Laboratory Animal Science

Establishment of in Vitro Primary Culture Method for Tree Shrews Corneal Endothelial Cells

2016, 33(05): 1-8.

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Abstract: Objective To establish an efficient and stable technique for primary isolation，culture and purification of tree shrew corneal endothelial cells( CECs) ，and provide a new experimental material for human ophthalmic corneal diseases． Method For the purpose of seeking the most advantageous way of acquiring tree shrew CECs，in our study，we compared the distrip method，double digests method and method Combining distrip and double digest． We used DMEM /F12，M199，Ham’s F12 /M199 cell culture mediums respectively to determine which is the most suitable medium for CECs cultivation． TGF-β inhibitor，low-serum culture method and gradient digestion were also employed to explore the feasibility of tree shrews CECs’purification． Using hematoxylin-eosin staining to observe the morphology of CECs． We designed and filtrated specific-primers according to tree shrews neuronspecific enolase ( NES) forecast sequences，utlised the PCＲ method to detect the expression of NES and NES antibody was also used to identify cell immunofluorescence．Ｒesult Cornea peeled off method is the most convenient and efficient way to obtain tree shrews CECs with high purificationin comparison with other methods． Ham's F12 /M199 medium is the most suitable medium for tree shrews CECs cultivation． Applying TGF-β inhibitor can help to purify CECs． Conclusion Optimized culture and purification method are more appropriate for in vitro tree shrew CECs culture，and the cells isolated by this manner more conform to CECs characteristics．

Measurement of the Body Weight，Organ Weight，Hematological and Blood Biochemical Parameters of B6 － Trp53tm1 /NIFDC after MNU Administration

2016, 33(05): 9-15.

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Abstract: Objective To determine body weight，organ weight，hematological and blood biochemical parameters of p53 + / － gene knockout mice，constructed by us and named as B6 － Trp53tm1 /NIFDC，after MNU administration of MNU and to provide background data for candidate animal models used as short-term in vivo alternative carcinogenicity study in the field of preclinical safety evaluation of drugs． Method There were four different groups，negative control group: wild type C57BL /6 mice treated with saline solution; vehicle control group: B6 －Trp53tm1 /NIFDC mice treated with citrate buffer solution; MNU group 1: B6 － Trp53tm1 /NIFDC mice treated with 75 mg /kg MNU and MNU group 2: wild type C57BL /6 mice treated with 75mg /kg MNU． Both negative control group and MNU group 2 have 20 wild type animals，10 male 10 female． Both vehicle control group and MNU group 1 have 20 B6 － Trp53tm1 /NIFDC mice，10 male 10 female． The body weight，absolute organ weight，relative organ weight，hematological and blood biochemical parameters were measured and carried out statistical analysis．Ｒesult The decrease of the body weight of MNU group 1 and MNU group 2 compared with negative and vehicle control groups was statistically significant ( P ＜ 0. 05) ． There was statistically significant difference ( P ＜ 0. 05) of the absolute weight of heart，liver，spleen，lung，thymus and mandibular salivary gland of MNU group 1 and 2 compared with those of the negative and vehicle control groups． Furthermore，there was also statistically significant difference ( P ＜ 0. 05) of the relative weight of heart，liver，spleen，lung，kidney，brain，thymus and mandibular salivary gland of MNU group 1 and 2 compared with those of the negative and vehicle control groups． When the hematological parameters of MNU group 1 and 2 were compared with those of the 2 control groups，there were statistically significant difference ( P ＜ 0. 05) in 15 parameters，including NEU，LYM%，LUC%，ＲBC，HGB， HCT，MCV，MCHC，ＲDW，HDW，CHCM，CHDW，PDW，MPV and PLT． When the blood biochemical parameters of MNU group 1 and 2 were compared with those of the 2 control groups，there were statistically significant difference ( P ＜ 0. 05) in 7 parameters，including TP，ALB，CＲEA，UＲEA，TCHO，TG and CA． AST was only statistically significant higher in MNU group 2 compared with that of the 2 control groups ( P ＜ 0. 05) ．There was no statistically significant difference between MNU group 1 and MNU group 2． Conclusion The body weight，organ weight，hematological and blood biochemical parameters of B6 － Trp53tm1 /NIFDC mice and wild type mice treated with MNU or citrate buffer solution were measured and analyzed in the paper． The model might be used as a short-term in vivo alternative carcinogenicity study in the field of preclinical safety evaluation of drugs in the future．

Bioactive Constituents Studies on Gentianopsis paludosa against UC Fibrosis Based on the Pharmacodynamics Indexes

2016, 33(05): 16-20.

