤总碱低剂量组成年斑马鱼(P=0.01)脑内的NR2B表达有显著性差异,钩藤总碱高剂量组成年斑马鱼(P=0.083)和氯胺酮组成年斑马鱼(P=0.105)脑内的NR2B表达无显著差异,与空白组成年斑马鱼相比,模型组成年斑马鱼脑内的GluR2表达有显著性差异(P=0.04),钩藤总碱高剂量组成年斑马鱼(P=0.334)和氯胺酮组成年斑马鱼(P=0.130)脑内的GluR2表达无显著差异,与甲基苯丙胺模型组成年斑马鱼相比,钩藤总碱高剂量组成年斑马鱼脑内的GluR2表达有显著差异(P=0.003),钩藤总碱低剂量组成年斑马鱼脑内的GluR2表达无显著性差异(P=0.698)。
3.转基因结果显示,与正常斑马鱼幼鱼相比,显微注射TH—FP质粒后的斑马鱼幼鱼头部出现明显荧光。在绿色荧光下,与空白组斑马鱼幼鱼相比,甲基苯丙胺(60mg/L)模型组斑马鱼幼鱼头部荧光变化明显,甲基苯丙胺(6mg/L)模型组斑马鱼幼鱼头部荧光变化明显,甲基苯丙胺(0.6mg/L)模型组斑马鱼幼鱼头部荧光变化明显,而甲基苯丙胺(0.06mg/L)模型组斑马鱼幼鱼头部荧光变化则不明显。将大叶钩藤水提物(1g/mL)组斑马鱼幼鱼,大叶钩藤水提物(0.33g/mL)组斑马鱼幼鱼,大叶钩藤水提物(0.033g/mL)组斑马鱼幼鱼,大叶钩藤水提物(0.0033g/mL)组斑马鱼幼鱼,大叶钩藤水提物(0.00033g/mL)组斑马鱼幼鱼放入相应浓度的水提物中24h后,大叶钩藤水提物(1g/mL)组斑马鱼幼鱼,大叶钩藤水提物(0.33g/mL)组斑马鱼幼鱼,大叶钩藤水提物(0.033g/mL)组斑马鱼幼鱼和大叶钩藤水提物(0.0033g/mL)组斑马鱼幼鱼死亡,大叶钩藤水提物(0.00033g/mL)组斑马鱼幼鱼游动正常。在共聚焦显微镜下,与空白组相比,甲基苯丙胺(0.6mg/L)模型组斑马鱼幼鱼头部荧光明显增强。与甲基苯丙胺(0.6mg/L)模型组相比,大叶钩藤水提物(0.00033g/mL)组斑马鱼幼鱼头部荧光有所减弱。
结论
1.甲基苯丙胺可以使成年斑马鱼产生明显的位置偏爱效应,成年斑马鱼在伴药箱中停留时间明显延长,证明成年斑马鱼可以作为模式生物进行药物依赖方面的研究。高剂量钩藤总碱(100μg/g)和氯胺酮(150μg/g)可显著抑制甲基苯丙胺依赖成年斑马鱼的位置偏爱效应,使之与甲基苯丙胺模型组成年斑马鱼有显著差异。而低剂量钩藤总碱(501μg/g)对甲基苯丙胺依赖成年斑马鱼的位置偏爱效应没有显示出明显的抑制效果。
2.甲基苯丙胺依赖的形成会使正常成年斑马鱼脑内TH、 NR2B、 GluR2三种蛋白表达显著上调。高剂量钩藤总碱(100μg/g)和氯胺酮(150μg/g)可显著抑制这种改变,使甲基苯丙胺依赖的成年斑马鱼脑内的TH、 NR2B、 GluR2三种蛋白表达下调,而低剂量钩藤总碱(50μg/g)对这种改变没有明显作用。
3.可以利用TH—GFP转基因斑马鱼,在显微镜下通过绿色荧光强弱来直观的观察斑马鱼甲基苯丙胺依赖情况,以及药物对其的干预效果：0.6mg/L的甲基苯丙胺溶液就能使TH—FP转基因斑马鱼头部荧光发生明显增强,而浓度为0.00033g/mL大叶钩藤水提物可以降低这种荧光的改变。
注：本论文中的所有数据均采用SPSS13.0进行分析。所有数据以(x±SD)表示,各组成年斑马鱼在伴药箱中的停留时间比较采用单向方差分析(One-Way ANOVA)或者协方差分析(analysis of covarinance,ANCOVA),各组均数的多重比较采用最小显著差值法(least significant different, LSD),不满足方差齐性要求的,采用Dunnett's T3法。Western blotting结果以每张转印膜上目的蛋白条带光密度值,进行单因素方差分析。检测水平α=0.05。
BACKGROUD
At present, the rapid momentum of amphetamine-type stimulants (ATS) abuse is increasing, the number of stimulant abuse has exceeded the number of heroin and cocaine abuse and was global spreading in the21st countries of five continents. A survey of seven European countries is that the ATS has become the second largest category of the most commonly abused addictive substances. In China, amphetamine-type stimulant abuse situation is grim, it can damage to the central nervous system and cardiovascular system seriously, prone to herd riots, personal attacks and other violence, causing social and public health problem, seriously damaging the social climate. Therefore, development of the treatment for its dependence and drug withdrawal symptoms is significance. The history of Chinese medicine in drug treatment has been more than200years, it has accumulated a wealth of experience, the formation of a unique theory and effective way. The treatment of ideology in Chinese medicine drug treatment is strengthening body resistance and eliminate pathogenic factor, highlighting the diagnosis and treatment characteristics. Traditional Chinese Medicine therapy has the unique advantages: low toxicity, low cost, a wide range source of drugs and few of addiction, pay attention to starting from the disease and syndrome, dialectical treatment by traditional Chinese medicine, so the development of new drugs or drug treatment from Chinese medicine is likely to a breakthrough on the problem of withdrawal.
A large number of research results show that the central glutamic acid nervous system and drug dependence are closely linked, there are many reports about NMDA receptors and AMPA receptors and drug dependence related, but tyrosine hydroxylase (TH) as a key enzyme in dopamine synthesis also plays an important role in the process of drug dependence, there is few reports about it. Marking TH With green fluorescent protein(GFP) is a new molecular biology techniques, it makes the TH expression changes become more intuitive. GFP is a light-emitting protein expression in living cells without exogenous substrates or cofactors, can issue the green fluorescence by blu-ray excitation. GFP as a fluorescent marker has both sensitive tag detection rate and no radioactive hazards.
