Abstract:Objective The allergic purpura animal model was successfully established, which will provide reference for evaluation of disease treatment and development of new drugs. Methods The model rabbits received oral thermal drug and intraperitoneal injection of ovalbumin and Freund’s complete adjuvant saline mixture, then ear marginal vein injection and subcutaneous injection of ovalbumin saline to stimulate allergic reactions; the rabbits in the control group received equivalent of normal saline in the same way. During the experimental process, several evaluations were performed: the observation of general symptom; the average amount measurement of daily diet, drinking water and body temperature; the detection of blood routine(BRT), urine routine（URT）, Feces Occult Blood(FOB) test; the pathological examination of skin and kidney. Then, we compared the above indicators of rabbit models with clinical patients in the parts of symptom, laboratory examination and pathological changes. Results After the test, Compared with the control group , the rabbits in the model group performed skin ecchymosis; Less eating, more drinking (p<0.01); Increased temperature (p<0.05); the number of WBC increased and RBC decreased (p<0.01), the content of HGB, MCHC reduce (p<0.01) and NEU, NEU%,EOS,EOS% increased (p<0.05); URT test showed 67 percent rabbits of model group exist urine protein ( PRO), urine erythrocyte and 70 percent of them appear Feces Occult Blood (FOB); Pathological manifestation displayed subcutaneous vascular dilatation and congestion, hemorrhage, dermal edema, inflammatory cell infiltration; glomerular focal chronic nephritis, sac protein exudation, vascular dilatation and congestion, mesangial matrix increased, mesangial thickening, red blood cell tube type, infiltration of inflammatory cell; joint cavity extravasated blood, connective tissue necrosis, inflammatory cell infiltration; skin and renal IgA immunoglobulin deposit; gastric mucosal hemorrhage, necrosis and exfoliation; small intestinal villus vascular dilatation and congestion, and shedding of epithelial cells; Lung congestion, mast cells degranulation; hepatic focal inflammatory cells infiltration etc. The above results are similar to human disease. Conclusions Through the establishment of heat syndrome body in rabbits, with continuous antigenic stimulation, vein and intradermal antigen impact, we found that their symptom, pathology and auxiliary examination results are similar to human allergic purpura lesions. As a result, a more appropriate rabbit model of allergic purpura. is expected to be established in the future study.

Abstract:Objective:To determine the contents of the amino acid neurotransmitters in insomnia rats’ brain tissue. Methods: Copying the drug-induced insomnia rats model,platform in water environment-induced rats model and stimulation-induced rats model,using the HPLC-FD as the testing tool,Agilent ZORBAX SB-Aq (250mm x 4.6mm, 5μm) as chromatographic column which temperature was 25℃,excitation wavelength was 357nm,emission wavelength was 455nm,using methanol-sodium acetate buffer(50mmol/L,pH 6.5) as mobile phase and adopting gradient elution way,to establish the determination methods of the glutamate(Glu), glycine(Gly), γ-aminobutyric(γ-GABA) and taurine (Tau) in the control group rats and the three kinds of insomnia model rats. Results: The standard curve of Glu,Gly,γ-GABA and Tau were linear in 10.06~0.0503,10.13~0.0506,10.05~0.0502,10.03~0.0501μg/mL (the correlation coefficient were 0.99995,0.99995,0.99985 and 0.99990).Contents of Glu,Gly,γ-GABA and Tau in drug-induced insomnic rats were 0.2042?.0145,0.0086?.0005,0.0919?.0024,0.0421?.0011μg. Contents of Glu,Gly,γ-GABA and Tau in platform in water environment-induced insomnic rats were 0.2144?.0159,0.0085?.0004,0.0966?.0035,0.0433?.0012μg.Contents of Glu,Gly,γ-GABA and Tau in stimulation-induced insomnic rats were 0.1818?.0043,0.0084?.0005, 0.0824?.0033,0.0414?.0018μg.While Contents of Glu,Gly,γ-GABA and Tau in control group rats were 0.1744?.0038,0.0085?.0004,0.0791?.0022, 0.0406?.0012μg,respectively n=8.Conclusions:This method can satisfy the demands of the simultaneous determination of Glu,Gly,Tau and γ-GABA in rats’ brain tissue. There may be exist certain relationship among Glu, Tau , γ-GABA and insomnia,the three kinds of insomnia animal model can reflect the changes in amino acid neurotransmitters in the brain well。

