斑马鱼药物依赖模型与啮齿类动物模型的比较研究

发布时间：2018-05-01 08:42

本文选题：斑马鱼 + 小鼠　；参考：《南方医科大学》2014年博士论文

【摘要】：背景药物滥用是公共卫生的主要威胁之一。苯丙胺类兴奋剂(Amphetamine-type stimulants,ATS)等是近十年才开始被普遍滥用的一种新型毒品,甲基苯丙胺(冰毒)是一个广泛滥用苯丙胺类物质。在2011年,全球在15-64岁之间的人群0.7%的人(约3380万)使用苯丙胺类药物。而甲基苯丙胺仍然是苯丙胺类使用最多的,2011年占全球的71%。苯丙胺类兴奋剂的滥用不仅导致滥用者身心健康严重损害,还因药物作用的影响,滥用者在极度兴奋下极易发生各种违法犯罪行为,这对社会公共安全造成极大威胁。因此,对于苯丙胺成瘾的干预是一项刻不容缓的医学任务和社会任务。高效低毒无成瘾性戒毒药物的开发成为药物依赖干预的重大难题,中药戒毒有着悠久的历史,中药资源极其广泛,每种中药又含有数十至数百种活性成分,由于传统药物依赖动物模型的限制,使得戒毒药物的高通量筛选难以开展。国内甲基苯丙胺药物依赖动物模型研究主要以神经细胞和大、小鼠动物模型为主,由于试验经费和试验室条件的限制,国内大多数试验室都未能建立药物依赖猴模型。斑马鱼作为一种理想的模式生物在生命科学领域被广泛的应用,特别在人类疾病模型和药物高通量筛选方面,已成为斑马鱼研究的热点,但国内外对甲基苯丙胺药物依赖斑马鱼模型报道都很少。在戒毒中药和活性成分高通量筛选方面,现有的大小鼠模型,乃至恒河猴模型由于试验周期长、用药量大、费用高昂等因素,使戒毒药物的高通量筛选难以开展,而斑马鱼模型的特性使之成为寻找有效药物的最佳选择。与啮齿类动物甲基苯丙胺药物依赖模型相比,斑马鱼模型的研究起步较晚,整体生物学体征及神经机制有待进一步研究。实验目的1.建立小鼠和成年斑马鱼的甲基苯丙胺CPP动物模型,观察小鼠和成年斑马鱼CPP效应,从行为学方面来对比小鼠和成年斑马鱼两种模型的依赖效果,同时用中药有效成分钩藤碱进行干预,对比观察形成依赖后不同动物模型的行为变化。2.采用免疫组化法,观测甲基苯丙胺条件性位置偏爱小鼠和成年斑马鱼脑内TH、NR2B、GluR1阳性细胞数目的改变及中药活性成分钩藤碱对其干预后变化情况的对比。3.采用蛋白免疫印迹(Western blotting)方法,观测甲基苯丙胺条件性位置偏爱小鼠和成年斑马鱼脑内TH、NR2B、GluR1受体蛋白表达的改变及中药活性成分钩藤碱对其干预后变化情况的对比。4.通过对两种动物模型不同组别及指标结果的分析,对比两种模型造模及给药后不同指标的改变趋势。实验方法1.参照课题组前期研究成果建立小鼠和成年斑马鱼条件性位置偏爱模型,从行为学方面来对比小鼠和成年斑马鱼两种模型的CPP效果取符合实验条件的50只小鼠,按随机原则分为5个组:即①空白对照组,②甲基苯丙胺模型组,③钩藤碱低剂量(40mg/kg)组,④钩藤碱高剂量(80mg/kg)组,⑤氯胺酮(15mg/kg)组,每组10只小鼠。②～⑤组每天上午(8:00)皮下注射甲基苯丙胺4 mg/kg,连续4天;①组注射同体积生理盐水,其它处理同②组;③和④组从第二天起每天上午(7:30,即注射甲基苯丙胺前30min)灌胃相应剂量的钩藤碱,连续3天;⑤组从第二天起在注射甲基苯丙胺之前15min,腹腔注射氯胺酮(15 mg/kg),连续3天。各组下午(16:00,间隔8h)均给予生理盐水(0.15 ml,sc) 1次。将箱体中间的隔板置于中间,第一天上午注射生理盐水或甲基苯丙胺后立即将小鼠置于白箱,下午注射生理盐水后立即将小鼠置于黑箱,均放置1h,连续4天。24 h后(第5天)进行位置偏爱检测,记录小鼠5 min内在白箱中停留的时间。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,③钩藤碱低剂量组(50μg/g),④钩藤碱高剂量组(1OOμg/g),⑤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(15 min内),并且用Noldus动物行为学分析系统来跟踪其路线图(5 min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)45 min。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钩藤碱溶液,高剂量组注射100μg/g钩藤碱溶液,氯胺酮组注射150μg/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)45 min。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.采用免疫组化法,观测甲基苯丙胺条件性位置偏爱小鼠和成年斑马鱼脑内TH、NR2B、GluR1阳性细胞数的改变及中药活性成分钩藤碱对其干预后变化情况的对比。试验最后一天将各组试验动物处死,将各组动物组织(小鼠取整个脑组织,斑马鱼取整个头部)于4%多聚甲醛(PFA)中固定,常规石蜡包埋,切片,免疫组化染色,显微镜下进行观察,拍片,观察阳性颗粒在细胞内的分布,细胞中出现棕黄色颗粒为阳性。采用Image-Proplus6.0图像分析软件,测定阳性细胞的积分光密度(IOD)。3.采用蛋白免疫印迹(Western blotting)方法,观测甲基苯丙胺条件性位置偏爱小鼠和成年斑马鱼脑内TH、NR2B、GluR1表达的改变及中药活性成分钩藤碱对其干预后变化情况的对比。试验最后一天将各组试验动物处死,取脑,提取脑总蛋白、测定蛋白含量。配制好实验所需的10×TBS、10%SDS、1.0MTris-HCl(PH=8.3)、1.0M Tris-HCl(PH=8.8)、1.0M Tris-HCl(PH=6.8)、1XTBST 以及 Transfer Buffer 等。配制凝胶、SDS-PAG电泳、转膜、一抗、二抗的孵育、显影、定影,用Metamorph软件分析TH、NR2B、GluR1受体每个特异条带的OD值。4.统计学方法本论文中的所有数据均采用SPSS 13.0进行分析。所有数据以(x±s)表示,如果各组比较方差齐采用单向方差分析(One-Way ANOVA),方差不齐采用了 Welch检验,方差齐多重比较采用最小显著差值法(least significant different,LSD),方差不齐,采用Dunnett's T3法。采用相关性分析方法,用相关系数(r)方法考察两种动物模型及给予中药活性成分钩藤碱干预后不同指标之间的相关性的情况。实验结果1、CPP实验结果给予一定量甲基苯丙胺后,小鼠及成年斑马鱼药物依赖模型均产生CPP效应,与空白组对比有统计学意义(P0. 01),通过钩藤碱干预均有调节作用。斑马鱼与小鼠的伴药箱中逗留的时间存在正相关r =0.593, P=0.000。2、TH实验结果免疫组化结果:小鼠及成年斑马鱼甲基苯丙胺药物依赖模型脑内TH阳性细胞数均显著增加(P0.01),通过钩藤碱干预有改善作用。斑马鱼与小鼠的脑内TH阳性细胞数存在正相关(r=0.408, P=0.000)。western结果:两种动物模型脑内TH蛋白表达显著加强(P0.01),通过钩藤碱干预均有改善作用。斑马鱼与小鼠脑内脑内TH蛋白表达呈正相关(r=0.337, P=0.000)。3、NR2B实验结果免疫组化结果:小鼠及成年斑马鱼甲基苯丙胺药物依赖模型脑内NR2B阳性细胞数均增加(P0.01),通过钩藤碱干预均有改善作用。斑马鱼与小鼠脑内NR2B阳性细胞数呈正相关(r =0.803, P=0.000)。western结果:小鼠及成年斑马鱼甲基苯丙胺药物依赖模型脑内NR2B蛋白表达加强(P0.01),通过钩藤碱干预均有改善作用。斑马鱼与小鼠NR2B蛋白表达呈正相关(r =0.502,P=0.000)。4、GluR1实验结果免疫组化结果:小鼠及成年斑马鱼甲基苯丙胺药物依赖模型脑内GluR1阳性细胞数均增加(P0.01),通过钩藤碱干预均有改善作用。