Conditioned place preference(CPP) is usually used to study on drug-dependence for Evaluation of drug psychological dependence potential. The experimental animals on drug psychological dependence studies is mostly rodents: rats and mice, but With the emergence of increasingly diverse drug, We need to find new available model organism as a complement to traditional model organisms for the field of addiction medicine.
Zebrafish as a new model animal, compared with rats or mice and other mammals, their mating behavior is affected by photoperiod control, the amount of spawning, the fertilized egg in vitro fertilization, the reproductive cycle fast, easy feeding; and their early embryo is transparent as a whole, observation in vivo easily, The application prospects of the behavior of the zebrafish are gradually being understood by researchers.
OBJECTIVE
1. To establish methamphetamine CPP animal models of adult zebrafish, observe the CPP effects in adult zebrafish, assess the dependence of the effect of adult zebrafish from behavior. At the same time, to treat by Chinese medicine active ingredients and to observe the behavioral changes of the methamphetamine-dependent adult zebrafish.
2. To establish the model of the adult zebrafish methamphetamine conditioned place preference, Observe the NR2B receptor, the GluR2receptor and the TH receptor expression changes in the methamphetamine conditioned place preference adult zebrafish brain and change of expression after treatment by the as Chinese medicine active ingredients by western blotting.
3. To mark a key enzyme—H in the dopamine synthesis with GFP, then cultivate the TH-GFP transgenic zebrafish by transgenic technology, determine its lever of methamphetamine dependence by the fluorescence in zebrafish Larvae head. Preliminary groping for the effective concentration of Uncaria macrophylla leaf aqueous extract about the zebrafish larvae.
4. To provide experimental evidence for confirming that the zebrafish is an ideal model to study amphetamine-type stimulants dependence, Further study of methamphetamine-dependent mechanism of the zebrafish and mechanism of Traditional Chinese Medicine treatment.
METHODS
1. To establish conditioned place preference model in adult zebrafish:According to the literature and pre-experimental results, they shows that the natural preference of adult zebrafish box is brown box, so to definie the transparent box with the kits.20 adult zebrafish which passed the determination of nature place preference were randomly divided into①ormal control group,②odel group of methamphetamine. The fish were moved to the behavior room under maintenance conditions and feeding schedule identical to the fish facility at least2days before each assay. The water level was kept to5cm from the tank bottom to minimize stress. On the third day, the fish was recorded for a15-min trial using Noldus system, The preferred compartment was defined as the compartment in which a fish spends most of its time during the measurement on day3, and the place preference(PP) is calculated as the percentage of time that the fish spends in the preferred compartment. On day4, day6and day8, anesthetized in tricaine for a few seconds. It was immobilized by hand into a Petri dish containing tank water, and then received methamphetamine (40μg/g) by intraperitoneal injection using syringe. After waking up, the fish was confined to the non-preferred compartment for45min. The restriction to this compartment was achieved using a transparent slider. Thus, visual contact with the preferred compartment remained, and the difference between the conditioning and recording conditions was minimized. The experimental tank and conditions were otherwise identical to the ones used for place preference determination, and each fish was tested separately. After45min, the fish was removed from the experimental tank and kept in a1.5L tank on a color-neutral background. On day5and day7, fish was injected intraperitoneally with a saline solution, then restricted for45min into the preferred compartment. Between each injection session, the experimental tank was cleaned with70%ethanol and rinsed with fish facility water. Place preference was then measured again on day9and compared the difference of stay-time in the prefer department.
2. The impact of Uncaria alkaloids for Methamphetamine-dependent adult zebrafish:
2.1The impact of Uncaria alkaloids for CCP of Methamphetamine-dependent adult zebrafish:50adult zebrafish which passed the determination of nature place preference were randomly divided into①normal control group,②odel group of methamphetamine,③odel group+low dose of group(50μg/g),④odel group+high dose of group(100μg/g),⑤model group+ketamine group (150μg/g) The fish were moved to the behavior room under maintenance conditions and feeding schedule identical to the fish facility at least2days before each assay. The water level was kept to5cm from the tank bottom to minimize stress. On the third day, the fish was recorded for a15-min trial using Noldus system, The preferred compartment was defined as the compartment in which a fish spends most of its time during the measurement on day3, and the place preference(PP) is calculated as the percentage of time that the fish spends in the preferred compartment. On day4, day6and day8, anesthetized in tricaine for a few seconds. It was immobilized by hand into a Petri dish containing tank water, and then received methamphetamine (40μg/g) by intraperitoneal injection using syringe, except control group; control group received equal volume of normal saline. After waking up, the fish was confined to the non-preferred compartment for45min. The restriction to this compartment was achieved using a transparent slider. Thus, visual contact with the preferred compartment remained, and the difference between the conditioning and recording conditions was minimized. The experimental tank and conditions were otherwise identical to the ones used for place preference determination, and each fish was tested separately. After45min, the fish was removed from the experimental tank and kept in a1.5L tank on a color-neutral background. After12hours, except control group, injected equal volume of normal saline in model group; injected low dose of in low dose of rhynchphylla total alkaloids group; injected high dose of rhynchphylla total alkaloids in high dose of rhynchphylla total alkaloids group; injected ketamine in ketamine group. On day5and day7, fish was injected intraperitoneally with a saline solution, then restricted for45min into the preferred compartment. After12hours, the steps are as similar as day4. Between each injection session, the experimental tank was cleaned with70%ethanol and rinsed with fish facility water. Place preference was then measured again on day9and compared the difference of stay-time in the prefer department.
2.2The impact of Uncaria alkaloids for expression of NR2B receptor, GluR2receptor and TH receptor in the brain of Methamphetamine-dependent adult zebrafish: to extract total protein in adult zebrafish brain and to computer protein content. According to the protocol of the SCT1111Lab in Hong Kong Baptist University, finished the western blotting.
3. To establish methamphetamine-dependent animal model of zebrafish larvae and to groping for the effective concentration of Uncaria macrophylla leaf aqueous extract about the zebrafish Larvae:microinjected the TH—FP plasmid into yoke of one cell stage zebrafish eggs. After5days,12TH—FP transgenic zebrafish larvae which is alive were randomly divided into①model group (60mg/L),②model group (6mg/L),③model group (0.6mg/L),④model group (0.06mg/L). Zebrafish larval of each group into corresponding concentrations of methamphetamine solution in freely moving with fasting. After3days, to observe the fluorescence intensity in of TH—FP transgenic zebrafish larvae brain.
