本文選題：斑馬魚 + 小鼠�。� 參考：《南方醫(yī)科大學》2014年博士論文

【摘要】：背景藥物濫用是公共衛(wèi)生的主要威脅之一。苯丙胺類興奮劑(Amphetamine-type stimulants,ATS)等是近十年才開始被普遍濫用的一種新型毒品,甲基苯丙胺(冰毒)是一個廣泛濫用苯丙胺類物質。在2011年,全球在15-64歲之間的人群0.7%的人(約3380萬)使用苯丙胺類藥物。而甲基苯丙胺仍然是苯丙胺類使用最多的,2011年占全球的71%。苯丙胺類興奮劑的濫用不僅導致濫用者身心健康嚴重損害,還因藥物作用的影響,濫用者在極度興奮下極易發(fā)生各種違法犯罪行為,這對社會公共安全造成極大威脅。因此,對于苯丙胺成癮的干預是一項刻不容緩的醫(yī)學任務和社會任務。高效低毒無成癮性戒毒藥物的開發(fā)成為藥物依賴干預的重大難題,中藥戒毒有著悠久的歷史,中藥資源極其廣泛,每種中藥又含有數十至數百種活性成分,由于傳統(tǒng)藥物依賴動物模型的限制,使得戒毒藥物的高通量篩選難以開展。國內甲基苯丙胺藥物依賴動物模型研究主要以神經細胞和大、小鼠動物模型為主,由于試驗經費和試驗室條件的限制,國內大多數試驗室都未能建立藥物依賴猴模型。斑馬魚作為一種理想的模式生物在生命科學領域被廣泛的應用,特別在人類疾病模型和藥物高通量篩選方面,已成為斑馬魚研究的熱點,但國內外對甲基苯丙胺藥物依賴斑馬魚模型報道都很少。在戒毒中藥和活性成分高通量篩選方面,現有的大小鼠模型,乃至恒河猴模型由于試驗周期長、用藥量大、費用高昂等因素,使戒毒藥物的高通量篩選難以開展,而斑馬魚模型的特性使之成為尋找有效藥物的最佳選擇。與嚙齒類動物甲基苯丙胺藥物依賴模型相比,斑馬魚模型的研究起步較晚,整體生物學體征及神經機制有待進一步研究。實驗目的1.建立小鼠和成年斑馬魚的甲基苯丙胺CPP動物模型,觀察小鼠和成年斑馬魚CPP效應,從行為學方面來對比小鼠和成年斑馬魚兩種模型的依賴效果,同時用中藥有效成分鉤藤堿進行干預,對比觀察形成依賴后不同動物模型的行為變化。2.采用免疫組化法,觀測甲基苯丙胺條件性位置偏愛小鼠和成年斑馬魚腦內TH、NR2B、GluR1陽性細胞數目的改變及中藥活性成分鉤藤堿對其干預后變化情況的對比。3.采用蛋白免疫印跡(Western blotting)方法,觀測甲基苯丙胺條件性位置偏愛小鼠和成年斑馬魚腦內TH、NR2B、GluR1受體蛋白表達的改變及中藥活性成分鉤藤堿對其干預后變化情況的對比。4.通過對兩種動物模型不同組別及指標結果的分析,對比兩種模型造模及給藥后不同指標的改變趨勢。實驗方法1.參照課題組前期研究成果建立小鼠和成年斑馬魚條件性位置偏愛模型,從行為學方面來對比小鼠和成年斑馬魚兩種模型的CPP效果取符合實驗條件的50只小鼠,按隨機原則分為5個組:即①空白對照組,②甲基苯丙胺模型組,③鉤藤堿低劑量(40mg/kg)組,④鉤藤堿高劑量(80mg/kg)組,⑤氯胺酮(15mg/kg)組,每組10只小鼠。②～⑤組每天上午(8:00)皮下注射甲基苯丙胺4 mg/kg,連續(xù)4天;①組注射同體積生理鹽水,其它處理同②組;③和④組從第二天起每天上午(7:30,即注射甲基苯丙胺前30min)灌胃相應劑量的鉤藤堿,連續(xù)3天;⑤組從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),連續(xù)3天。各組下午(16:00,間隔8h)均給予生理鹽水(0.15 ml,sc) 1次。將箱體中間的隔板置于中間,第一天上午注射生理鹽水或甲基苯丙胺后立即將小鼠置于白箱,下午注射生理鹽水后立即將小鼠置于黑箱,均放置1h,連續(xù)4天。24 h后(第5天)進行位置偏愛檢測,記錄小鼠5 min內在白箱中停留的時間。取經過自然位置偏愛測定合格的成年斑馬魚50條,按隨機分組原則分為:①正常對照組,②甲基苯丙胺模型組,③鉤藤堿低劑量組(50μg/g),④鉤藤堿高劑量組(1OOμg/g),⑤氯胺酮組(150μg/g)。整個實驗需要進行9d,d1將斑馬魚置于獨立的用于訓練的CPP箱,每個CPP箱的水位不低于5cm,以保證足夠的水壓,適應性喂養(yǎng)至少2d。d3測試正常狀態(tài)下,所有斑馬魚的位置偏愛箱(15 min內),并且用Noldus動物行為學分析系統(tǒng)來跟蹤其路線圖(5 min內)。在d4、d6、d8,將除開空白組以外的所有斑馬魚浸入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),然后將其置于非偏愛箱(伴藥箱)45 min。之后的訓練步驟與建立模型的訓練步驟相同。12h后,將除開空白組以外的所有斑馬魚再次浸入200mg/L tricaine methanesulfonate溶液中麻醉,用微量注射器迅速腹腔注射相應的處理藥物(模型組注射等體積生理鹽水,低劑量組注射50μg/g鉤藤堿溶液,高劑量組注射100μg/g鉤藤堿溶液,氯胺酮組注射150μg/g氯胺酮溶液),之后將其移至較大的有藍色環(huán)境的魚缸�？