斑马鱼与小鼠GluR1阳性细胞数呈正相关(r =0.626, P=0.000)。western结果:小鼠及成年斑马鱼甲基苯丙胺药物依赖模型脑内GluR1表达加强(P0.01),通过钩藤碱干预均有改善作用。斑马鱼与小鼠GluR1蛋白表达呈正相关(r =0.875,P=0.000)。实验结论1、给予一定量甲基苯丙胺后,小鼠及成年斑马鱼药物依赖模型均产生CPP效应,通过相关性分析,两种模型动物CPP效果显著相关,提示与成熟的啮齿类动物(小鼠)药物依赖模型相比,斑马鱼药物依赖模型是成功的、稳定的、可复制的。2、通过对TH、NR2B、GluR1三个指标免疫组化和western结果的分析,小鼠及成年斑马鱼药物依赖模型两组数据间都存在正相关,提示两种动物模型分子生物学作用机制相似,斑马鱼动物模型在很大程度上与啮齿类动物模型相似。3、通过钩藤碱干预后,对小鼠及成年斑马鱼药物依赖模型行为学及TH、NR2B、GIuR1三个指标均有调节作用,两组数据间基本都存在正相关,提示钩藤碱对两种模型效果相近,斑马鱼模型由于具有周期短、用药少、体型小等生物学优势,更有利于进行药物研究及药物筛选。
[Abstract]:Background drug abuse is one of the major threats to public health. Amphetamine-type stimulants (ATS) is a new type of drug that has been widely abused for nearly ten years. Methamphetamine (methamphetamine) is a widespread abuse of amphetamines. In 2011, 0.7% of people around 15-64 years of age (about 33 million 800 thousand) Methamphetamines are used. Methamphetamine is still the most commonly used amphetamine, and the abuse of 71%. amphetamine type stimulants worldwide in 2011 not only causes serious physical and mental health damage to abusers, but also because of the effect of drug effects, abusers are vulnerable to all kinds of criminal offenses under extreme excitement, which is a social security for public safety. It is a great threat. Therefore, the intervention of amphetamine addiction is an urgent medical task and social task. The development of high efficiency and low toxicity and unaddictive drug addiction has become a major problem in drug dependence intervention. The traditional Chinese medicine has a long history, and the resources of traditional Chinese medicine are extremely wide, and each kind of Chinese medicine contains dozens to hundreds of active forms. Due to the limitation of traditional drug dependence on animal models, the high throughput screening of drug addicts is difficult to carry out. The domestic animal model of methamphetamine drug dependence is mainly based on neural cells and large, mouse model. Due to the limitation of test funds and laboratory conditions, most laboratory laboratories in China have failed to establish drugs. Zebrafish, as an ideal model organism, is widely used in the field of life science, especially in the human disease model and high throughput screening, which has become a hot spot in the study of zebrafish. However, the drug dependence of methamphetamine on zebrafish models at home and abroad is very few. In screening, the existing rat model, and even the Ganges RIver monkey model, is difficult to carry out because of the long test period, large dosage and high cost, and the characteristics of the zebrafish model make it the best choice for finding effective drugs. Compared with the model of the rodent methamphetamine drug dependence model, zebra zebra is the same as the zebra horse model. The study of fish model started late, and the overall biological signs and neural mechanisms need to be further studied. Objective 1. to establish the model of methamphetamine CPP in mice and adult zebrafish, to observe the CPP effect of mice and adult zebrafish, and to compare the dependence effect of the two models of mice and adult zebrafish from the behavioral aspects. An effective component of the active component of rascarine was intervened, and the behavior changes of different animal models after the formation of dependent.2. were observed by