After microinjection5days,21transgenic zebrafish larvae were randomly divided into①Uncaria macrophylla leaf aqueous extract group (1g/mL),②Uncaria macrophylla leaf aqueous extract group (0.33g/mL),③Uncaria macrophylla leaf aqueous extract group (0.033g/mL),④Uncaria macrophylla leaf aqueous extract group (0.0033g/mL),⑤Uncaria macrophylla leaf aqueous extract group (0.00033g/mL),⑥model group (0.6mg/L),⑦control group. Zebrafish larval of each group into0.6mg/L methamphetamine solution in freely moving with fasting, except①group. After3days, Zebrafish larval of each group into corresponding concentrations of Uncaria macrophylla leaf aqueous extract, Zebrafish larval of⑥group into water in freely moving with fasting, except①group. Next day, to observe the fluorescence intensity in of TH—FP transgenic zebrafish larvae brain.
RESULTS
1. The experimental results shown that compared with control group and model group by two independent sample t-test (t=-11.791, P=0.000), the difference of stay-time is significant. According the map of zebrafish in the prefer department, after modeling, the track of model group zebrafish is reduced significantly than before; and the change of control group is not significant.
2. The experimental results shown that compared with control group, the difference of model group stay-time is significant (P=0.000), compared with model group, the difference of high dose of rhynchphylla total alkaloids group(P=0.000)stay-time and ketamine group (P=0.000) are significant, but the difference of low dose of rhynchphylla total alkaloids group stay-time is not significant (P=0.949)
The western blotting results shown that compared with control group, the difference of the TH receptor expression in model group adult zebrafish brain is significant (P=0.001), the difference of high dose of rhynchphylla total alkaloids group (P=0.777) the TH receptor expression and ketamine group (P=0.546) the TH receptor expression are not significant; compared with model group, the difference of high dose of rhynchphylla total alkaloids group (P=0.001) the TH receptor expression and ketamine group (P=0.007) the TH receptor expression are significant, the difference of low dose of rhynchphylla total alkaloids group the TH receptor expression is not significant (P=0.113); compared with control group, the difference of the NR2B receptor expression in model group (P=0.001) and low dose of rhynchphylla total alkaloids group (P=0.01) the NR2B receptor expression are significant, the difference of high dose of rhynchphylla total alkaloids group (P=0.083) the NR2B receptor expression and ketamine group (P=0.105) the NR2B receptor expression are not significant; compared with control group, the difference of the GluR2receptor expression in model group (P=0.04) is significant, the difference of high dose of rhynchphylla total alkaloids group (P=0.334) the GluR2receptor expression and ketamine group(P=0.130)the GluR2receptor expression are not significant, compared with model group, the difference of the GluR2receptor expression in high dose of rhynchphylla total alkaloids group (P=0.003)is significant, the difference of the GluR2receptor expression in low dose of rhynchphylla total alkaloids group (P=0.698) is not significant.
3. The experimental results shown that compared with normal zebrafish larvae, the fluorescence of transgenic zebrafish larvae is brighter. Compared with control group, the fluorescence of model group (60mg/L) and model group (6mg/L) and model group (0.6mg/L) are brighter. After zebrafish larval of each group into corresponding concentrations of Uncaria macrophylla leaf aqueous extract, Uncaria macrophylla leaf aqueous extract group (1g/mL), Uncaria macrophylla leaf aqueous extract group (0.33g/mL),Uncaria macrophylla leaf aqueous extract group (0.033g/mL) and Uncaria macrophylla leaf aqueous extract group (0.0033g/mL) are dead, but Uncaria macrophylla leaf aqueous extract group (0.00033g/mL) is alive.
CONCLUSION
1. Methamphetamine can make adult zebrafish have obvious place preference in the medicine cabinet, prolonging the stay-time in the drug-paired department. It confirmed that adult zebrafish can be used as a model organism for the study of drug dependence. High doses of rhynchphylla total alkaloids (100μg/g) and ketamine (150μg/g) could significantly inhibit methamphetamine dependent adult zebrafish place preference. Low dose of rhynchphylla total alkaloids (50μg/g) on methamphetamine dependence in adult zebrafish place preference shows no obvious inhibition effect.
2. Methamphetamine-dependent formation can make the expression of normal adult zebrafish brain TH, NR2B, GluR2was significantly higher. High doses of rhynchphylla total alkaloids (100μg/g) and ketamine (150μg/g) could significantly inhibit the change, make the expression of TH, NR2B, GluR2in methamphetamine-dependence adult zebrafish brain is down regulated, while the lower dose of rhynchphylla total alkaloids(50μg/g) had no obvious effect on the change.
3. We can use the TH—GFP transgenic zebrafish, under the microscope by green fluorescence intensity, to observe the zebrafish methamphetamine-dependence, and the effects of the drug treatment.0.6mg/L TH—GFP methamphetamine solution can make transgenic zebrafish head have obvious fluorescence enhancement, and the concentration of0.00033g/mL Uncaria macrophylla leaf water extract can to reduce this fluorescence change.