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Abstract: Objective To study the bioactive constituents of Tibetan medicine Gentianopsis paludosa against UC fibrosis． Method Active fragment were obtained by silica gel and TLC from ethyl acetate active part which was extracted from Tibetan Gentianopsis paludosa． With the method of qＲT-PCＲ，CollagenI，collagenⅢ，α-SMA，and E-cad were detected after treating model rats with active isolates to screen the active components．Ｒesult After Purified and identified from ethyl acetate part using spectroscopy，four monomer compounds ( 1，8-hydroxy-3，7- dimethoxy xanthones; 1-hydroxy-3，7，8-trimethoxysilane xanthones; 1，7-hydroxy-3，8-dimethoxy xanthones and 1-hydroxy-3，7-dimethoxy-xanthones) ，had significant bioactive on UC fibrosis． Conclusion The material basis of Gentianopsis paludosa against fibrosis concentrated in parts of ethyl acetate，and the active ingredient mainly owed to four xanthones．

Zebrafish as an Animal Model for Assessing Hepatotoxicity of Five Chinese Medicines

2016, 33(05): 21-27.

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Abstract: Objective To determine the hepatotoxicity of 5 known mammalian hepatotoxic drugs ( Zhuangguguanjie ［ZGGJ］pill，Yi Shen Wu Fa oral liquid，Niuhuang Jiedu Pian，Tripterygium Glycosides ［TWP］，saikosaponin d) in larval zebrafish and to validate the practicability of zebrafish model for rapid assessment of drug hepatotoxicity． Method Zebrafish at 3 dpf ( days past fertilization) were treated with Chinese medicines at series of concentration for 2 days． At the end of treatment，zebrafish from each group were randomly selected and images were acquired under a fluorescent stereomicroscope and quantitatively assessed in larval zebrafish using three specific phenotypic endpoints of hepatotoxicity: liver degeneration，changes in liver size and yolk sac retention． Zebrafish liver degeneration was originally screened visually，quantified using an image-based morphometric analysis．Ｒesult ZCGJ pill induced liver degeneration and reduced liver size in larval zebrafish，Yi Shen Wu Fa oral liquid induced liver degeneration and increased liver size in larval zebrafish，Niuhuang Jiedu Pian reduced liver size in larval zebrafish，TWP and saikosaponin d increased liver size in larval zebrafish． All the tested mammalian hepatotoxic drugs delayed yolk sac absorption in larval zebrafish． Conclusion Chinese medicines could induce the liver damage in larval zebrafish． Zebrafish phenotypic assay is a highly predictive animal model for rapidly in vivo assessment of compound hepatotoxicity．

Zebrafish Model for Assessing Neurotoxicity of Six Drugs

2016, 33(05): 28-32.