Abstract:Abstract: Aim To set up primary dysmenorrheal model in mice with progynova and oxytocin; Methods Administrated different doses of progynova in mice continuously and after last dosage peritoneal injected oxytocin to induce writhing response, recorded the writhing latency period and frequency. Results: The optimum dose of progynova was 0.5 mg?kg-1and oxytocin 2U.1hour after administrated progynova was the optimal time to observe writhing response. From the third day writhing frequency begin to increase and maintain high level from the fourth day. Conclusion: progynova combined with oxytocin could build primary dysmenorrheal model.

Abstract:Objective G-protein-coupled receptor kinase 4 (GRK4) negatively regulates sodium balance in the proximal tubule of the kidney by desensitizing the dopamine-1-receptor. It is reported that mutation of GRK4 gene has been linked to impaired natriuresis and hypertension. In the current paper, we establish GRK4γ wild-type and three GRK4γvariant transgenic mice and analyze dynamic blood pressure using this transgenic mouse model. Methods The transgenic vector was constructed by inserting the human GRK4γ wild-type, GRK4γR65L, GRK4γA142V and GRK4γA486V gene into the down steam of chickenβ-actin promoter respectively. The transgenic mice were created by the method of microinjection. The genotype of transgenic line was identified by PCR and the expression level of target gene was determined by Western blot. Dynamic blood pressure was measured by noninvasive blood pressure meter. Results Each line of human wild-type, GRK4γR65L, GRK4γA142V and GRK4γA486V C57BL/6J transgenic mice with high levels of GRK4 expression in heart, kidney and adrenal gland tissues was established. Mice overexpressing the human GRK4γ wild-type and GRK4γR65L gene are normotensive, whereas mice overexpressing the human GRK4γA142V are hypertensive even on a normal NaCl intake. In contrast, human GRK4γA484V transgenic mice become hypertensive only after an increase in sodium intake. Conclusion GRK4γA142V transgenic mice could be an animal model for spontaneous hypertension, whereas GRK4γA486V transgenic mice could be an animal model for salt-sensitive hypertension.

Abstract:In order to apply the animal models for the study of etiopathogenisis, pathogenesis and therapy of endometriosis(EM),this article summarize various kinds of animal models through the choice of animals ,the methods of establishing the traditional and the special animal models and analyzing the advantages and disadvantages of them.It will be beneficial for the study of endometriosis model.

Abstract:Although the manned space flight has made great progress, but the problems that the astronauts adapt to weightlessness on the outerspace and the readjustment after their return to the earth, has not to conquer no matter in theory or in practice. The microgravity flight syndrome occurrence mechanism and the countermeasure remain an important task of aerospace medicine. Nowadays, it is unable to create long-term weightlessness environment on the ground, while we can realize the ground-based simulated weightlessness experiments according to the physiological effects of microgravity on the body. This paper summarizes the ground-based simulation techniques on microgravity in human and animals, in order to provide references on the researches of ground-based simulated microgravity.

