Table of Content

28 April 2019, Volume 36 Issue 02

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Homology Analysis and Prokaryotic Expression of Mongolian Gerbil CST3

2019, 36(02):  1. 

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Objective To analyze the sequence homology of CST3 and establish a prokaryotic expression system of CST3 protein in vitro for producing anti-Mongolian gerbil Cystatin C (CST3) antibodies and perform function studies in future. Method After cloning and homology analysis, the condon optimization of Cst3 cDNA sequences of Mongolian gerbils were conducted. The recombinant CST3 protein expression vector was constructed by inserting the optimized sequences into pET28a vectors, which were then transformed into competent cells. The prokaryotic expression of CST3 protein was induced by Isopropyl β-D-Thiogalactoside (IPTG) and verified by SDS-PAGE gel electrophoresis and Western blotting. Result Both nucleotide and amino acid sequences of CST3 in Mongolian gerbils displayed low identity with human or mice CST3. After codon optimization, the Cst3 expression sequences of Mongolian gerbils were inserted into the pET28a plasmids, and a large amount of CST3 protein was obtained after induced expression with 1 mmol/L IPTG at 37 ℃ for 12 h as an optimized condition. Conclusion We successfully established a prokaryotic expression system of CST3 protein in vitro.

Modeling of Systemic Parkinsonism and In Vivo Dynamic Evaluationof Clinical Progress in Cynomolgus Monkeys

2019, 36(02):  9. 

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Objective To model the systemic parkinsonism and in vivo dynamically evaluate the clinical characteristics by using behavioral and molecular imaging method in cynomolgus monkeys, and provide a reliable Parkinson's disease(PD)model for preclinical studies of drug and stem cell and translational research. Method Seven healthy middle aged cynomolgus monkeys with age between 10-15 years were intravenously injected of MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)(0.2 mg/kg)to induce a systemic PD model, continuously observe the evolution of clinical symptoms up to 3 months followed by L-Dopa intervention. Behavior analysis including rating scale for evaluating the severity of clinical symptoms, the EthoVision tracking system for measuring the moved distance, motion trajectory, 18F-AV-133 the Positron emission tomography(PET)imaging for detecting the functional integrity of striatal dopaminergic system. Result All animals showed the typical PD symptoms including resting tremor, rigidity, and bradykinesia etc. at 14 days or so when MPTP injection was stopped. The clinical score reached peak at 1 month(21.43±5.35), and then the symptoms remain stabilized and continued. The clinical scores were (14.43±1.90) at the end of MPTP injection, (18.43±3.87) in the 2 months and (18.14±3.53) in 3 months post MPTP respectively. The moved distance was significantly lower at 3 months(809.77±401.15)cm than the baseline(8627.46±5751.04)cm(P<0.01). The Specific uptake rate(Sur)of the 18F-AV-133 was significantly reduced at 3 months(0.16±0.03)than the baseline(1.66±0.58)(P<0.01). PD symptoms were significantly improved after L-Dopa intervention, the clinical score(12.86±3.63)was significantly decreased(P<0.05)while the moved distance was significantly increased(P<0.05)than that before. Conclusion A systemic PD is modeled with continuously stable clinical symptoms with no spontaneous recovery, and the damage of functional integrity of striatal dopaminergic system, and the positive response to L-Dopa intervention, which closely simulates the clinical features of PD and provides a stable and reliable PD model for preclinical translational studies.

Effect of Ginseng Decoction on HSP70 Expression of Livers and Kidneys in Laboratory Red Crucian Carps Irradiated by 137Cs

2019, 36(02):  16. 