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Abstract: Objective To explore the neurotoxicity of six kinds of known positive drugs，Mycophenolate Mofetil， Isoniazid，Ciclosporin A，Carbamazepine，Dilantin，Ethyl Alcohol，using Zebrafish embryos． Method We selected normally developed 5 dpf Wild － type AB line Zebrafish，and then Mycophenolate Mofetil ( 4． 5，15，45 and 51 μmol /L) ，Isoniazid ( 1 460，4 866，14 599 and 15 328 μmol /L) ，Carbamazepine ( 17. 5，58． 3、175 and 187 μmol /L) ，Dilantin( 16． 7，55． 7，167 and 185 μmol /L) ，Ethyl Alcohol ( 0． 18%，0． 63%，1． 81% and 2. 0%) and Ciclosporin A( 5，16． 7 and 50 μmol /L) were added to the artificial seawater． After 24 h，Zebrafish total movement distance was acquired by using the Zebralab larvae behavior video tracking system，and then 6 kinds of drug’s effect on Zebrafish movement dynamic was evaluated according to the research results． We selected normally developed 1 dpf motor neurons transgenic zebrafish，and then Mycophenolate Mofetil ( 0． 15、0． 5、1． 5 and 5． 5 μmol /L) ，Isoniazid ( 10 949、36 496、109 708 and 111 825 μmol /L) ，Carbamazepine ( 45． 9、153、459 and 484 μmol /L) ，Dilantin( 200、667 and 2 000 μmol /L) ，Ethyl Alcohol ( 0． 19%、0． 62%、1． 86% and 1． 98%) and Ciclosporin A( 5、16． 7 and 50 μmol /L) were added to the artificial seawater． After 48 h, Zebrafish’s motor neuron axons length was acquired by analyzing motor neurons transgenic Zebrafish fluorescent images，and then 6 kinds of drug’s effect on Zebrafish’s motor neuron axons growth was evaluated according to the Zebrafish’s axon length．Ｒesult After exposed by Mycophenolate Mofetil，Ethyl Alcohol and Carbamazepine，With the increase of concentration，Zebrafish movement distance increased after decreased，produce a typical dose dependent reverse “U”type; After exposed by Isoniazid and Dilantin，with the increase of concentration，the inhibiting effect on Zebrafish increased; After exposed by Ciclosporin A，with the increase of concentration，Zebrafish movement distance increased gradually，produce a excited effect; the inhibiting effect on Zebrafish increased; 6 kinds of drug all can inhibit the growth of motor neuron axons under the experimental doses． Conclusion Zebrafish model can be used to evaluate nerve toxicity of drugs preliminary．

The Effects of PSP on Inflammation and Oxidative Stress in Acute Myocardial Infarction Ｒats

2016, 33(05): 33-38.

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Abstract: Objective To observe the effects of Polygonatum sibiricum polysaccharides( PSP) on myocardial injury and the possible mechanism in acute myocardial infarction rats． Method The AMI models were established by closing the left anterior descending coronary artery with SD rats，sham group wearing without ligation，then the successful model rats were randomly divided into model group，PSP high，medium and low dose group ( 900 mg· kg － 1、450 mg·kg － 1、225 mg·kg － 1 ) ，positive group ( Shexiang protect myocardial pills，12. 2 mg·kg － 1 ) ． 10 rats in each group． Gastric infusion one time every day for 28 days． ECG S-T were measured before surgery and immediately after surgery，1，3，7，10，14，21，28 d in each group; the concentration serum inflammatory cytokines IL-6，TNF-α，and GSH-Px，MDA levels were measured by ELISA，myocardium tissue morphology was measured by the microscope．Ｒesult Compared with model group，PSP inhibited ST segment rat model of myocardial infarction; decreased serum levels of TNF-α， IL-6，MDA( p ＜ 0. 05 or 0. 01) ，increased serum levels of GSH-Px( p ＜ 0. 05 or 0. 01) ，myocardial tissue morphology was improved． Conclusion PSP could improve the myocardial injury in acute myocardial infarction rats，its mechanism may be related to alleviate inflammation， improve the oxygen radical scavenging ability，reduce lipid peroxidation damage．

The Ability of Spontaneous and Exploratory Behavior was observed in Chronic Fatigue Syndrome Mice

2016, 33(05): 39-42.

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Abstract: Objective To study the differences of space exploration ability between chronic fatigue syndrome mice and normal mice． Method First of all，the Chronic Fatigue Syndrome ( CFS) mouse model is copied by using compound stimulus method，and then to explore the activity in the open field devices between chronic fatigue syndrome mice and normal mice．Ｒesult The total distance moved and average movement speed in the open field devices was decreased in chronic fatigue syndrome group compared with the normal group; The distance in the out ring was significant decreased in chronic fatigue syndrome group compared with the normal group ( P ＜ 0. 05) ; However the duration of movement and distance in the area was significant higher than control group( P ＜ 0. 05) ． Conclusion The ability of spontaneous and exploratory behavior was reduced in CFS mice，which the levels of avoid tropism and anxiety was increased．

Effects of Acupoint Catgut Embedding Therapy Combined Scopolamine on the Levels of 5-Hydroxytryptamin of Duodenum in Morphine Dependent Ｒats

2016, 33(05): 43-47.