Abstract:Objective：to study the pathogenesis of autoimmune disease, transgenic zebraifsh overexpressing human BAFF is constructed . Method：855bp BAFF cDNA was cloned by RT-PCR from human lymphoma cell line to construct the BAFF overexpression recombinant plasmid Tol2-hBAFF. The protein expression in Hela cell-line was detected by western blot after invitro transfection. The zebrafish embryo was injected with recombinant BAFF plasmids and screened by GFP tracing. The GFP positive embryos were selected to analyze the early immune-related genes expression by qPCR. Result: BAFF-GFP fusion protein was successfully expressed, which proved an effective method to produce the transgene zebrafish for future study. Besides, qPCR results demonstrated that TCRAC signficantly high expressed in 1dpf transgenic embryos while the Ikaros gene was down-regulated. It suggests that BAFF overexpression in zebrafish embryo may reslult in immuno-related gene premature expression in early lymphatic system. All this results proved that transgenic zebrafish can be obtained by microinjection of the Tol2-hBAFF recombinant plasmids in zebrafish embryo, which supply an effective method to explore the human SLE disease and its antagonist on animal models.

Abstract:[Abstract]Objective To make the childhood simple obesity animal model by adjusting the litter size of offspring and compare with the obesity model by high fat diet.Methods After 48 female KM mice farrowing, the litter size of half of them remain 14-16, the other adjust to 6 (3 male and 3 female).Pups were fed with basic diet and high fat at 4 or 9 week old and continued to the fifteen week.Weight, body length, waist circumference, weight of genital organ, fat weight including perirenal fat and genital organ fat were weighed and the ratio of body fat were calculated.Results ①There was significantly difference in the female or male body weight between BD1 and BD2 group feeding the basic diet for 15 weeks (P<0.05), and the body weight of BD2 were over 26.3%(female), 20%(male). The ratio of body fat of female in each group were higher than male’s under the same treatment methods.② No matter when we changed the diet or whether we adjusted the litter size or not, the body weight of newborn rats feeding HFD, both male and female, were higher than BD1 group (all P<0.05).Female and male offspring rats body weight in HFD4 group had significant higher than BD2 group (all P<0.05).Conclusion Childhood simple obesity animal model could be successfully prepared by adjusting the litter size.They began to intake high fat diet at adult stage,who got childhood simple obesity,would make the body store more fat.

Abstract:[Abstract] Objective To establish drug screening model with primary cultured prostatic epithelial cells from rats in vitro. Methods The ventral prostate was freed from adult SD rat under sterile conditions and weighted, then cut into 1mm3 slices, after being digested with collagenase 2 for 1 h, the prostate cells were harvested through filtering and centrifuging. All cells were seeded into 24-well plate and cultured for cell morphology and immunohistochemistry detection. The prostatic epithelial cells were seeded respectively into 96-well plate and 24-well plate, treated with epristeride （0.1-1000µM）and tamoxifen （0.1-1000µM）for 72 h after being cultured for 12 d. The prostatic epithelial cell survival rate was detected by CCK-8 method, and the IC50 value of epristeride and tamoxifen was calculated, then further observed their effects on the growth and cell morphology of prostatic epithelial cells. Results According to cell morphology and immunohistochemical detection result, all cells from rat prostate displayed typical epithelial cell shape, and their expressions of cytokeratin antibody and prostate specific antigen were positive, such results suggested that the harvested cells were prostatic epithelial cell. After the prostatic epithelial cell being treated with epristeride for 72 h, its growth significantly been inhibited, and the IC50 value of epristeride was 42.7µM. Cell morphology results further showed that epristeride didn’t significantly changed the shape of prostatic epithelial cell, but it could reduce the survival prostatic epithelial cells number. While tamoxifen didn’t inhibit the cell growth, and had no obvious effect on cell number and cell shape. Conclusion Benign prostatic hyperplasia drug screening model could be successfully establish with primary cultured prostatic epithelial cells from rats in vitro.

Abstract:Making reference to the priciple of data classification in“the Common Description Criteria of laboratory animal resources”issued by China, the article classifys laboratory animals according to five kinds of data of biological characteristics including essential information，genetic data，physiological data，biochemical data and anatomical data, aiming at exploring a set of flexible and extendible method of classification and coding of laboratory animal's biological characteristics by using the structure of administrative level, which plays a major role in saving, sharing and managing data resources of laboratory animal.

