| BACKGROUDDrug dependence,is a social problem of common concern at home and abroad.Drug addiction can also cause a range of social issues.The main reason is that drug addiction is drug abuse arising.And drug abuse mainly refers to opium,heroin,methamphetamine(ice),the other can make morphine,marijuana,cocaine and under the control of the country addicted to narcotic drugs and psychotropic substances of abuse.Many drugs can produce dependence,can basically be divided into ten categories,namely:alcohol,opiates,cannabinoids,sedative hypnotics,cocaine,other stimulants,including caffeine,hallucinogens category;tobacco,volatile solvents;other psychoactive substances.The domestic research of drug dependence mainly nerve cells and large and small mouse animal models,this research built on morphine dependence zebrafish model,based on the comparative study of mouse models clearly different animal models of drug dependence in morphine behavioral variation and its mechanism of,this research is based on the zebrafish model of morphine dependence on the comparative study of mouse model of morphine dependence,clear zebrafish and mouse morphine dependent model features changes in behavior and its mechanism of.Zebrafish and mouse model of morphine dependence morphine dependent model for drug dependence research provides a basis for a new model,and has become one of the options zebrafish animal model of drug dependence.The main indicators of the current judgment of animals on their addiction is to observe whether the change in the performance of behavior,conditioned place preference model(CPP)is currently one of the most effective means of measuring the rewarding effects of animals,but also an effective way to evaluate the drug psychological dependence.However,the present domestic use CPP drug dependence mainly in rodent models of rats and mice,etc.,where there is little use of other models for drug dependence research reports.According to domestic and morphine model and test data,the present study is to replicate the morphine-dependent position preference zebrafish and mouse model,and with the active ingredients of traditional Chinese medicine Sinomenine zebrafish and mouse model of morphine dependence position preference to intervene and sinomenine observed dependence on zebra fish and mice morphine and related protein in the model,the zebrafish model and a mouse model of drug comparative study to determine the zebrafish model of morphine dependence scientific,rational,feasible sex,practical,and draw their own advantages and disadvantages of the model zebrafish and mouse model of morphine dependence.OBJECTIVE1.Establishment of an animal model of morphine CPP mice and adult zebrafish,mice and adult zebrafish were observed CPP effects,from behavioral aspects to compare dependent effects in mice and adult zebrafish two models,while the active ingredients with herbal sinomenine intervention,compared to observe changes in the behavior of different animal models after the formation of dependence.2.The use of PCR method to detect morphine-induced zebrafish and mouse brain opioid receptor level changes were observed after the Chinese intervention,compared to the zebrafish brain-related changes in receptor levels.3.Using immunohistochemical methods to detect morphine-induced changes in zebrafish and mouse brain TH and NR2B receptor expression,and to observe the effect of post-intervention,whether related receptor expression levels change positive change in the number of cells and medicine active ingredient sinomenine its intervention after the change of contrast.4.By analyzing the results of the two different groups of animal models and indicators,the comparison of the two models,and changing trends in modeling different indicators after administration.According to statistical theory,using correlation analysis method to study the two animal models with correlation coefficients(r)method and the active ingredient in traditional Chinese medicine after giving sinomenine intervention correlation between different indicators of the situation.METHODS1.Referring to build on previous study of mice and adult zebrafish conditioned place preference model to compare the behavioral aspects of mice and adult zebrafish of rely CPP effect.Take in line with the experimental conditions 50 mice were randomly divided into five groups:namely?blank control group,? model morphine hydrochloride(9 mg/kg)group,? sinomenine low dose(40mg/kg)group,? sinomenine high dose(80mg/kg)group,? methadone(15mg/kg)group,10 mice per group,male and female.??? group every morning(8:00 am)subcutaneous injection of morphine hydrochloride 4mg/kg,continuous 4d;? group injected with the same volume of saline,the other group treated like ? group;? and ? group every morning(7:30,before that morphine hydrochloride injection 30min)gavage doses corresponding Sinomenine for 3 days;? group from the next day before the injection of morphine hydrochloride 15min,intraperitoneal injection of methadone(15mg/kg),continuous 3d.Each group in the afternoon(16:00,interval 8h)were given saline(0.15ml,sc)1 times.The partition in the middle of the box,the first morning injections of saline or morphine hydrochloride immediately the mice were placed in a white box,in the afternoon after the mice were injected with saline immediately placed a black box,are placed lh,continuous 4 days.24h(Day 5)place preference testing,white box mouse 5min inherent residence time record.CPP home made aquarium(a cylinder of transparent,a cylinder of brown),brown box section for adult zebrafish preference box,transparent housing portion non?preference box(with kits).