Table of Content

29 April 2017, Volume 34 Issue 02

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The Expression of Spink3 was Ｒegulated through EＲK Pathways in Mouse Hepatic Tumors

2017, 34(02):  1-6. 

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Abstract: Objective To investigate the molecular mechanisms of the regulation to Spink3 expression in mouse hepatic tumors． Method The mＲNA levels of SPINK1 in human hepatocellular carcinoma and Spink3 in hepatic tumors of H-ras12V transgenic mice were detected by ＲT-qPCＲ． The protein levels of p-EＲK and p-mTOＲ and mＲNA levels of Spink3 in primary cultured hepatic tumor tissues and tumor-adjacent normal tissues of H-ras12V transgenic mice were detected by Western blot and ＲT-qPCＲ． In vivo inhibition of the activities of p-EＲK and p-mTOＲ was performed to investigate their influence on the mＲNA levels of Spink3． Ｒesult Compared with tumor-adjacent normal tissues，the mＲNA levels of SPINK1 and Spink3 were significantly higher expressed in hepatic tumor tissues ( P ＜ 0. 05) ． In the primary cultured tumor and normal liver tissues of transgenic mice，along with the cultured time ( 6，12. 24. 48 h) ，the protein levels of p-EＲK and p-mTOＲ are significantly decreased compared to the 0 h． Consistently，the mＲNA levels of Spink3 were also significantly decreased ( P ＜ 0. 01) ． Inhibition of the activities of p-EＲK significantly reduced the mＲNA levels of Spink3 ( P ＜ 0. 05) ． However， Inhibition of the activities of p-mTOＲ had no influence on the mＲNA levels of Spink3． Conclusion SPINK1 and Spink3 are significantly higher expressed in hepatic tumor tissues and could be used as potential biomarkers for clinical diagnosis． The EＲK pathway may play important roles in up-regulating the expression of Spink3．

Establishment and Genotyping of Pumilio1 and Pumilio2 Double Conditional Knockout Mouse Strain

2017, 34(02):  7-10. 

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Abstract: Objective To establish and genotype a Pumilio1 ( Pum1) and Pumilio2 ( Pum2) double conditional knockout ( dCKO ) mice， in order to further study the biological function of PUF family in mammalian spermatogenesis． Method Homozygtes of dCKO offspring were produced from crossbreeding of Pum1 conditional knockout ( CKO) mice and Pum2 CKO mice． The pups were identified using toe-marking system at postnatal day 10 ( P10) ，and weaned at P21． Genomic DNA was extracted，and genotyping was performed using PCＲ，agarose gel electrophoresis and gel-imaging． Ｒesult Pum1 and Pum2 dCKO mice were obtained and reproduced normally． PCＲ products showed correct size of each allele as expected． Conclusion Pum1 and Pum2 dCKO mice are established successfully，providing an useful conditional knockout animal model to study roles of PUF family at different stages and different cell types for spermatogenesis as well as for any other physiological process．

Culture and Identification of Intestinal Epithelial Cells Isolated from Tree Shrew in vitro

2017, 34(02):  11-15. 

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Abstract: Objective To isolate intestinal epithelial cells( IECs) from the tree shrew，to establish a cellular model for drug mechanism research，and to lay the foundation for establishment of intestinal epithelial cell lines． Method Collagenase type XI，and neutral protease type I and DTT were used for digestion of tree shrew small intestine． Cells morphology was observed by phase contrast microscopy，identified by cytokeratin Immunofluorescence assay．Ｒesult Isolated intestinal epithelial crypts attachment were performed in 24 h，confluent in 6 d． Epithelial outgrowth from isolated crypts were formed with confluent monolayers cobblestone and multi-angular morphology． Cells were passaged every 2 ～ 3 d from the nest generation，and between the third and sixth day from the day when cells were plated the cells got into the logarithmic growth phase． The cells were stained positive for cytokeratin immunofluorescence staining． Conclusion Intestinal epithelial cells were successfully isolated from the tree shrew， and the method of isolation for tree shrew’s intestinal epithelial cells was established．

Ｒemoving Signal Peptide Accumulate Chicken Interleukin-10 in the Cytoplasm of Mammal Animal Cell Line

2017, 34(02):  16-20. 

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Abstract: Objective To analyze the effect of signal peptide of chicken interleukin-10 ( chIL-10) on expressing in CHO-K1 cell line，and to establish a stable chIL-10 expressing cell line． Method Whole chIL-10F and signal peptide-deleting genetic fragment of chIL-10 were amplified with two pairs of primers and inserted into pEGFP-N1 vector． The obtained recombinant plasmids of N1-chIL-10F and N1-chIL-10M were transfected into CHO-K1 cells under G418 screening． Ｒesult Two stable cell lines of CHO-chIL-10F and CHO-chIL-10M were obtained， respectively，confirmed by ＲT-PCＲ and western blot assays． Confocal examination showed that the reporter protein GFP was expressed in the cytoplasm of CHO-chIL-10M and stronger than in the CHO-chIL-10F． Conclusion Ｒemoving signal peptide of chIL-10 improves expression of chIL-10 in the cytoplasm in vitro．

Study on the Screening Methods of Technology on Small Complex Prescription of Chinese Medicine for Treatment of Insomnia

2017, 34(02):  21-25. 