Abstract:Objective By using loop-mediated isothermal amplification (LAMP) assay, to found the fast, convenient, sensitive and specific method of enterohaemorrhagic escherichia coli (ETEC), and evaluating the specificity and sensitivity of the method, for detection and diagnosis of bacterial diarrhea in laboratory animals. Methods According to the published LT toxin gene sequences (S60731.1) to design PCR and LAMP primers, the LAMP specificity and sensitivity compared with PCR. Results the LAMP method established detection limit was 100 pg, sensitivity is more 10 times than PCR and has the high specificity. Using PCR and LAMP to detect 27 feces samples of monkey diarrhea, we had the same results of LAMP (within 60 minutes) and PCR. And LAMP(within 90 min) has more sensitive than PCR. Conclusion we had established the ETEC LAMP detection method, this method is strong specificity, high sensitivity, fast and convenient, suitable for rapid detection of ETEC clinical.

Abstract:【Abstract】 Objective To establish an effective way for intratracheal siRNA delivery to the mouse airway. Methods Female BALB/c mice were used to establish asthma model by egg albumin hypersensitivity. Asthma mice were then randomly divided into control group and siRNA group. mice in siRNA group were intratracheally adminstrated with NF-κB p50 siRNA (20uM，100ul/per mouse) by aerosolizer while mice in control group were administrated negative control siRNA.after administration, lung tissue were collected for analysis of NF-κB expression by Real time PCR. Results The expression of NF-κB gene was reduced by 1.743 fold after intratracheal aerosol administration of NF-κB siRNA compared to control group（P=0.0035）. Conclusion Intratracheal aerosol administration of siRNA was an effective method to inhibit gene expression in mice airway.

Abstract:Objective:To establish type 1 diabetes model in transgenic mice expressing human TCRα gene for investigating the pathogenesis of type 1 diabetes. Methods: 20 TCR transgenic mice were randomly divided into two groups. One was experimental group induced by intraperitoneal injection of STZ (100 mg/kg/times), the other was control group injected with the same amount of saline. Two groups were injected two times with an interval of 48h. The blood glucose levels and weight were detected weekly. The mice were killed when they had significant clinic appearance or 8 weeks after STZ injection, followed by pathologic studies and serum insulin，cytokine assays. Results:The incidence of experimental group was 10/10 whereas that of control group was 0/10. The level of CD3 ,CD4 ,CD8 ,the relation between CD4 and CD8 T-cells were changed significantly between experimental group and control group. The amounts of insulin，IFN-γ，TFN-? were changed significantly between experimental group and control group. The amount of IL-2 of experimental group was higher than control group, but there was no significant difference. Conclusion:Model mice of type 1 diabetes can be established in transgenic mice expressing human TCRα gene during the 2~ 8 week after i.p injection with STZ.

Abstract:Objectives: To establish the cultured protocol on Mongolian Gerbil hepatocytes. Method: The hepatocytes were isolated from male Mongolian Gerbil by the methods of tissue digestion and collagenase perfusion. The yield and viability were determined by trypan blue exclusion. The isolated hepatocytes were identified by Periodic acid-Schiff reaction(PAS). The morphology was evaluated with using microscope. Then the medium was repalced with various kinds of cytokines. Results: In sequence of tissue digestion, collagenase perfusion in situ, the hepatocyte’s yield per Mongolian Gerbil was respectively (1.33?.34)?07, (3.97?.15)?07; survival ratio was respectively (29.4?.05)%, (80.3?.56)%. Significant differences were revealed in the yield and viability between the two methods. Numerous glycogenosomes were found in cultured hepatocytes and revealed redness by PAS staining. Significant changes were discovered on morphology after the hepatocytes inoculated in 72 hours. Conclusions: The collagenase perfusion isolation method was economical and high efficiency for separating Mongolian Gerbil hepatocytes. The presence of cytokines in medium could promote the Mongolian Gerbil hepatocyte growth and maintain its function. Successfully establishing the primary cultured protocol of Mongolian Gerbil hepatocytes and it could be used for the reseach on the liver disease and drug exploitation in the future.