immunohistochemistry. The changes of the number of TH, NR2B, GluR1 positive cells in the brain of methamphetamine conditioned conditioned mice and adult zebrafish were observed and the changes of the survival rate of the active components of the Chinese herbal medicine were observed. Comparison of.3. using Western blotting method to observe the changes of TH, NR2B, GluR1 receptor protein expression in the brain of methamphetamine conditioned conditioned mice and adult zebrafish and the comparison of the changes of the activity of the active ingredient of Chinese medicine on the dry prognosis of the Chinese medicine..4. through the analysis of the results of different groups and indexes of two animal models. Comparison of the two models and the changing trend of different indexes after administration. Method 1. the model of conditioned place preference of mice and adult zebrafish was established with reference to the previous research results of the experimental group. From the aspect of behavior, 50 mice of the CPP effect of two models of mice and adult zebrafish were compared, and the random principles were divided according to the random principles. 5 groups were: (1) blank control group, (2) methamphetamine model group, (40mg/kg) group, high dose (80mg/kg) group, ketamine (15mg/kg) group, 10 mice in each group, and group 5 was subcutaneously injected with methylphenylpropanamine 4 mg/kg every morning (8:00); (1) group injection of same volume of saline, the other treated the same Group (2); (3) and group 4 from second days (7:30, before injection of methamphetamine 30min) to the corresponding dose of hohoo base for 3 days, and the fifth group from second days before the injection of methamphetamine 15min, intraperitoneal injection of ketamine (15 mg/kg) for 3 days. All groups (16:00, interval 8h) were given saline (0.15 ml, SC) 1 times. The partition board in the middle of the box was placed in the middle. The mice were immediately placed in the white box after the first day of injection of saline or methamphetamine. The mice were placed in the black box immediately after the afternoon injection of normal saline. The mice were placed in the black box immediately after 4 days of.24 H (fifth days). The time of stay in the white box in the 5 min of the mice was recorded. 50 qualified adult zebrafish were determined by position preference: (1) normal control group, (2) methamphetamine model group, (3) low dose group (50 g/g), high dose group (1OO mu g/g) and ketamine group (150 mu g/g). The whole experiment required 9D and D1 to put zebrafish in independent CPP box for training. The water level of the CPP box is not less than 5cm to ensure sufficient water pressure. Under the normal condition of the adaptive feeding at least 2d.d3 test, all zebrafish positions prefer the box (15 min), and the Noldus animal behavior analysis system is used to track its roadmap (5 min). In D4, D6, D8, all zebrafish other than the blank group will be immersed in 200mg/L Tricaine. In the methanesulfonate solution, the methamphetamine (40 g/g) was intraperitoneally injected with a micro syringe and then placed after the training step of the non preferred box (with the medicine box) 45 min., and after the same.12h of the training step of the model, all zebrafish other than the blank group would be immersed in 200mg/L Tricaine methanesulfonate