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Abstract: Objective To observe the effects of acupoint catgut embedding therapy combined western medicine scopolamine on the levels of 5-hydroxytryptamin ( 5-HT) of duodenum in morphine dependent rats． Method The rats were randomized into Control group，Spontaneous withdrawal group，Scopolamine group，Acupoint catgut embedding group，and Acupoint catgut embedding combined scopolamine group． Morphine dependent rat model was induced by administration ( ip) of morphine． Withdrawal syndromes were evaluated after different treatments．Ｒats were sacrificed after treated for 7 and 14 days respectively． Compare morphological changes，5-HT levels of duodenum among different groups．Ｒesult Withdrawal syndromes appeared in morphine dependent model group obviously with increased 5-HT expression in duodenum and tissue homogenate． All detecting items of Acupoint catgut embedding combined scopolamine group were improved significantly after treated for 7 days，there was no significant difference compared to Control group． Detecting items of Scopolamine group were close to the Control group after treated for 14 days ( P ＞ 0. 05) ． Conclusion Both western medicine scopolamine and acupoint catgut embedding therapy could reverse withdrawal syndromes，5-HT levels of duodenum in morphine dependent rats．However，combined two therapies could shorten the time of treatment with better effect．

The Effect of Dahuang Mudan Decoction to Pancreatic Gland Microcirculation of Acute Pancreatitis Model Ｒat

2016, 33(05): 48-51.

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Abstract: Objective To study the effect of Dahuang Mudan Decoction to pancreatic gland microcirculation of acute pancreatitis model rat，investigate the function of minicirculation alteration in acute pancreatitis． Method Divided 60 SD rats randomly into 6 groups as drug action 4 hours SO group，model group，ＲPDP group ，and drug action 8 hours SO group，model group，ＲPDP group，there were 10 rats in each group．Ｒats in the model group and ＲPDP group，3． 5 % Sodium Cholate was retrogradedly injected into the pancreatic duct to produce the AP model．ＲPDP 40 g /kg intragastric administration 1 times in the ＲPDP group． 4 h and 8 h after intragastric administration，to get the blood and ascites，detect the content of amylase in the blood and ascites． Use fluorescence microscopy flow rate test method，observe the change of pancreatic gland microcirculation 4 h and 8 h after administration．Ｒesult There are blood flow rate difference of pancreatic gland microcirculation in the model group，and the blood flow rate in ＲPDP group are coincidence． Compared with the SO group，the serum-amylase and ascites-amylase were all significantly elevated in the model group． Compared with the model group，the serumamylase and ascites-amylase were all significantly depress ( P ＜ 0． 01 ) ． Conclusion AP was concerned with pancreatic gland microcirculation disturbance， pancreatic gland edema and pancreatic gland necrosis were simultaneous in AP．ＲPDP could improve pancreatic gland microcirculation disturbance in AP modle rats．

Determination of Hematological and Visceral Weight Parameters of p53+ /－ Gene Knockout Mice

2016, 33(05): 52-56.