目前,苯丙胺类中枢兴奋剂(amphetamine-type stimulants,ATS)滥用增长势头迅猛,世界五大洲21个国家的兴奋剂滥用人数已超过海洛因和可卡因滥用人数之和,呈全球蔓延之势。欧洲7个国家的一项调查表明,ATS已成为第二大类最常滥用的成瘾物质。在我国,苯丙胺类中枢兴奋剂等新型毒品滥用形势严峻,又因其严重损害中枢神经系统、心血管系统等器官功能,易发生群体淫乱、人身攻击等暴力,引起社会和公共卫生问题,严重败坏社会风气,因而研制针对其依赖及其戒断症状的治疗药物意义深远。在我国中医药戒毒已经有200多年历史,积累了丰富的经验,形成了一套特有的理论和方法。中医药戒毒以扶助正气,排解烟毒为治疗思想,突出辨证论治的特色。中药疗法具有低毒价廉、药源广泛、无成瘾性等独特的优势,注重从病证出发,辨证施治,因此戒毒中药很可能在戒毒问题上取得突破性进展。
大量研究结果表明中枢谷氨酸神经系统与药物依赖有着密切的联系,有关NMDA受体和AMPA受体与药物依赖的报道较多,而TH作为多巴胺合成的关键酶在药物依赖形成过程中也起着十分重要的作用,但相关的报道不多。用绿色荧光蛋白(Green Fluorescent Protein,GFP)标记TH是一种新的分子生物学技术,它使TH表达的变化变得更加直观。GFP是一种能够在活细胞中表达的发光蛋白,不需要外源底物或辅助因子,在蓝光的激发下能发出绿色荧光。GFP作为荧光标记分子,既具有敏感的标记检测率,又没有放射性的危害。
条件性位置偏爱(conditioned place preference,CPP)实验是一种通过测定实验动物对体验药物效应的位置是否产生偏爱,从而评价药物精神依赖性潜力的有效方法。目前关于药物精神依赖的研究的实验动物,多集中啮齿类的大鼠、小鼠等,而随着越来越多样化的毒品的出现,我们需要寻找新的可用的模式生物作为传统模式生物的补充,应用于成瘾医学领域。
斑马鱼作为一种新型模式动物,与大鼠、小鼠等哺乳动物相比,由于其交配行为受光周期控制、产卵量多、受精卵在体外受精、繁殖周期快、易于饲养；而且前期胚胎整体透明,易于活体观察等优点,斑马鱼在行为学中的应用前景逐渐被研究者所认识。
实验目的
1.拟建立成年斑马鱼的甲基苯丙胺CPP动物模型,观察成年斑马鱼CPP效应,从行为学方面来评估成年斑马鱼的依赖效果,同时用中药有效成分进行干预,观察形成依赖后成年斑马鱼的行为变化。
2.采用蛋白免疫印迹(Western blotting)方法,观察甲基苯丙胺条件性位置偏爱成年斑马鱼脑内NR2B受体、GluR2受体和TH受体表达的改变及中药活性成分钩藤总碱对其干预后是否发生变化。
3.用GFP来标记多巴胺合成的关键酶—TH,然后利用转基因技术培育出TH-GFP转基因斑马鱼,通过观察转基因斑马鱼幼鱼头部荧光的强弱来判断其甲基苯丙胺依赖程度,同时初步摸索对于斑马鱼幼鱼大叶钩藤水提物的有效浓度。
4.证实斑马鱼是一个研究苯丙胺类兴奋剂依赖的理想模型和进一步研究斑马鱼的甲基苯丙胺依赖的作用机理以及中药对其的干预机制提供实验证据。
实验方法
1.成年斑马鱼条件性位置偏爱模型的建立：根据文献以及预实验结果可知成年斑马鱼的天然偏爱箱为褐色箱,故选择透明箱作为伴药箱。给药前用动物行为学分析系统对实验成年斑马鱼进行位置偏爱测定,剔除不符合天然偏爱习惯的动物。测定前将实验箱中间隔板抽出,当成年斑马鱼处于两个箱体之间时开始计时,逐条观察15min,以成年斑马鱼头部为准,记录成年斑马鱼在白箱中的停留时间。取经过自然位置偏爱测定合格的成年斑马鱼20条,按随机分组原则分为：①正常对照组,②甲基苯丙胺模型组。d1将斑马鱼置于独立的用于训练的CPP箱,每个CPP箱的水位不低于5cm,以保证足够的水压,适应性喂养至少2d。于d3测试正常状态下,所有斑马鱼的位置偏爱箱(15min内),并且用Noldus动物行为学分析系统来跟踪其路线图(5min内)。d4、d6、d8,将甲基苯丙胺模型组斑马鱼用tricaine麻醉后,置于柔软粗糙面上,用微量注射器,迅速腹腔注射甲基苯丙胺(40μg/g),将其置于伴药箱45min,伴药箱与偏爱箱之间用一个透明挡板隔开,使鱼不能游过去,但又不会遮挡其视线。45min后,将鱼移至较大的有蓝色环境的鱼缸,并且用70%的乙醇清洁CPP箱,最后用系统水冲洗。空白组注射等体积鱼用生理盐水,其他处理与模型组一致。d5、d7,在与d4注射的同一时间,斑马鱼用相同方法注射等体积的鱼用生理盐水,按相同的步骤,对斑马鱼进行训练。最后一次注射24h后(即d9),测试所有斑马鱼的伴药箱停留时间以及在鱼缸的路线图,比较它们在伴药箱前后停留时间的差值。
2.钩藤总碱对甲基苯丙胺依赖成年斑马鱼的影响：
2.1钩藤总碱对甲基苯丙胺依赖成年斑马鱼CPP的影响：取经过自然位置偏爱测定合格的成年斑马鱼50条,按随机分组原则分为：①正常对照组,②甲基苯丙胺模型组,③模型+钩藤总碱低剂量组(50μg/g),④模型+钩藤总碱高剂量组(100μg/g),⑤模型+氯胺酮组(150μg/g)。整个实验需要进行9d,d1将斑马鱼置于独立的用于训练的CPP箱,每个CPP箱的水位不低于5cm,以保证足够的水压,适应性喂养至少2d。d3测试正常状态下,所有斑马鱼的位置偏爱箱(15min内),并且用Noldus动物行为学分析系统来跟踪其路线图(5min内)。在d4、d6、d8,将除开空白组以外的所有斑马鱼浸入200mg/L tricaine methanesulfonate溶液中麻醉,用微量注射器迅速腹腔注射甲基苯丙胺(40μg/g),然后将其置于非偏爱箱(伴药箱)45min。之后的训练步骤与建立模型的训练步骤相同。12h后,将除开空白组以外的所有斑马鱼再次浸入200mg/L tricaine methanesulfonate溶液中麻醉,用微量注射器迅速腹腔注射相应的处理药物(模型组注射等体积生理盐水,低剂量组注射50μg/g钩藤总碱溶液,高剂量组注射100μg/g钩藤总碱溶液,氯胺酮组注射150gg/g氯胺酮溶液),之后将其移至较大的有蓝色环境的鱼缸。空白组斑马鱼用200mg/L tricaine methanesulfonate溶液麻醉后注射等体积鱼用生理盐水,其他处理与模型组一致。d5和d7在与d4注射的同一时间,给予斑马鱼注射等体积的鱼用生理盐水,然后将其置于偏爱箱(非伴药箱)45min。按上述相同的步骤,对斑马鱼进行训练。最后一次注射24h后(即d9),测试所有斑马鱼的伴药箱停留时间以及在鱼缸的路线图,比较它们在伴药箱前后停留时间的差值。