瞻捉M斑馬魚用200mg/L tricaine methanesulfonate溶液麻醉后注射等體積魚用生理鹽水,其他處理與模型組一致。d5和d7在與d4注射的同一時間,給予斑馬魚注射等體積的魚用生理鹽水,然后將其置于偏愛箱(非伴藥箱)45 min。按上述相同的步驟,對斑馬魚進行訓練。最后一次注射24h后(即d9),測試所有斑馬魚的伴藥箱停留時間以及在魚缸的路線圖,比較它們在伴藥箱前后停留時間的差值。2.采用免疫組化法,觀測甲基苯丙胺條件性位置偏愛小鼠和成年斑馬魚腦內TH、NR2B、GluR1陽性細胞數的改變及中藥活性成分鉤藤堿對其干預后變化情況的對比。試驗最后一天將各組試驗動物處死,將各組動物組織(小鼠取整個腦組織,斑馬魚取整個頭部)于4%多聚甲醛(PFA)中固定,常規(guī)石蠟包埋,切片,免疫組化染色,顯微鏡下進行觀察,拍片,觀察陽性顆粒在細胞內的分布,細胞中出現棕黃色顆粒為陽性。采用Image-Proplus6.0圖像分析軟件,測定陽性細胞的積分光密度(IOD)。3.采用蛋白免疫印跡(Western blotting)方法,觀測甲基苯丙胺條件性位置偏愛小鼠和成年斑馬魚腦內TH、NR2B、GluR1表達的改變及中藥活性成分鉤藤堿對其干預后變化情況的對比。試驗最后一天將各組試驗動物處死,取腦,提取腦總蛋白、測定蛋白含量。配制好實驗所需的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。配制凝膠、SDS-PAG電泳、轉膜、一抗、二抗的孵育、顯影、定影,用Metamorph軟件分析TH、NR2B、GluR1受體每個特異條帶的OD值。4.統(tǒng)計學方法本論文中的所有數據均采用SPSS 13.0進行分析。所有數據以(x±s)表示,如果各組比較方差齊采用單向方差分析(One-Way ANOVA),方差不齊采用了 Welch檢驗,方差齊多重比較采用最小顯著差值法(least significant different,LSD),方差不齊,采用Dunnett's T3法。采用相關性分析方法,用相關系數(r)方法考察兩種動物模型及給予中藥活性成分鉤藤堿干預后不同指標之間的相關性的情況。實驗結果1、CPP實驗結果給予一定量甲基苯丙胺后,小鼠及成年斑馬魚藥物依賴模型均產生CPP效應,與空白組對比有統(tǒng)計學意義(P0. 01),通過鉤藤堿干預均有調節(jié)作用。斑馬魚與小鼠的伴藥箱中逗留的時間存在正相關r =0.593, P=0.000。2、TH實驗結果免疫組化結果:小鼠及成年斑馬魚甲基苯丙胺藥物依賴模型腦內TH陽性細胞數均顯著增加(P0.01),通過鉤藤堿干預有改善作用。斑馬魚與小鼠的腦內TH陽性細胞數存在正相關(r=0.408, P=0.000)。western結果:兩種動物模型腦內TH蛋白表達顯著加強(P0.01),通過鉤藤堿干預均有改善作用。斑馬魚與小鼠腦內腦內TH蛋白表達呈正相關(r=0.337, P=0.000)。3、NR2B實驗結果免疫組化結果:小鼠及成年斑馬魚甲基苯丙胺藥物依賴模型腦內NR2B陽性細胞數均增加(P0.01),通過鉤藤堿干預均有改善作用。斑馬魚與小鼠腦內NR2B陽性細胞數呈正相關(r =0.803, P=0.000)。western結果:小鼠及成年斑馬魚甲基苯丙胺藥物依賴模型腦內NR2B蛋白表達加強(P0.01),通過鉤藤堿干預均有改善作用。斑馬魚與小鼠NR2B蛋白表達呈正相關(r =0.502,P=0.000)。4、GluR1實驗結果免疫組化結果:小鼠及成年斑馬魚甲基苯丙胺藥物依賴模型腦內GluR1陽性細胞數均增加(P0.01),通過鉤藤堿干預均有改善作用。斑馬魚與小鼠GluR1陽性細胞數呈正相關(r =0.626, P=0.000)。western結果:小鼠及成年斑馬魚甲基苯丙胺藥物依賴模型腦內GluR1表達加強(P0.01),通過鉤藤堿干預均有改善作用。斑馬魚與小鼠GluR1蛋白表達呈正相關(r =0.875,P=0.000)。實驗結論1、給予一定量甲基苯丙胺后,小鼠及成年斑馬魚藥物依賴模型均產生CPP效應,通過相關性分析,兩種模型動物CPP效果顯著相關,提示與成熟的嚙齒類動物(小鼠)藥物依賴模型相比,斑馬魚藥物依賴模型是成功的、穩(wěn)定的、可復制的。2、通過對TH、NR2B、GluR1三個指標免疫組化和western結果的分析,小鼠及成年斑馬魚藥物依賴模型兩組數據間都存在正相關,提示兩種動物模型分子生物學作用機制相似,斑馬魚動物模型在很大程度上與嚙齒類動物模型相似。3、通過鉤藤堿干預后,對小鼠及成年斑馬魚藥物依賴模型行為學及TH、NR2B、GIuR1三個指標均有調節(jié)作用,兩組數據間基本都存在正相關,提示鉤藤堿對兩種模型效果相近,斑馬魚模型由于具有周期短、用藥少、體型小等生物學優(yōu)勢,更有利于進行藥物研究及藥物篩選。
[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.