again. Anaesthesia in the liquid, using a micro syringe to intraperitoneally injected with the corresponding treatment drugs (model group injection of normal saline, low dose group injection of 50 g/g Hoo Carine solution, high dose group injection of 100 g/g rhoehoine solution, ketamine group injection of 150 g/g ketamine solution), then moved to a larger blue environment fish tank. Blank group spot The fish of the horse fish were injected with 200mg/L Tricaine methanesulfonate solution after the injection of physiological saline. The other treatments were in accordance with the model group,.D5 and D7 were given the same time as D4, given the zebrafish injection of the same volume of fish with physiological saline, and then placed them in the preferred box (non companion medicine box) 45 min. to the zebra fish according to the same steps. Training. After the last injection of 24h (D9), the residence time of all zebrafish and the route map of the fish tank were tested. The difference between the stay time of the drug box and the difference value.2. by immunohistochemistry was used to observe the changes in the number of TH, NR2B, GluR1 positive cells in the conditioned place of methamphetamine and the brain of the adult zebrafish. On the last day, the animal tissues of each group (the mice were taken from the whole brain, the whole head of zebra fish were taken from the 4% paraformaldehyde (PFA), and the paraffin was embedded, sectioning, immunohistochemical staining, observation and film under microscope. The distribution of the positive particles in the cells and the brown yellow granules in the cells were positive. The Image-Proplus6.0 image analysis software was used to determine the integral light density (IOD).3. of the positive cells using the Western blotting method to observe the conditioned place of methamphetamine and the TH, NR2B, GluR1 in the brain of adult zebrafish. The changes in the expression and the changes in the dry prognosis of the active components of the Chinese medicine. On the last day, the experimental animals were killed, the brain was taken, the total brain protein was extracted and the protein content was measured. The 10 x TBS, 10%SDS, 1.0MTris-HCl (PH=8.3), 1.0M Tris-HCl (PH=8.8), 1.0M Tris-HCl (PH=6.8), 1XTBST and Transfer needed for the experiment were prepared. Buffer et al. Preparation of gel, SDS-PAG electrophoresis, conversion film, one anti, two anti incubation, developing, fixing, and Metamorph software analysis of TH, NR2B, GluR1 receptor each specific band of.4. statistics method in this paper all data are analyzed with SPSS 13. All data are indicated by (x + s), if each group of comparison variance is unidirectional square Difference analysis (One-Way ANOVA), variance unhomogeneous using Welch test, variance homogeneous multiple comparison using the minimum significant difference (least significant different, LSD), variance is not homogeneous, the use of Dunnett's T3 method. Correlation analysis method, use correlation coefficient (R) method to examine the two animal models and to give Chinese Medicine active components of hochline after the prognosis. Results 1, experimental results 1, after a certain amount of methamphetamine was given to a certain amount of methamphetamine, the CPP effect was produced in both mice and adult zebrafish drug dependence models. The results were statistically significant (P0. 