2.2钩藤总碱对甲基苯丙胺依赖成年斑马鱼脑内NR2B、GluR2和TH表达的影响：完成CPP测定后,将成年斑马鱼冷冻处死,取脑。将取出的脑,放入EP管中,加入适量的Lysis Buffer,冰浴匀浆。之后于其后于4℃,14000rpm离心10min,取其上清液。每组取上清液2μ1,将其稀释20倍,从稀释液中取5¨1至96孔板,每组取3次,共15μ1。然后,在96孔板每孔中加入Protein Assay Reagent A25μl、 Protein Assay Reagent B200μl。避光,缓慢振摇30min后,测蛋白浓度,放至-80℃冻存。配制好实验所需的溶液,待用。配制凝胶：一、分离胶,取10ml塑料离心管一支,分别加入MilliQ2.85ml、1.0M Tris-HCl(PH=8.8)3ml,30%Acrylamide2.15ml、10%SDS40μl,10%APS40μl,灌胶前加入TEMED8μl,混匀,灌胶。二、浓缩胶,取10ml塑料离心管一支,分别加入MilliQ2.1ml、1.0M Tris-HC1(PH=6.8)0.375ml、30%Acrylamide0.375ml、10%SDS15μl10%APS22.5μl,待分离胶凝好后,加入TEMED4.5μl,混匀,灌胶。SDS-PAGE电泳：将电泳槽加入足够电泳液后,准备上样。每组蛋白按30μg上样,两端,分别上MagicMark和Dual Mark。前40min,以40V,130mA电泳。40min之后,Dual Mark会逐渐分开,将电压加大,以70V,130mA电泳。转膜：在电泳结束前约5min,配制好Transfer Buffer。剪一张与分离胶差不多大小的PVDF膜,剪去一角,用100%甲醇浸润。取2块Mini Trans Blot Filter paper,与浸润好的PVDF膜一起放入Transfer Buffer中。电泳结束后,将浓缩胶与分离胶小心剥离,然后将分离胶轻轻取出,放入Transfer Buffer中。从下至上,按照夹子黑色面一Filterpaper—分离胶-PVDF膜—Filter paper—夹子红色面的顺序做好“三明治”。将“三明治”放入转移槽中,加入足够的Transfer Buffer,以15V,110mA,室温,搅拌,电转过夜。一抗、二抗的孵育：取出己转好的PVDF膜,室温下,在摇床上用1×TBST洗3次,每次10min,之后再用1×TBS洗10min。洗完后,用5%的脱脂牛奶在摇床上封闭1h。将封闭好的PVDF膜正面向上,涂上稀释成适当浓度的一抗,室温下静置2h。2h后,取出PVDF膜,于室温下,在摇床上用1×TBST洗3次,每次5min,之后再用1×TBS洗5mmin。将洗好的PVDF膜正面向上,涂上稀释成适当浓度的二抗,室温下静置1h。按照洗一抗的方法,洗去二抗。显影、定影：在最后一遍洗涤时,准备好ECL发光液,将其置于保鲜膜上,将洗好的PVDF膜,慢慢地盖上(确保没有气泡),包好,放入X一光片夹。在暗室中,将1×的显影液和定影液分别倒入塑料托盘中,在红灯下取出X一光片,并剪成合适大小(比PVDF膜略大)。打开X一光片夹,将剪好的X一光片覆盖在PVDF膜上,合上X一光片夹,根据信号强弱来调整曝光时间。拿出曝光好的X一光片,浸入显影液中,直到出现明显条带后,取出,用清水漂洗后,放入定影液2-5min,取出再用清水清洗。扫描特异条带,用Metamorph软件分析每个特异条带的OD值。
3.斑马鱼幼鱼药物依赖模型的建立以及给药剂量的初步探索：选取10对繁殖正常的成年斑马鱼,于繁殖前一天晚上分别放入10个繁殖缸中,雄鱼和雌鱼中间用透明隔板分开,过夜。第二天上午,做好显微注射的准备之后,抽开中间隔板,迅速向卵黄囊中注射TH—FP质粒。然后,将注射完的鱼卵,放入恒温恒湿箱。12小时后,将普通egg water换成含PTU的egg water。5天后在激光扫描共聚焦显微镜下观察是否转基因成功。显微注射后第五天,取转基因成功并且已经能自由游动的斑马鱼幼鱼12条,按随机分组原则分为：①甲基苯丙胺(60mg/L)模型组,②甲基苯丙胺(6mg/L)模型组,③甲基苯丙胺(0.6mg/L)模型组,④甲基苯丙胺(0.06mg/L)模型组。将每组斑马鱼幼鱼放入相应浓度的甲基苯丙胺溶液中自由游动,禁食。3天后,取出。用PBST漂洗5min后,再用4%PFA固定,过夜。第二天,取出固定好的斑马鱼幼鱼,用PBST漂洗3次,每次5min。然后将固定好的斑马鱼幼鱼放入液态的低熔点琼脂糖中,调整好位置,待其凝固后,用荧光体视显微镜观察其荧光变化。
浓度摸索：显微注射后第五天,取转基因成功并且已经能自由游动的斑马鱼幼鱼21条,按随机分组原则分为：①大叶钩藤水提物(1g/mL)组,②大叶钩藤水提物(0.33g/mL)组,③大叶钩藤水提物(0.033g/mL)组,④大叶钩藤水提物(0.0033g/mL)组,⑤大叶钩藤水提物(0.00033g/mL)组,⑥甲基苯丙胺(0.6mg/L)模型组,⑦空白对照组。除第⑦组外,将每组斑马鱼幼鱼放入0.6mg/L的甲基苯丙胺溶液中自由游动,禁食。3天后,将第①组一第⑤组的斑马鱼幼鱼取出,放入相应浓度的大叶钩藤水提物中,第⑥组放入清水中,自由游动。24h后,将所有斑马鱼幼鱼取出,用PBST漂洗5mmin后,再用4%PFA固定,过夜。第二天,取出固定好的斑马鱼幼鱼,用PBST漂洗3次,每次5mmin。然后将固定好的斑马鱼幼鱼放入液态的低熔点琼脂糖中,调整好位置,待其凝固后,用激光扫描共聚焦显微镜观察其荧光变化。
实验结果
1.从实验结果可以看出,空白对照组成年斑马鱼和甲基苯丙胺模型组成年斑马鱼在非偏爱箱前后停留时间的差值,通过两独立样本t检验(P=0.000),表明两组成年斑马鱼在非偏爱箱前后停留时间的差值之间有显著性差异。斑马鱼在鱼缸的路线图显示,造模后,空白组造模前后的活动路线变化不显著,而甲基苯丙胺模型组斑马鱼在偏爱箱活动的活动轨迹与造模前相比,有明显减少。
2.从实验结果可以看出：模型组训练前后在伴药箱停留时间差与空白组训练前后在伴药箱停留时间差相比,有显著性差异(P=0.000),表明成年斑马鱼条件性位置偏爱模型建立成功。与模型组训练前后在伴药箱停留时间差相比,钩藤总碱高剂量组(P=0.000)和氯胺酮组(P=0.000)训练前后在伴药箱停留时间差有显著性差异,而钩藤总碱低剂量组训练前后在伴药箱停留时间差无明显差别(P=0.949)。
以适当比例稀释抗体进行Western blotting检测,以P-actin为内标。与空白组成年斑马鱼相比,模型组成年斑马鱼脑内的TH表达有显著性差异(P=0.001),钩藤总碱高剂量组成年斑马鱼(P=0.777)和氯胺酮组成年斑马鱼(P=0.546)脑内的TH表达无显著差异,与甲基苯丙胺模型组成年斑马鱼相比,钩藤总碱高剂量组成年斑马鱼(P=0.001)和氯胺酮组成年斑马鱼(P=0.007)脑内的TH表达有显著差异,钩藤总碱低剂量组成年斑马鱼脑内的TH表达无显著性差异(P=0.113),与空白组成年斑马鱼相比,模型组成年斑马鱼(P=0.001)和钩藤总碱低剂量组成年斑马鱼(P=0.01)脑内的NR2B表达有显著性差异,钩藤总碱高剂量组成年斑马鱼(P=0.083)和氯胺酮组成年斑马鱼(P=0.105)脑内的NR2B表达无显著差异,与空白组成年斑马鱼相比,模型组成年斑马鱼脑内的GluR2表达有显著性差异(P=0.04),钩藤总碱高剂量组成年斑马鱼(P=0.334)和氯胺酮组成年斑马鱼(P=0.130)脑内的GluR2表达无显著差异,与甲基苯丙胺模型组成年斑马鱼相比,钩藤总碱高剂量组成年斑马鱼脑内的GluR2表达有显著差异(P=0.003),钩藤总碱低剂量组成年斑马鱼脑内的GluR2表达无显著性差异(P=0.698)。