【學位授予單位】：南方醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R-332;R285.5

【參考文獻】

相關期刊論文 前10條

1 孫艷;馬寶苗;黃坤玉;黃嫻妮;楊澍均;劉昱;;大鼠甲基苯丙胺自身給藥行為模型構建[J];中國藥物依賴性雜志;2012年06期

2 王章姐;孫維礦;;鉤藤的化學成分及藥理作用研究進展[J];現代企業(yè)教育;2010年24期

3 林曉亮;湯偉;陳文倩;翁建霖;莫志賢;;鉤藤堿對甲基苯丙胺條件性位置偏愛大鼠AMPA受體蛋白改變的影響[J];中華行為醫(yī)學與腦科學雜志;2010年02期

4 霍塞虎;;多巴胺D2受體基因及其多態(tài)性與物質依賴的相關研究[J];健康研究;2009年03期

5 孔晗;劉彥玲;朱杰;閻春霞;魏曙光;張洪波;李生斌;陳騰;;甲基苯丙胺誘導的小鼠條件性位置偏愛及多巴胺D_3受體的調制作用[J];中國藥物依賴性雜志;2009年03期

6 武曉華;景強;;酒精依賴與多巴胺系統(tǒng)基因多態(tài)性研究進展[J];醫(yī)學研究雜志;2009年01期

7 周吉銀;莫志賢;;鉤藤堿對苯丙胺依賴大鼠神經核團中NR2B mRNA表達的影響[J];中國藥理學通報;2007年09期

8 周吉銀;莫志賢;;鉤藤堿對苯丙胺依賴大鼠腦內氨基酸類神經遞質含量的影響[J];中國藥物依賴性雜志;2007年02期

9 周吉銀;莫志賢;;鉤藤堿對苯丙胺依賴大鼠伏核和杏仁核中NR2B蛋白表達的影響[J];中國藥物依賴性雜志;2007年01期

10 周吉銀;莫志賢;;鉤藤堿對苯丙胺誘導的大鼠條件性位置偏愛的影響及機制探討[J];山東醫(yī)藥;2006年30期

相關會議論文 前1條

1 蔡志基;;全球藥物濫用現狀與主要動向[A];第一屆全國藥物濫用流行病學研討會論文摘要匯編[C];1998年

，

本文編號：1828607