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Objective To study the effects of 137Cs irradiation on the HSP70 expression of livers and kidneys in laboratory red crucian carps and explore the radioprotective effect of ginseng decoction on laboratory red crucian carp. Method The laboratory red crucian carps were randomly divided into 8 groups: blank control group, ginseng control group, 4.1Gy irradiation group, 4.1Gy ginseng prevention group, 4.1Gy ginseng treatment group, 8.2Gy irradiation group, 8.2Gy ginseng prevention group and 8.2Gy ginseng treatment group. Laboratory red crucian carps were exposed to 137Cs irradiation, or given ginseng decoction for 7 days before irradiation treatment, or treated with ginseng decoction for 7 days after irradiation treatment. Then the livers and kidneys of laboratory red crucian carps were collected separately, and the expression of HSP70 in each group was detected by Western blot method . Result Compared with the blank control group, the expression of HSP70 protein was increased after 4.1Gy or 8.2Gy irradiation. Compared with the irradiation groups, the expression of HSP70 was lower after the prevention or treatment of ginseng decoction. There was no significant influence on HSP70 expression just by treatment with ginseng decoction.It was found that there were no obvious differences in HSP70 protein expression between ginseng prevention groups and ginseng treatment groups. Conclusion 137Cs irradiation can increase the HSP70 expression of livers and kidneys of the laboratory red crucian carps, while the expression of HSP70 is reduced after the prevention or treatment of ginseng decoction.

Methodological Study on Establishing Mouse Learning and Memory Injury Model

2019, 36(02):  21. 

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Objective To establish an effective method for learning and memory impairment in mice by avoiding darkness and Morris water maze experiments. Method Scopolamine 1 mg/kg was intraperitoneally injected 20 min before the mice were protected from dark electric shock. Immediately after the end of the electric shock training, sodium nitrite 90 mg/kg was administered subcutaneously and the mice were given 35% ethanol 0.1 mL/10 g 15 minutes before the next shock test., establish a model of memory acquisition, memory consolidation and memory reconstruction disorder, and determine the indicators of avoidance darkness, number of errors and number of probes after 6 h, 24 h, 30 h, 48 h after shock training in each group. The sensitivity of the indicators of the memory impairment model method and the optimal test time. The mice were intraperitoneally injected with scopolamine 1 mg/kg and 35% ethanol 0.1 mL/10 g to cause learning and memory impairment. The Morris water maze positioning navigation test and space exploration test were used to test the spatial orientation and direction of the experimental animals (spatial positioning), the learning and memory ability, the establishment of a spatial learning and memory impairment mouse model evaluation method . Result The dark-avoidance experiment showed that compared with the blank group, the mice with memory impairment caused by scopolamine significantly shortened the dark-avoidance period at 6 h and 24 h after electric shock training, and the number of errors increased significantly (P0.05). Conclusion When using scopolamine and sodium nitrite to construct memory impairment and memory consolidation model in mice, the corresponding indicators should be detected at 24 h and 30 h after modeling. The model of memory reproduction disorder caused by ethanol should be 24 h after electric shock training. The mice were tested with ethanol after 15 min before the next test. The indicators were to avoid dark latency, number of errors and number of probes. Morris water maze experiments suggest that scopolamine can damage spatial learning and memory in mice, and can be used as a drug to construct a model of spatial learning and memory impairment in mice. Ethanol cannot establish a model of spatial memory impairment in mice.

Analysis of the Effects of N-acyl homoserine lactones on the Gut Microbes in Mice

2019, 36(02):  27. 

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Objective Study the effection of N-acyl homoserine lactones (AHLs) on gut microbes flora in mice so as to supply the theoretical basis of intestinal microecological disorder under infection status. Method After the mice were intraperitoneal injected with 3-oxo-C10-HSL for 24 h, samples of mice duodenum and jejuno-ileum contents were acquired with swab smear method . The sample 16S rDNA sequencing was detected by Illumina Hiseq 2 500 high-throughput sequencing platform, and the classification of microbial flora composition and diversity were analyzed. Result There were 22 phyla and 109 genera in all identified in the mice gut, and Firmicutes (>50%) and Proteobacteria (>20%) dominated the microbiota. The control group and 3-oxo-C10-HSL treatment group had obvious difference in the level of flora composition, for the former the dominant genera mainly included Staphylococcus, Escherichia-Shigella, galactococcus, Pseudomonas and Enterococcus, and for the latter the dominant genera mainly included Enterococcus, Escherichia-Shigella, and Staphylococcus. It was also revealed that the 3-oxo-C10-HSL treatment could improve the gut microbial diversity, and the abundance of dominant genera such as Escherichia-Shigella and jejuno-ileum microbiota significantly rose. Conclusion AHLs had little effect on gut microbiota in mice, but some specific microbial taxa would be significantly affected.