3.转基因结果显示,与正常斑马鱼幼鱼相比,显微注射TH—FP质粒后的斑马鱼幼鱼头部出现明显荧光。在绿色荧光下,与空白组斑马鱼幼鱼相比,甲基苯丙胺(60mg/L)模型组斑马鱼幼鱼头部荧光变化明显,甲基苯丙胺(6mg/L)模型组斑马鱼幼鱼头部荧光变化明显,甲基苯丙胺(0.6mg/L)模型组斑马鱼幼鱼头部荧光变化明显,而甲基苯丙胺(0.06mg/L)模型组斑马鱼幼鱼头部荧光变化则不明显。将大叶钩藤水提物(1g/mL)组斑马鱼幼鱼,大叶钩藤水提物(0.33g/mL)组斑马鱼幼鱼,大叶钩藤水提物(0.033g/mL)组斑马鱼幼鱼,大叶钩藤水提物(0.0033g/mL)组斑马鱼幼鱼,大叶钩藤水提物(0.00033g/mL)组斑马鱼幼鱼放入相应浓度的水提物中24h后,大叶钩藤水提物(1g/mL)组斑马鱼幼鱼,大叶钩藤水提物(0.33g/mL)组斑马鱼幼鱼,大叶钩藤水提物(0.033g/mL)组斑马鱼幼鱼和大叶钩藤水提物(0.0033g/mL)组斑马鱼幼鱼死亡,大叶钩藤水提物(0.00033g/mL)组斑马鱼幼鱼游动正常。在共聚焦显微镜下,与空白组相比,甲基苯丙胺(0.6mg/L)模型组斑马鱼幼鱼头部荧光明显增强。与甲基苯丙胺(0.6mg/L)模型组相比,大叶钩藤水提物(0.00033g/mL)组斑马鱼幼鱼头部荧光有所减弱。
结论
1.甲基苯丙胺可以使成年斑马鱼产生明显的位置偏爱效应,成年斑马鱼在伴药箱中停留时间明显延长,证明成年斑马鱼可以作为模式生物进行药物依赖方面的研究。高剂量钩藤总碱(100μg/g)和氯胺酮(150μg/g)可显著抑制甲基苯丙胺依赖成年斑马鱼的位置偏爱效应,使之与甲基苯丙胺模型组成年斑马鱼有显著差异。而低剂量钩藤总碱(501μg/g)对甲基苯丙胺依赖成年斑马鱼的位置偏爱效应没有显示出明显的抑制效果。
2.甲基苯丙胺依赖的形成会使正常成年斑马鱼脑内TH、 NR2B、 GluR2三种蛋白表达显著上调。高剂量钩藤总碱(100μg/g)和氯胺酮(150μg/g)可显著抑制这种改变,使甲基苯丙胺依赖的成年斑马鱼脑内的TH、 NR2B、 GluR2三种蛋白表达下调,而低剂量钩藤总碱(50μg/g)对这种改变没有明显作用。
3.可以利用TH—GFP转基因斑马鱼,在显微镜下通过绿色荧光强弱来直观的观察斑马鱼甲基苯丙胺依赖情况,以及药物对其的干预效果：0.6mg/L的甲基苯丙胺溶液就能使TH—FP转基因斑马鱼头部荧光发生明显增强,而浓度为0.00033g/mL大叶钩藤水提物可以降低这种荧光的改变。
注：本论文中的所有数据均采用SPSS13.0进行分析。所有数据以(x±SD)表示,各组成年斑马鱼在伴药箱中的停留时间比较采用单向方差分析(One-Way ANOVA)或者协方差分析(analysis of covarinance,ANCOVA),各组均数的多重比较采用最小显著差值法(least significant different, LSD),不满足方差齐性要求的,采用Dunnett's T3法。Western blotting结果以每张转印膜上目的蛋白条带光密度值,进行单因素方差分析。检测水平α=0.05。
BACKGROUD
At present, the rapid momentum of amphetamine-type stimulants (ATS) abuse is increasing, the number of stimulant abuse has exceeded the number of heroin and cocaine abuse and was global spreading in the21st countries of five continents. A survey of seven European countries is that the ATS has become the second largest category of the most commonly abused addictive substances. In China, amphetamine-type stimulant abuse situation is grim, it can damage to the central nervous system and cardiovascular system seriously, prone to herd riots, personal attacks and other violence, causing social and public health problem, seriously damaging the social climate. Therefore, development of the treatment for its dependence and drug withdrawal symptoms is significance. The history of Chinese medicine in drug treatment has been more than200years, it has accumulated a wealth of experience, the formation of a unique theory and effective way. The treatment of ideology in Chinese medicine drug treatment is strengthening body resistance and eliminate pathogenic factor, highlighting the diagnosis and treatment characteristics. Traditional Chinese Medicine therapy has the unique advantages: low toxicity, low cost, a wide range source of drugs and few of addiction, pay attention to starting from the disease and syndrome, dialectical treatment by traditional Chinese medicine, so the development of new drugs or drug treatment from Chinese medicine is likely to a breakthrough on the problem of withdrawal.