Abstract:Abstract: objective To amplify the xanthine dehydrogenase/oxidase (XDH/XO) gene fragment from liver tissue of rhesus monkey for further research． Metheds Total RNA was extracted from the liver tissue for amplification of XDH/XO gene fragment by two-step RT-PCR. The sequence of amplified nucleotide acids was analyzed by Multiple alignment of DNAMAN software after gel electrophoresis and sequencing. The sequence were comparised with the other species of Homo sapiens, Mus musculus, Rattus norvegicus, Sus scrofa from gene bank．Sequencing results by DNAMAN software predicted amino acid sequence. Conserved domains and protein three-dimensional structure of XDH/XO was analysised by tools of InterProScan and SWISS-MODEL. Results The expected 683bp fragment was obtained by two-step RT-PCR. This nucleotide acide was predicted that it encoded an open reading frame with 158 amino acids by DNAMAN software. The amplified fragment sequence comparison with the XDH/XO gene showed nucleotide homologies of 95.6% with Homo sapiens（U39487.1），85.2% with Mus musculus（NM0111723.2），84.3% with Rattus norvegicus（NM 017154.4）， 86.1% with Sus scrofa（JN896312.1）in GenBank. It has more similarity within difference species. The bioinformatic analysis showed that XDH/XO-encoding protein contained Aldehyde oxidase/xanthine dehydrogenase molybdopterin binding domain、Xanthine Dehydrogenase domain. Conclusion The XDH/XO gene fragment has been successfully amplified from liver tissue of rhesus monkey. It lay the foundation for further studies on pathogenic mechanism of hyperuricemia, on new effective medicine to treat hyperuricemia

Abstract:Objective This work is to construct a recombinant adeno-associated virus (rAAV) vector containing rat leptin gene, which mediates transduction of leptin into neurons in rat primary neuronal culture. Methods The leptin cDNA was obtained from rat adipose tissue by RT-PCR, cloned into pGEM-T vector, confirmed by sequencing, and then transferred into an AAV2 vector pTR-UF22 that carries CBA promotor. This vector, designated as pAAV2-CBA—Leptin, was packaged in human embryonic kidney (HEK) 293 cells using pDG as helper plasmid and purified with a single-step gravity-flow column. Copies of viral genome DNA were determined by quantitative PCR. Vector doses were expressed as viral genome copies (vg). The viral vectors AAV2-CBA-Leptin and AAV2-CBA-EGFP were transduced into primary cerebral cortical neuronal culture, and leptin expression determined using Western blotting. The cellular localization of EGFP in neuronal cultures was determined by immunocytochemistry. Results Restriction enzyme digestion and sequencing results demonstrate successful cloning of the amplified leptin cDNA into pGEM-T vector and subsequent subcloning into pTR-UF22. Incubation of cerebral cortical neuronal cultures with AAV2-CBA-leptin produced dose-dependent increases in leptin protein expression as compared to the background control with AAV2-CBA-EGFP. The AAV2-CBA-EGFP generated a high level of EGFP expression that colocalized with neuron-specific immunolabeling of NeuN, indicating a specificity of neuronal transduction by the vector. Conclusion We have developed a recombinant viral vector that efficiently transduces leptin into neurons. This vector offers a valuable bio-tool for studies of leptin CNS function in the control of body weight and diabetes.