Establishment of RPA and fluorescent RPA detection methods forSimian T-cell lymphotropic virus type 1

2019, 36(02):  33. 

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Objective In order to survey and investigate the epidemic character of simian T-cell lymphotropic virus type 1(STLV-1)transmission among wild and experimental monkeys, method of recombinase polymerase amplification (RPA) and fluorescent RPA to detect STLV-1 were established. Method Analysis conservative sequence of STLV-1 gag polyprotein genes separated from different countries and areas, RPA primers and fluorescent probe were designed and RPA methedes were tested by specificity, sensitivity and stability. Result Compared with other simian viruses, new RPA and fluorescent RPA mothedes had highly specific to STLV-1. The limit of detection of RPA and fluorescent RPA mothedes is similar with PCR method. The stability of RPA and fluorescent RPA was comfirmed by detection of 30 postive and negative STLV-1 samples. Conclusion The RPA and fluorescent RPA mothedes to detect STLV-1 were established and proved to be specific with high sensitivity and stability, which could be applied to survey STLV-1 infection among simian.

Effects of Different Concentrations of Tea Polyphenols on Intestinal Flora in Mice

2019, 36(02):  38. 

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Objective To study the effects of different concentrations of tea polyphenols on intestinal flora in mice. Method Twenty four C57BL/6 mice were randomly divided into 4 groups, control group(CK, no tea polyphenols added), low-dose tea polyphenols group(LTP, 0.4 g/L GTP added in drinking water), medium dose tea polyphenols group(MTP, 0.8 g/L GTP added in drinking water), high-dose tea polyphenols(TP, 1.6 g/L GTP added in drinking water), 4 groups are given regular feed, and tea polyphenols are replaced once a day for 14 consecutive days. Analysis of intestinal microbial diversity in mice by MiSeq sequencing and bioinformatics. Result High concentration of tea polyphenols could increase the diversity of intestinal flora. The content of Lactobacillus in medium dose group and high dose group was significantly higher than that in low dose and control group(P=0.038). Conclusion Tea polyphenols can increase the diversity of intestinal microorganisms, increase the proportion of probiotics and change the composition of flora.

Pathological Changes of Specific Myocardial Ischemia Induced by Isoproterenol in Rats Cardiomyocytes

2019, 36(02):  42. 

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Objective To observe the changes of HE staining, VEGF and FGF expression in rat myocardial tissue slices induced by subcutaneous injection of isoproterenol (1 mg/kg) in different days. Method Except for the natural control group, rats were injected subcutaneously with isoproterenol (1 mg/kg) 2-day group、4-day group， 6-day group. One week after injection, the rats were anesthetized, the ECG was measured, and the heart was taken. HE staining was used to observe the changes of rat myocardial cell damage. Immunohistochemical staining was used to observe the changes of VEGFand FGF expression. Result Compared with the natural control group, continuous subcutaneous injection of isoproterenol (1 mg/kg) at 6-day group showed significant differences in HE staining. Expression of VEGF (vascular endothelial growth factor) and FGF (fibroblast growth factor) in the model group was also significantly different from that in the natural control group. Conclusion Rats were injected subcutaneously with isoproterenol (1 mg/kg) 6 days can be used as an experimental animal model for evaluating the efficacy of heterosexual cardiomyopathy.

Pathological Study on Spontaneous Lesions of Sprague-Dawley Ratsin Different Week-old，Sexs，and Feeding Environments

2019, 36(02):  55. 