A large number of research results show that the central glutamic acid nervous system and drug dependence are closely linked, there are many reports about NMDA receptors and AMPA receptors and drug dependence related, but tyrosine hydroxylase (TH) as a key enzyme in dopamine synthesis also plays an important role in the process of drug dependence, there is few reports about it. Marking TH With green fluorescent protein(GFP) is a new molecular biology techniques, it makes the TH expression changes become more intuitive. GFP is a light-emitting protein expression in living cells without exogenous substrates or cofactors, can issue the green fluorescence by blu-ray excitation. GFP as a fluorescent marker has both sensitive tag detection rate and no radioactive hazards.
Conditioned place preference(CPP) is usually used to study on drug-dependence for Evaluation of drug psychological dependence potential. The experimental animals on drug psychological dependence studies is mostly rodents: rats and mice, but With the emergence of increasingly diverse drug, We need to find new available model organism as a complement to traditional model organisms for the field of addiction medicine.
Zebrafish as a new model animal, compared with rats or mice and other mammals, their mating behavior is affected by photoperiod control, the amount of spawning, the fertilized egg in vitro fertilization, the reproductive cycle fast, easy feeding; and their early embryo is transparent as a whole, observation in vivo easily, The application prospects of the behavior of the zebrafish are gradually being understood by researchers.
OBJECTIVE
1. To establish methamphetamine CPP animal models of adult zebrafish, observe the CPP effects in adult zebrafish, assess the dependence of the effect of adult zebrafish from behavior. At the same time, to treat by Chinese medicine active ingredients and to observe the behavioral changes of the methamphetamine-dependent adult zebrafish.
2. To establish the model of the adult zebrafish methamphetamine conditioned place preference, Observe the NR2B receptor, the GluR2receptor and the TH receptor expression changes in the methamphetamine conditioned place preference adult zebrafish brain and change of expression after treatment by the as Chinese medicine active ingredients by western blotting.
3. To mark a key enzyme—H in the dopamine synthesis with GFP, then cultivate the TH-GFP transgenic zebrafish by transgenic technology, determine its lever of methamphetamine dependence by the fluorescence in zebrafish Larvae head. Preliminary groping for the effective concentration of Uncaria macrophylla leaf aqueous extract about the zebrafish larvae.
4. To provide experimental evidence for confirming that the zebrafish is an ideal model to study amphetamine-type stimulants dependence, Further study of methamphetamine-dependent mechanism of the zebrafish and mechanism of Traditional Chinese Medicine treatment.
METHODS
1. To establish conditioned place preference model in adult zebrafish:According to the literature and pre-experimental results, they shows that the natural preference of adult zebrafish box is brown box, so to definie the transparent box with the kits.20 adult zebrafish which passed the determination of nature place preference were randomly divided into①ormal control group,②odel group of methamphetamine. The fish were moved to the behavior room under maintenance conditions and feeding schedule identical to the fish facility at least2days before each assay. The water level was kept to5cm from the tank bottom to minimize stress. On the third day, the fish was recorded for a15-min trial using Noldus system, The preferred compartment was defined as the compartment in which a fish spends most of its time during the measurement on day3, and the place preference(PP) is calculated as the percentage of time that the fish spends in the preferred compartment. On day4, day6and day8, anesthetized in tricaine for a few seconds. It was immobilized by hand into a Petri dish containing tank water, and then received methamphetamine (40μg/g) by intraperitoneal injection using syringe. After waking up, the fish was confined to the non-preferred compartment for45min. The restriction to this compartment was achieved using a transparent slider. Thus, visual contact with the preferred compartment remained, and the difference between the conditioning and recording conditions was minimized. The experimental tank and conditions were otherwise identical to the ones used for place preference determination, and each fish was tested separately. After45min, the fish was removed from the experimental tank and kept in a1.5L tank on a color-neutral background. On day5and day7, fish was injected intraperitoneally with a saline solution, then restricted for45min into the preferred compartment. Between each injection session, the experimental tank was cleaned with70%ethanol and rinsed with fish facility water. Place preference was then measured again on day9and compared the difference of stay-time in the prefer department.
2. The impact of Uncaria alkaloids for Methamphetamine-dependent adult zebrafish:
2.1The impact of Uncaria alkaloids for CCP of Methamphetamine-dependent adult zebrafish:50adult zebrafish which passed the determination of nature place preference were randomly divided into①normal control group,②odel group of methamphetamine,③odel group+low dose of group(50μg/g),④odel group+high dose of group(100μg/g),⑤model group+ketamine group (150μg/g) The fish were moved to the behavior room under maintenance conditions and feeding schedule identical to the fish facility at least2days before each assay. The water level was kept to5cm from the tank bottom to minimize stress. On the third day, the fish was recorded for a15-min trial using Noldus system, The preferred compartment was defined as the compartment in which a fish spends most of its time during the measurement on day3, and the place preference(PP) is calculated as the percentage of time that the fish spends in the preferred compartment. On day4, day6and day8, anesthetized in tricaine for a few seconds. It was immobilized by hand into a Petri dish containing tank water, and then received methamphetamine (40μg/g) by intraperitoneal injection using syringe, except control group; control group received equal volume of normal saline. After waking up, the fish was confined to the non-preferred compartment for45min. The restriction to this compartment was achieved using a transparent slider. Thus, visual contact with the preferred compartment remained, and the difference between the conditioning and recording conditions was minimized. The experimental tank and conditions were otherwise identical to the ones used for place preference determination, and each fish was tested separately. After45min, the fish was removed from the experimental tank and kept in a1.5L tank on a color-neutral background. After12hours, except control group, injected equal volume of normal saline in model group; injected low dose of in low dose of rhynchphylla total alkaloids group; injected high dose of rhynchphylla total alkaloids in high dose of rhynchphylla total alkaloids group; injected ketamine in ketamine group. On day5and day7, fish was injected intraperitoneally with a saline solution, then restricted for45min into the preferred compartment. After12hours, the steps are as similar as day4. Between each injection session, the experimental tank was cleaned with70%ethanol and rinsed with fish facility water. Place preference was then measured again on day9and compared the difference of stay-time in the prefer department.