Abstract:Objective To investigate the morphological changes in the ovaries and the serum levels of several related hormones, insulin and glucose in the polycystic ovary syndrome ( PCOS) mouse model induced by D-galactose, and to discuss the significance. Methods 20-week-old female ICR mice were injected daily (i. p) with D-galactose for up to 8 weeks. ELISA was used to measure the serum levels of follicle-stimulating hormone (FSH), estrogen (E2), testosterone (T) and insulin (INS). HE staining was used to examine the morphological changes of the ovaries. The stages of the estrous cycle were verified by vaginal cytology. Results The ovaries of the D-galactose-treated mice showed almost polycystic changes with ovary cysts and atretic follicles. Abnormal estrous cycles were induced in D-galactose-treated mice. Comparing with the normal group, the serum estrogen (E2) and testosterone (T) were increased and the serum FSH was reduced in D-galactose group, with a statistically significant difference (P <0.001 or P<0.0001). The level of serum insulin was significantly higher in the D-galactose-treated mice than in the normal group (P < 0.01). In comparison with the normal group, the value of 1/ (INS×G) was significantly decreased in the D-galactose-treated mice (P < 0.05). Conclusions The level of serum sex hormones and the ovarian morphology of the D-galactose-treated mice were similar to those of the patients with PCOS, and insulin resistance was observed. Our results indicate that the D-galactose-treated mouse model may be a useful animal model for the future experimental research of polycystic ovary syndrome.

Abstract:Objective To detect microsatellite instability(MSI) in transgenic(Tg) mice and spontaneously mutated (SM) inbred mice. MethodsAccording to the references, we selected198 microsatellite loci from 19 autosomes and X chromosomes of mice in GenBank. These loci are rich in polymorphic of mice. Then the comparative analysisof microsatellite DNA polymorphism in 6 Tg strain and 5 SM strain of mice and wild-typecontrol mice was conducted through PCR amplified. The analysis of microsatellite DNA polymorphism was undergone through 1.5% agarose gel electrophoresis and STR scanning. Results We found 40 microsatellite loci showed MSIafter the genetic modification compared to the wild type control mice.In the spontaneously mutated mice, 55.6% (10/18) changed from homozygous to heterozygous (type I), whereas three loci (16.6%) of these mice experienced homozygous mutations (type II). Five loci (27.8%) had both types of mutations. In the Tg animals, however, most of the CMP genotypes (87.5%, 28/32) were changed from homozygous to heterozygous mutations (type I), whereas only two loci (6.2%) of the micepossessed homozygous mutations (type II). Moreover, two loci harbored both types of mutations. Conclusion We primarily confirmed that gene-modify can result in MSI. Some microsatellite loci are more sensitive to genetically modification

Abstract:AIM To Explore the relationship between vitamin D3 and inflammation of hypertension. METHOD Twenty spontaneous hypertensive rats were randomly divided into two groups. The rats in experimental group received vitamin D3 3μg/kg treatment twice a week for three months; and the rats in control group were injected with propylene glycol. Blood pressure of rats were measured once a week during the experiment period.Serum 25 (OH) D3, calcium, IL-6,MMP-9 concentration were detected by Enzyme-linked immunosorbent. The kidney / body weight ratio and heart / body weight ratio were calculated. Kidney, heart and artery were done the biopsy for HE staining, observation of the rats pathological change. RESULT Blood pressure between the experimental group and control group before the intervention was no significant difference (P＞0.05).Three months later, the mean systolic blood pressure in the experimental group was (157 ?9) mmHg, which was(173 ?8) mmHg in control rats, difference was significant (P＜0.05). 25 (OH) D3 concentration was higher in experimental group than that in the control group (P＜0.05), IL-6, MMP-9 in the experimental group were lower than that in the control group (P＜0.05).The heart / body weight ratio in the experimental group was less than that in control group (P＜0.05). Biopsy showed that there was significant hypertensive inflammation performance with the kidney, heart and aorta of the control group, while the experimental group was much better than the control group . CONCLUSION The law of vitamin D3 medication could regulate the blood pressure by suppressing inflammatory factors IL-6 and MMP-9 and inhibiting inflammatory response.