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Objective To observe the spontaneous lesions of SD Rats of different week-old, sexs,and feeding environments,and to provide the background database for pathological evaluation in drug safety study. Method SPF SD rats of normal control groups in drug safety tests，male and female included，separately kept under the center barrier and the ordinary environment，were killed at respectively 19，23，32 and 36 weeks time points．Tissues were examined under optical microscope，as well as animal disease type and disease incidence were counted．Result Compared with barrier environment，the incidence of spontaneous lesions of respiratory system was markedly increased in SD rats under ordinary environment．In addition，the incidence of spontaneous lesions of SD rat trended to increase with age in both feeding environments．Meanwhile，there were obvious sex differences in a few lesions，especially in progressive cardiomyopathy and mineralization in kidney．Spontaneous lesions consisted of progressive cardiomyopathy，focal inflammatory cell infiltrate (granulomatous inflammation included) in liver，spontaneous altered hepatocellular focus，vacuolation of the hepatic cell，focal inflammatory cell infiltrate in lung，aggregates of alveolar macrophages，mineralization in kidney，interstitial inflammatory cell infiltrate in kidney，atrophy in pancreas， inflammatory cell infiltrate in trachea，interstitial inflammation in prostate，atrophy of seminiferous tubules in testis，exfoliation of germ cell in testis，inflammation in endometrium and inflammation in vagina and so on．Conclusion The present study enriched the existing pathology background information of laboratory animal，and provided the reference for relevant technical personnels．

A Model of Hemorrhagic Shock in Beagle Dogs

2019, 36(02):  55. 

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Objective To establish a model of hemorrhagic shock in Beagle dogs for the experimental study of drug evaluation and research. Method Fifteen Beagle dogs of common grade were bled until the arterial pressure (SBP) reached 50 mmHg, which was then maintained as best as possible. The criteria of the model were 9～11 mmol/L of Lac and 12 mmol/L of BE in arterial blood, less than 60% PO2 in venous blood. When the model was established, the changes of general physiological indexes, blood-gas parameters and blood routine indexes were observed. Result Twelve animals survived, the success rate of the model was 80%. The changes of indexes were in accordance with the characteristics of hemorrhagic shock. Conclusion The model was able to simulate the clinical characteristics of hemorrhagic shock objectively and accurately, and is suitable for the efficacy evaluation of blood products and blood substitutes.

The Embryo-fetal Developmental Toxicity Test of IntravenousCyclophosphamide on New Zealand Pregnant Rabbits

2019, 36(02):  58. 

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Objective To observe the embryo-fetal developmental toxicity of intravenous cyclophosphamide on pregnant rabbits, and provide basis for using cyclophosphamide as a positive control in embryo-fetal developmental toxicity test. Method Pregnant New Zealand rabbits were randomly divided into control group (0.9% Sodium chloride injection, GD11, i.v), and cyclophosphamide group(cyclophosphamide 30 mg/kg, GD11, i.v). At termination (GD28), the uterine content was removed by caesarean section. Live fetuses were examined for external, visceral and skeletal malformations and variations. Result There were no noticeable changes in the reproductive function and embryogenesis of rabbits by cyclophosphamide. But cyclophosphamide could cause the teratogenic in external, visceral and skeletal malformations. The rate of cleft palate was 32.95%, the rate of visceral variation was 10.23%, and the rate of skeletal malformation was 23.37%. Conclusion Inject cyclophosphamide (30 mg/kg) into the New Zealand rabbits can cause fetal malformation. Type and ratio of fetal malformations are proportional. Cyclophosphamide can be used as a positive control for embryo-fetal developmental toxicity test in rabbits.

Comparison of Test Method for Histamine-like Substance and Depressor Substance in Hepatocyte Growth-promoting Factors for Injection

2019, 36(02):  63. 

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Objective To study the feasibility of histamine-like substance test in hepatocyte growth-promoting factors for injection safety. Method Histamine-like substance and depressor substance in three batches of hepatocyte growth-promoting factors was test according to the test method in the appendix of Chinese Pharmacopoeia (edition 2015) and the result of the tests were compared. Result The test result of histamine-like substance test in all three batches of hepatocyte growth-promoting factors were consistent with the test result of depressor substance test and met the requirement of Chinese Pharmacopoeia. While positive result were observed in depressor substance test, but negative result observed in histamine-like substance test. Conclusion Hepatocyte growth-promoting factors for injection may cause drop of blood pressure without histamine-like substance, and in vivo experiments cannot be extrapolate simply by the result of in vitro experiments.