01) compared with the blank group (P0. 01). There was a positive correlation between positive correlation R =0.593, P=0.000.2, and TH experimental results: the number of TH positive cells in the brain of the mice and adult zebrafish methamphetamine drug dependent models increased significantly (P0.01), and improved by the intervention of Hoo Carine. There was a positive correlation between the number of TH positive cells in the brain of zebra fish and mice (r=0.408, P=0.000).Western results. The expression of TH protein in the brain of the two animal models was significantly enhanced (P0.01). The expression of TH protein in the brain of the brain was positively correlated with the mouse brain (r=0.337, P=0.000).3. The results of immunohistochemistry in NR2B experiment results: the number of NR2B positive cells in the brain of mice and adult zebrafish methamphetamine drug dependent models increased. Adding (P0.01) was improved by the intervention of Hoo Carine. Zebrafish had a positive correlation with the number of NR2B positive cells in the brain of mice (R =0.803, P=0.000).Western results: the expression of NR2B protein in the model brain of mice and adult zebrafish was enhanced (P0.01) and improved by the intervention of hoo Carine. Zebrafish and mice NR2B eggs The white expression was positive correlation (R =0.502, P=0.000).4, and the results of immunohistochemistry in GluR1 experimental results: the number of GluR1 positive cells in the brain of the mice and adult zebrafish methamphetamine drug dependence model increased (P0.01), and improved by the intervention of Hoo Carine. The number of zebra fish was positively correlated with the number of GluR1 positive cells in mice (R =0.626, P=0.000).Western junction Fruit: GluR1 expression in the brain of the mice and adult zebrafish methamphetamine drug dependence model enhanced (P0.01) and improved by the intervention of Hoo Carine. Zebrafish and mouse GluR1 protein expression was positively correlated (R =0.875, P=0.000). Experimental conclusion 1, after a certain amount of methylphenylpropanamine was given, the mice and adult zebrafish drug dependence models produced CP P effect, by correlation analysis, the effect of the two models of animal CPP is significantly correlated, suggesting that the zebrafish drug dependence model is a successful, stable, replicable.2, compared with the mature rodent (mouse) drug dependence model, through the analysis of the three indications of immunofluorescence and Western results of TH, NR2B, GluR1, mice and adult zebrafish. There was a positive correlation between the two groups of drug dependence models, suggesting that the mechanism of molecular biology of the two animal models was similar. The zebrafish animal model was similar to the rodent model to a large extent.3. By the prognosis of the hooked Hoo base, the behavior of the drug dependence model of mice and adult zebrafish, and the three indexes of TH, NR2B, and GIuR1 were all adjusted. There is a positive correlation between the two groups of data, suggesting that the effect of hooked Carine is similar to the two models. The zebrafish model has the biological advantages of short period, less drug use and small size, which is more beneficial to drug research and drug screening.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R-332;R285.5

【参考文献】