(1)Grouping:Take a baseline measurement of qualified zebrafish 50,weighed,were randomly divided into normal saline group,morphine(40 ?g · g-1)model group,morphine + methadone group(40 ?g · g-1),morphine +sinomenine low-dose group(40 ?9 · g-1),morphine + sinomenine high-dose group(80 ?g · g-1),10 ? each group,male and female.(2)observed:determination within the zebrafish 15 min(non-preference box)active time for CPP box transparent side in a formal experiment dl,and track zebrafish in CPP box trajectory(5 min)with Noldus EthoVision XT 8.5 software.(3)Training:in experiments d2,d4,d6 will zebrafish dipped 50 mg · L-1 tricaine methanesulfonate anesthesia solution in each group were injected morphine 40 ?g · g-1(the control group injected with saline)and then put it in the water system,30 s,wait until after recovery from anesthesia,put it in the medicine cabinet with 40 min,while separated by a transparent bezel training after the end of the zebrafish has moved to a larger blue aquarium environments,and clean CPP box;d3,d5 at the same time in zebrafish injected saline the same volume as morphine,put preference box 40 min.(4)administration:Sinomenine high and low-dose groups and methadone treatment group zebrafish 12h after training daily intraperitoneal injection of various concentrations of the drug,injected saline group and model group with normal saline.(5)observed:12h after the last dose determination d7 zebrafish box again in non-preference tank of the activity time,simultaneous analysis of zebrafish in a non-preference tank trajectory.2.Immunohistochemical staining was observed morphine conditioned placepreference in mice and adult zebrafish brain TH,NR2B-positive cell count change and medicine for its active ingredient Sinomenine contrast changes after interventionTest on the last day of animals in each group were sacrificed,each group of animal tissues(mouse whole brain tissue taken,zebrafish the entire head taken)in 4%paraformaldehyde(PFA)in a fixed,regular paraffin embedded,sliced,immunohistochemical staining was observed under the microscope,film,positive particles observed in cells distributed,brown granules in cells as positive.Using Image-Proplus 6.0 image analysis software to measure positive cells integrated optical density(IOD).3.Sinomenine on morphine in mice and zebrafish brain opioid receptor expression in comparisonAfter each group of zebrafish behavioral indicators detect zebrafish rapid freezing to death,brain tissue under a microscope.Using PCR to detect brain tissue of mice in each group of mor,dorRNA expression and zebrafish brain tissue zfinor,zfdorl,zfdor2 mRNA expression.4.Statistical MethodsStatistical results are expressed in the form ± s,using statistical software for processing spsslg.0,Multiple sets of data were analyzed using one-way ANOVA with LSD method when homogeneity of variance,variance missing when using Dunnett,s T3 tested,the same group of data using paired sample t test.P<0.05 was considered statistically different.RESULTS1.Behavior resultsBehavior results showed:After giving a certain amount of morphine hydrochloride,adult mice and zebrafish models of drug dependence are produced CPP,eomparison with the control group was statistically significant(P<0.01),by Sinomenine were on morphine-induced location preference for regulation.And time of mice and zebrafish to stay with the medicine chest in the presence of a positive correlation(r =0.606,P?0.000).2.TH resultsImmunohistochemical results showed that:the mice with adult zebrafish brain morphine drug dependency model TH-positive cells were significantly increased(P<0.01),Sinomenine had some improvement.TH-positive cells in the brain of zebrafish and mice there is a positive correlation(r =0.677,P=0.000).3.NR2B resultsImmunohistochemistry showed:mice and adult zebrafish brain morphine drug dependency model NR2B-positive cells were increased(P<0.01),Sinomenine had some improvement.Zebrafish and mouse brain NR2B-positive cells was positively correlated(r =0.429>P=0.000).4.PCR resultsPCR results showed that the relative expression of mouse brain ?-opioid receptor(mor)and ?-opioid receptor(dor)compared with the control group,were significantly lower(P<0.01).The relative expression of the model group zebrafish brain ?-opioid receptor(zfmor)and ?-opioid receptors(zfdorl and zfdor2)compared with the control group,were significantly lower(P<0.05).After intervention by Sinomenine are on the ?-opioid receptor(zfmor)and ?-opioid receptors(zfdorl and zfdor2)have a certain amount of improvement relative expression,and the two receptors in zebrafish and mice relative expression levels were positively correlated(r =0.328,P=0.000).CONCLUSIONS1 After giving a certain amount of morphine hydrochloride,mice and adult zebrafish of morphine dependence model produced CPP effect,and the correlation analysis,zebrafish and mice results in two animal models CPP significantly correlated,indicating that mature mice morphine dependent model compared to morphine dependence zebrafish model is reproducible,stable and successful.2 For TH,NR2B two indicators immunohistochemical analysis of the results,zebrafish and mouse model of morphine dependence between the two sets of data are basically there is a positive correlation,suggesting that zebrafish and mice in two animal models of biological neural mechanisms of action similar to Tip zebrafish animal models to some extent can replace rodent models.3.After intervention by sinomenine,dependence on morphine in mice and adult zebrafish model of behavior,TH,NR2B,PCR(mor and dor)three indicators have a regulatory role,between the two sets of data are basically there is a positive correlation,suggesting sinomenine for the effect of the two models are similar,because of the zebrafish model has a short cycle,less medication,small size,and low cost relative biological advantage,conducive to drug research and drug screening. |
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