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Abstract: Objective By using pharmacodynamical and acute poison experiment，the best extraction technology was determined by comprehensive score method． Method The experimental design was to prolong the sleep time of mice with sodium hypnosis and the threshold hypnotic dose，and together with acute toxicity experiment in mice．Ｒesult NO． 7 drug could shorten substantially the sleep latent period of rats． Compared with the control group there was significant difference ( P ＜ 0. 05) ． No． 2，No． 3，No． 5，No． 7 and No． 9 groups could enhance the number of asleep mice ( P ＜ 0. 01，P ＜ 0. 01，P ＜ 0. 05，P ＜ 0. 05，P ＜ 0. 01) ; acute toxicity experiment in mice identified the LD50 of all the reagent． The results showed that the combined score of NO． 7 drugs was the highest． Conclusion NO． 7 group extraction technology was the best．

Population Genetic Quality Analysis of 2 outbred BA-MAminiature pigs in Beijing

2017, 34(02):  26-30. 

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Abstract: Objective To analyze and evaluate the population genetic quality of 2 outbred BA-MA miniature pigs in Beijing． Method Twenty-five pairs of microsatellite primers were used in A group and B group outbred BA-MA miniature pigs according to the local standard DB11 /T828. 3 － 2011． And software Popgen32 was used to process the data． Ｒesult Twenty-five microsatellite loci shared 126 and 160 alleles in A group and B group outbred BAMA miniature pigs，respectively． The average heterozygosity was 0. 6541 and 0. 6973，respectively，while the average polymorphism information content ( PIC) was 0. 6037 and 0. 6605，respectively． The genetic distance between the two groups was 0. 2650． Conclusion B group BA-MA miniature pigs have adaptive genetic stability and genetic diversity，which satisfied with the genetic characteristics of closed colony laboratory animal．

Histology Section Preparation and Hematoxylin and Eosin Staining of Gobiocypris rarus

2017, 34(02):  31-34. 

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Abstract: Objective Gobiocypris rarus，a freshwater fish with high prolificacy，distributes in Sichuan Province． In order to became a laboratory animal，the fish has been investigated its geographical distribution，morphology， habita，ontogenesis，reproduction，growth，taxonomy，semitiviqto ecological factors，karyotype and so on by Chinese Academy of Science since 1990s． However，there was few histological background of Gobiocypris rarus until now． Here we introduce a practical way to make sections of Gobiocypris rarus． Method The healthy individuals were fixed in the Bouin’s fluid，washed with running water slightly，and put into decalcifying fluid． After finishing decalcification，the fish was cut into several small pieces，the specimens were dehydrated and infiltrated，respectively，and then waxed with paraffin． The specimens in solid paraffin were allowed to cut into serial slices． The slices were dyed by Hematoxylin and Eosin ( H＆E) staining according to the routine protocol． Transverse and sagittal section pictures were collected by a optical microscope． Ｒesult The sections were scaned under microscope and the pictures of different kinds of organs and tissues were taken． Those high quality pictures can set up the histological background of Gobiocypris rarus． Conclusion Histology can detect features of disease which are difficult to recognize from clinical symptoms． The histological background of Gobiocypris rarus tissues and organs is the foundation of pathology knowledge． The method of making sections of Gobiocypris rarus may contribute to the research of aquatic animal．

The Effect of Xueluofuxin Granule on Blood Coagulated Activity and Platelet Aggregation in Ｒats

2017, 34(02):  35-38. 

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Abstract: Objective To study the effect of Xueluofuxin granule on blood-coagulated activity and platelet aggregation in rats． Method SD rats were randomly divided into 6 groups，the tested groups were intragastricly administrated with Xueluofuxin granule for 7 d，and the blood was sampled by abdominal aorta to measure the Thrombin Time ( TT) ，Prothrombin Time ( PT) ，Activated Partial Thromboplastin Time ( APTT) ，fibrinogen content ( Fib) and the platelet aggregation rate induced by adenosine diphosphate ( ADP) ． Ｒesult Xueluofuxin granule prolonged PT、TT and APTT，but reduced the content of Fib and platelet aggregation rate induced by ADP in rats． Conclusion Xueluofuxin inhibited obviously blood coagulation and platelet aggregation induced by ADP.

Ｒesearch on Effect of FGF21 on Ｒeducing Myocardial Fibrosis in Type 1 Diabetic Mice

2017, 34(02):  39-43. 