2.2The impact of Uncaria alkaloids for expression of NR2B receptor, GluR2receptor and TH receptor in the brain of Methamphetamine-dependent adult zebrafish: to extract total protein in adult zebrafish brain and to computer protein content. According to the protocol of the SCT1111Lab in Hong Kong Baptist University, finished the western blotting.
3. To establish methamphetamine-dependent animal model of zebrafish larvae and to groping for the effective concentration of Uncaria macrophylla leaf aqueous extract about the zebrafish Larvae:microinjected the TH—FP plasmid into yoke of one cell stage zebrafish eggs. After5days,12TH—FP transgenic zebrafish larvae which is alive were randomly divided into①model group (60mg/L),②model group (6mg/L),③model group (0.6mg/L),④model group (0.06mg/L). Zebrafish larval of each group into corresponding concentrations of methamphetamine solution in freely moving with fasting. After3days, to observe the fluorescence intensity in of TH—FP transgenic zebrafish larvae brain.
After microinjection5days,21transgenic zebrafish larvae were randomly divided into①Uncaria macrophylla leaf aqueous extract group (1g/mL),②Uncaria macrophylla leaf aqueous extract group (0.33g/mL),③Uncaria macrophylla leaf aqueous extract group (0.033g/mL),④Uncaria macrophylla leaf aqueous extract group (0.0033g/mL),⑤Uncaria macrophylla leaf aqueous extract group (0.00033g/mL),⑥model group (0.6mg/L),⑦control group. Zebrafish larval of each group into0.6mg/L methamphetamine solution in freely moving with fasting, except①group. After3days, Zebrafish larval of each group into corresponding concentrations of Uncaria macrophylla leaf aqueous extract, Zebrafish larval of⑥group into water in freely moving with fasting, except①group. Next day, to observe the fluorescence intensity in of TH—FP transgenic zebrafish larvae brain.
RESULTS
1. The experimental results shown that compared with control group and model group by two independent sample t-test (t=-11.791, P=0.000), the difference of stay-time is significant. According the map of zebrafish in the prefer department, after modeling, the track of model group zebrafish is reduced significantly than before; and the change of control group is not significant.
2. The experimental results shown that compared with control group, the difference of model group stay-time is significant (P=0.000), compared with model group, the difference of high dose of rhynchphylla total alkaloids group(P=0.000)stay-time and ketamine group (P=0.000) are significant, but the difference of low dose of rhynchphylla total alkaloids group stay-time is not significant (P=0.949)
The western blotting results shown that compared with control group, the difference of the TH receptor expression in model group adult zebrafish brain is significant (P=0.001), the difference of high dose of rhynchphylla total alkaloids group (P=0.777) the TH receptor expression and ketamine group (P=0.546) the TH receptor expression are not significant; compared with model group, the difference of high dose of rhynchphylla total alkaloids group (P=0.001) the TH receptor expression and ketamine group (P=0.007) the TH receptor expression are significant, the difference of low dose of rhynchphylla total alkaloids group the TH receptor expression is not significant (P=0.113); compared with control group, the difference of the NR2B receptor expression in model group (P=0.001) and low dose of rhynchphylla total alkaloids group (P=0.01) the NR2B receptor expression are significant, the difference of high dose of rhynchphylla total alkaloids group (P=0.083) the NR2B receptor expression and ketamine group (P=0.105) the NR2B receptor expression are not significant; compared with control group, the difference of the GluR2receptor expression in model group (P=0.04) is significant, the difference of high dose of rhynchphylla total alkaloids group (P=0.334) the GluR2receptor expression and ketamine group(P=0.130)the GluR2receptor expression are not significant, compared with model group, the difference of the GluR2receptor expression in high dose of rhynchphylla total alkaloids group (P=0.003)is significant, the difference of the GluR2receptor expression in low dose of rhynchphylla total alkaloids group (P=0.698) is not significant.
3. The experimental results shown that compared with normal zebrafish larvae, the fluorescence of transgenic zebrafish larvae is brighter. Compared with control group, the fluorescence of model group (60mg/L) and model group (6mg/L) and model group (0.6mg/L) are brighter. After zebrafish larval of each group into corresponding concentrations of Uncaria macrophylla leaf aqueous extract, Uncaria macrophylla leaf aqueous extract group (1g/mL), Uncaria macrophylla leaf aqueous extract group (0.33g/mL),Uncaria macrophylla leaf aqueous extract group (0.033g/mL) and Uncaria macrophylla leaf aqueous extract group (0.0033g/mL) are dead, but Uncaria macrophylla leaf aqueous extract group (0.00033g/mL) is alive.
CONCLUSION
1. Methamphetamine can make adult zebrafish have obvious place preference in the medicine cabinet, prolonging the stay-time in the drug-paired department. It confirmed that adult zebrafish can be used as a model organism for the study of drug dependence. High doses of rhynchphylla total alkaloids (100μg/g) and ketamine (150μg/g) could significantly inhibit methamphetamine dependent adult zebrafish place preference. Low dose of rhynchphylla total alkaloids (50μg/g) on methamphetamine dependence in adult zebrafish place preference shows no obvious inhibition effect.
2. Methamphetamine-dependent formation can make the expression of normal adult zebrafish brain TH, NR2B, GluR2was significantly higher. High doses of rhynchphylla total alkaloids (100μg/g) and ketamine (150μg/g) could significantly inhibit the change, make the expression of TH, NR2B, GluR2in methamphetamine-dependence adult zebrafish brain is down regulated, while the lower dose of rhynchphylla total alkaloids(50μg/g) had no obvious effect on the change.
3. We can use the TH—GFP transgenic zebrafish, under the microscope by green fluorescence intensity, to observe the zebrafish methamphetamine-dependence, and the effects of the drug treatment.0.6mg/L TH—GFP methamphetamine solution can make transgenic zebrafish head have obvious fluorescence enhancement, and the concentration of0.00033g/mL Uncaria macrophylla leaf water extract can to reduce this fluorescence change.
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