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Abstract: Objective To determine the normal range of hematological and visceral weight parameters of 4 － week and 8 － week old，male and female p53+ /－ gene knockout mice and analysis the influence of gender and week age． Method Body weight of 4 － week old and 8 － week old p53+ /－ gene knockout mice ( half male and half female，n = 20 in each group) were measured，then were sacrificed and the main viscera were weighted，and blood samples were collected to test the physiological and biochemical parameters．Ｒesult Some of the organ weights， blood physiological and biochemical parameters of different ages and sexes were significant differences． When the parameters of 4 － week old and 8 － week old Trp53+ /－ mice were compared，there were significant differences in 21 parameters ( body weight，heart，liver，left kidney，right kidney，left testis，right testis，WBC，ＲBC，HGB，HCT，MCV，MCHC，ＲDW，LYM，AST，ALB，ALP，CＲEA and Tbili) ( P ＜ 0. 01 ) ，and P ＜ 0. 05 in 8 parameters ( left adrenal，right adrenal，thymus，left ovary，right ovary，LYM%，NEUT% and TP) ． When male and female 4 － week Trp53+ /－ mice were compared，there were significant differences only in 2 parameters ( ＲDW and CHO) ( P ＜ 0. 01) ，and P ＜ 0. 05 for lung weight． For male and female 8 － week old Trp53+ /－ mice，there were significant differences in 16 parameters ( body weight，heart，liver，left kidney，right kidney，WBC，ＲBC，HGB，MCV，MCHC，ＲDW，LYM，AST，CA，P and CHO) ( P ＜ 0. 01) ，and P ＜ 0. 05 in 9 parameters ( HCT，PLT，PCT，LYM%，NEUT%，ALB，ALP，BUN and GLU) ． Conclusion The normal range of hematological and visceral weight parameters of 4 － week and 8 － week old male and female p53 + /－ gene knockout mice are determined． Our study provides a background data for rational use and application of the model．

The Anesthesia Effects of Ketamine and Sumianxin Ⅱ on Bama MiniPig

2016, 33(05): 57-60.

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Abstract: Objective To study the anesthesia effect of Ketamine and Sumianxin Ⅱ on minipigs，to investigate the effects of two kinds of anesthetic on physiology of animal． Method Ketamine and Sumianxin Ⅱ were mixed together，the volume ( between 0． 3 to 0． 5 mL /kg) of the mixture was used for anesthesia，then the anesthetic effect and physiology index were recorded．Ｒesult With the increasing of anesthesia dose，the length of anesthesia effect － act，muscle relaxant and eyelid reflex disappearing were decreasing． The heart rate and respiratory rate of animals in high dose ( 0. 4—0. 5 mL /kg) group higher than low dose ( 0. 3—0. 4 mL /kg) group． The mean arterial pressure and the oxygen saturation were similar between two groups． Conclusion 0. 3—0. 4 mL /kg could be used safely for anesthesia on minipig ，this dose could offered not only good anesthesia effect but also little impact on physiology indexes．

Ｒesearch Progress in Animal Models of Inflammatory Bowel Disease

2016, 33(05): 61-63.

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Abstract: Inflammatory bowel disease ( IBD) is a chronic and non-specific inflammatory disease which comprises Crohn's disease( CD) and ulcerative colitis( UC) ． The typical clinical manifestations are abdominal pain，diarrhea and bloody mucopurulent stool． The disease is caused by the interaction of a number of factors，generally including infected，genetic，environmental， immunologic abnormalities and other factors． The experimental models of IBD have been proven to be important tools to investigate the potential therapeutic agents． This review is intended to cover recent advances in suitable animal models and describe its features of IBD animal models．

Copy before Animal Model of Constipation and Evaluation

2016, 33(05): 64-67.

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Abstract: Constipation is the most common clinical，but also very complex gastrointestinal symptoms，constipation animal model is the mechanism of constipation and laxative drug discovery tools，this paper summarizes the methods and models to replicate features constipation animal model for the conduct of intestines laxative pharmacodynamics experiments and related research reference．

The Ｒesearch Progress of Animal Model of Alzheimer＇s Disease

2016, 33(05): 68-70.

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Abstract: Alzheimer’s disease is a kind of central nervous system neurodegenerative disease． Because of its complex pathogenesis and various etiology， restricting the development of animal models． In this paper，the animal model of alzheimer＇s disease is divided into transgenic animal model and the non-transgenic animal models，and the author concretely introduces the APP transgenic mice model，APP /PSI transgenic mice model，Tau /APP /PSI transgenic mice model ，and the AD animal models induced by Aβ，cholinergic nerve injury animal model and other． At the same time ， the advantages and disadvantages of the animal models are summarized，and the future development trend is forecasted．

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