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Abstract: Objective To investigate the effects of FGF21 on myocardial fibrosis in type 1 diabetic mice induced by STZ． Method Thirty-two 8 weeks old male C57 /BL6J mice were randomly divided into four groups，ten mice received STZ treatment，ten mice received STZ and FGF21siＲNA treatment， six mice were treated with FGF21siＲNA，and another six mice remain untreated． Type 1 diabetes was induced by STZ in concentration of 150mg / kg． Collagen fiber was observed by Masson staining and electron microscope． The expression of type 1 collagen mＲNA ( Col I mＲNA) ，type Ⅲ collagen mＲNA ( Col Ⅲ mＲNA) ，transforming grow factor β mＲNA ( TGF － β mＲNA) and FGF21mＲNA of myocardium were detected by Ｒeal-time PCＲ． Ｒesult The expression of Col I mＲNA，ColⅢ mＲNA and TGF － βmＲNA in myocardium of type 1 diabetes mice treated with FGF21siＲNA increased more than that of untreated ones，cardiac collagen fiber proliferation was worse than those of untreated ones． Conclusion FGF21 may be able to protect myocardium from fibrosis in mice with type 1 diabetes induced by STZ．

Establishment of Novelty Searching and Verification System for the Local Standards of Laboratory Animals and Application of these Standards

2017, 34(02):  44-50. 

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Abstract: The local standard of laboratory animals is an important part of the standard system for laboratory animals in China． Under the guidance and support of the sci-tech department and the quality inspection department，the research work on the local standards of laboratory animals in China has developed rapidly． It is very significant to establish mechanism for novelty searching and verification system of local standards of laboratory animals，to convince its effectiveness，to ensure the scientific supervision for laboratory animal administration，and to ensure laboratory animal quality and accurate result of animal experiment．

Study of In Vitro EpiDerm Skin Irritation Test

2017, 34(02):  51-53. 

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Abstract: Objective To assess the sensitivity，specificity and accuracy of an in vitro EpiDerm Skin Irritation Test． Method Thirty μL of the test substance was added into three single EpiDerm skin tissues，and negative control ( NC) and positive control ( PC) were introduced． Each group contained three parallel tissues． After the 60 min test substance exposure，MTT viability test and isopropanol extraction were done． Ｒead A value in a 96 － well plate spectrophotometer using a wavelength at 570nm，relative viability was calculated． skin irritation potential of the test material was predicted if the relative viability was below 50% ． The results of acute skin irritation test were compared with that in vivo． Ｒesult Comparing with acute skin irritation test in vivo，the sensitivity，specificity and accuracy of in vitro EpiDerm Skin Irritation Test were 100% ． And the results were objective，the workload was small． The issue of animal ethics was completely avoided． Conclusion In vitro EpiDerm Skin Irritation Test is the most ideal method of in vitro skin irritation．

Design and Application of Laboratory Animal Management Information Platform of Universities

2017, 34(02):  54-56. 

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Abstract: Laboratory animals are important experimental objects and tools in teaching and research work in the universities． In order to improve the safety， standardization and service quality of the laboratory animal management，we constructed the laboratory animal management information platform according to the characteristics of laboratory animal management in medical colleges and universities． This paper mainly introduces the design idea，function of the system， technical characteristics and operation procedures of the laboratory animal management information platform． After application of the laboratory animal management information platform，the standardization and service quality of laboratory animal management were further improved．

The Ｒesearch Progress of Depression-like Ｒodent Models by Ｒeserpine

2017, 34(02):  57-61. 

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Abstract: Depression is a high prevalence，morbidity，and suicidal chronic mental illness，which symptoms are pessimism and despair，severe cases can affect their daily life． At present，with the deepening of the research on depression，depression animal models are widely used in experimental studies． Among them，the reserpine-induced depression-like model can be replicated in some pathological features of depression． This paper reviews the reserpine-induced depression-like animal model studies，by observing the general situation，the behavior，neural system，immune system，analyzing the characteristics，applicability and limitations of the model，providing a reference model for the researchers who used reserpine depression-like model．

Animal Experiment Technology of Emotional Syndrome

2017, 34(02):  62-65. 

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Abstract: In nowadays society，with the accelerating pace of life as people increasingly competition，as people life rhythm accelerate ceaselessly，competition is becoming increasingly fierce． Negative emotional stimulation is a growing threat to people＇s health，and emotional diseases research has gradually become a hot spot． In the emotional diseases research，animal experiments are increasingly carried out，but the experimental technology is crucial． The aim of the paper is to evaluate animal experiment technology of the emotional syndrome and to contribute a better study of modern emotional diseases．

Preparation of Post-myocardial Infarction Heart Failure Ｒats and Evaluation of Cardiac Function

2017, 34(02):  66-70. 

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Abstract: Heart failure ( HF) is the result of various cardiovascular diseases，which seriously threatens human health． The model of post-myocardial infarction ( post-MI) heart failure rats has provided the experimental basis for the further study of the pathophysiology of heart failure． The methods of heart failure induced by myocardial infarction in rats include coronary artery ligation， coagulation， coronary microembolization， cryoinjury， radiofrequency ablation，etc． Hemodynamic，echocardiography and BNP were the main techniques for screening qualified heart failure models． This review introduces the post-MI heart failure model and the evaluation of its cardiac function to promote the study of heart failure．