Making and Application of Laser Locator for LaboratoryAnimal Magnetic Resonance Imaging System

2019, 36(02):  69. 

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Objective To design and develop a simple rat and mouse laser locator to provide a safe, convenient, accurate and affordable experimental tool for medical NMR experiments. Method Selected plexiglass plates and laser locator combination assembled into small animal laser locator. Result Compared with other similar products, the mouse laser locator has the advantages of simple and safe manufacture, basically meeting the experimental requirements and low price. Conclusion The small animal laser locator plays an active role in the nuclear magnetic inspection of small animals, which greatly shortens the experiment time and improves the accuracy of the placement of experimental animals, providing a powerful guarantee for the next step of nuclear magnetic resonance scanning of experimental animals.

The Present Situation and Thinking of the Regulation on Humane Endpoint and Euthanasia of Laboratory Animal

2019, 36(02):  72. 

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Laboratory animal welfare is currently followed by 3R principle. Humane endpoint and euthanasia are the important part of “refinement” of 3R principle, and has been formed a perfect system of laws and regulations in the international community. China need further improvement on the relevant laws, regulations, and standards. This paper introduces the main laws and regulations on humane endpoint and euthanasia, and hopes to provide reference for the development of related work in China.

A Rapid Method for Removing Synovial Membrane of Rat Knee Joint

2019, 36(02):  76. 

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Objective To study a more rapid and feasible method than the traditional method for removing knee joint synovial tissue of rats. Method The traditional method of extracting synovial tissue of the rat knee joint were studied by consulting the literature, and a large number of anatomical maps of the knee joint of the rats were examined. In the experiment, 60 successful adjuvant arthritis rats were randomly divided into two groups: the synovial tissue of the knee joint of 30 rats and the synovial tissue of the knee joint of the other 30 rats were taken from the synovial tissue of the knee joint of the rats by the traditional method , and the synovial tissue of the knee joint of the other 30 rats was obtained by a new method . Result Twenty-three rats were taken out of the synovial tissue in the traditional method . The synovial tissue of knee joint was taken out completely in 30 rats by the new method , and the average time consuming of unilateral synovial tissue was about (15.26±3.41) min in the traditional method, and (10.12 ±2.34) minutes in the new method . There was a significant difference between the two method (P<0.01). Conclusion The new method is more rapid and feasible than the traditional method in removing synovial tissue of rat knee joint.

Research Progress of Miniature Pig inbred Line Identification

2019, 36(02):  78. 

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This paper is reviewed a identification method of the experimental research and the progress appraisal for miniature pig inbred line, including: skin allograft transplantation,physiological and biochemical marker，isozyme protein identification, DNA genetic marker, SNP analysis method and so on.The comprehensive analysis is show that the technology of allogeneic skin grafts is the best identification method for a large mammal inbred line, which is a scientific basis for development and utilization of innovation resources on pig inbred line in China. It will be provided the scientific testing technology and method for identification of large mammals inbred line and make our country is a international leading position in large mammals inbred line study also.

Preparation and Comparison of Facial Paralysis Animal Model

2019, 36(02):  83. 

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Objective To search the relevant literature of different models of facial paralysis, analyze the advantages and disadvantages of various models, and explore the production method suitable for different types of models, so as to provide experimental evidence for making animal models of facial paralysis. Method The related literatures about animal models of facial paralysis have been searched in the past decade, and various experimental animals and modeling method have been summarized. Result At present, most animals used in facial paralysis model are rats, mice and Japanese white rabbits. The method of facial nerve transection, compression and traction are the method of modeling. Conclusion There are many ways to make facial paralysis model, each has its own advantages and disadvantages. According to different research purposes, it is possible to explain the acupuncture treatment of facial paralysis.