Objective To screen genotypes of small body in Tibet minipigs by single nucleotide polymorphism analysis (SNP) of growth hormone (GH) gene.Methods The PCR products are sequenced.According to the results, SNP analysis are applied in GH gene 5′fragment of 108 Tibet minipigs .Results Five mutation points were found by comparison.The polymorphism information content of the T45C locus is highest.The results of growth traits of different genotypes showed that the abdomen of 6~8 month old Tibet minipigs of TC genotype at T 45C mutation site is small; the weight and height of 3~5 month old Tibet minipigs of AA genotype at G 84A mutation site is small; the body length and height of 6~8month old Tibet minipigs of GG genotype at G 93A mutation site is small.Conclusion The AA TC, GG genotypes of T45C, G84A, G93A mutation sites in GH gene may be associated with small body .We also found that genetic mutations always occur in the above sites with high heterozygosity and genetic diversity , which can provide rich material for breeding research .

Objective To observe the therapeutic effect of acupuncture therapy for the rat model of type 2 diabetes mellitus (T2DM) combined with renal hypertension and try to explore its mechanism. Methods We randomly select 10 Wistar rats as the blank group and 40 rats were used to make the model groups, which were divided into simple diabetes group, simple renal hypertension group, the compound group with electroacupuncture and the compound group without elec?troacupuncture, with 10 rats in each group. After a high fat and sugar diet for 4 weeks, the Wistar rats were given strepto?zotocin i. p. injection to establish models of type 2 diabetes mellitus. Renal hypertension was developed by the“2K1C” im?proved method to make unilateral renal artery ligation?induced renal artery stenosis. Then, electroacupuncture treatment was performed on the rats for 2 weeks except the compound group without electroacupuncture. The changes of values of BP, FBG, Cr, BUN, glycated hemoglobin, renin and Ang II were recorded and analyzed. Results The values of BP, FBG, Cr, BUN, glycated hemoglobin, renin and Ang II in the compound group with electroacupuncture showed a significant re?duction compared with the compound group without electroacupuncture after 2 weeks (P<0?01), but there was no obvious changes in the values of Cr and BUN(P>0?05). Conclusions The blood glucose and blood pressure in the rat model of compound group can be reduced to a normal level with continuous electroacupuncture at bilateral acupoints Zusanli, and it can also be kept at a stable level after single electroacupuncture for 2-3 days. The acupuncture therapy is more suitable for early clinical treatment and can be used in basic research with advantages of economic, safe, no side effect and so on.

Objective To detect and analyze the HBV?related indexes in the urine of HBV transgenic mice and further understand the biological characteristics of transgenic mice, and to clarify the tissue sources of HBV?related indexes. Methods HBV?related indexes in the urine of transgenic mice were tested using enzyme?linked immune sorbent assay ( ELISA ) and fluorescence quantitative PCR ( real?time RCR ) . The tissue sources were confirmed by several experiments, i. e. hydrodynamic transfection of mice, RNA interference to inhibit HBV?expression in the transgenic mice, and to infect normal mice with HBV?positive serum from patients. Results Expression of HBsAg, HBeAg and HBV?DNA was present in the urine of transgenic mice, of which the HBsAg expression level was high (6674 ± 619?8 IU/mL), but lower than that in the serum (16470 ± 2704 IU/mL). The level of HBsAg expression in the urine of male mice was higher than that in female mice. The level of HBeAg expression in the urine was lower and the HBeAg positive rate of urine was higher than that of blood, and the levels of HBeAg expression showed significant inter?individual and inter?sexual differences. HBV?DNA level reached 103 -105 copy/mL in the urine, but no related antibody expression was detected. The experiments such as hydrodynamic infection test indicated that the HBV?related indexes in the urine are derived from replication in the kidneys rather than secreted from the liver, entered into the blood circulation, and discharged from the urine. The kidneys are an independent expression site of HBV. Conclusions The expression of HBV?related indexes is present in the urine of transgenic mice and it is a long?term expression along with the age in months, of which the expression levels of HBsAg and HBV?DNA are rather high and stable. HBsAg titer in the urine of the male mice is higher than that of female mice. HBeAg expression level in the male mice is more stable compared with that in female mice. No expressions of various kinds of antibodies have been found in the urine. The kidneys are an independent expression site of HBV.

Objective To simulate the process of hypoxic?ischemic brain injury at high altitude in a simulated cabin with plateau low pressure environment, and to prepare a rat model of cerebral injuries at different high altitudes. Method Thirty?two 0?day?old neonatal SD rats were divided into four groups, namely group A ( control group) and three test groups:group B (2000 m group), group C (4000 m group), and group D (6000 m group). The rats of control group were reared in a barrier environment. The rats of test groups were placed in a simulated cabin with plateau low pressure environment, and to prepare neonatal cerebral ischemia?hypoxia model by sport activities. The sport movements were carried out in the cabin in a swimming groove 60 min/d, and not less than 20 hours a day at high altitude low pressure environment. Zea Longa 5 point scale standard was used to determine the behavioral scores at the 3 th 7 th 11 th 15 th days, and samples were collected on the 15th day to observe red blood cell morphology using HE and 2, 3, 5?triphenyltetrazolium chloride ( TTC ) staining and ultrastructure by scanning electron microscopy. Result ( 1 ) The neurological scores of the test groups A, B, C were significantly different from that of the control group (P<0?05), and the scores of test group D and control group were very significantly different ( P <0?01 ) . ( 2 ) The histopathological examination using HE staining showed inflammatory cell infiltration in all rats of the test groups, and the extent of inflammatory cell infiltration was positively correlated with the increase of altitude. ( 3 ) The histopathology with TTC staining revealed prominent ischemia in the cerebral cortex of rats in the plateau hypoxic environment. ( 4 ) Scanning electron microscopy showed that the rat erythrocytes were cap?like in the group B, irregular in the group C, and zigzag shape in the group D. Conclusions In this study, a rat model of neonatal hypoxic?ischemic encephalopathy ( HIE) is successfully established by hypoxic cabin combined with sport activity. This model is stable, reliable, more closely mimicking the pathogenesis and clinical manifestation of neonatal HIE than models prepared with other methods, therefore, may be used in related research.

Comparative medicine is a new crossing frontier branch subject based on laboratory animal science,and the content,aim and request of its research have been remarkably different from that of laboratory animal science.Acting as the methods,means and support conditions of advancment and development of medicine research,comparative medicine is an important platform ensuring the sustainable development of medicine,and is imperative for the medico to be educated with systemic comparative medicine.

Objective To provide original reference data for oral ecosystem research, Tibet minipigs, beagle dogs, rhesus monkey, New Zealand white rabbits and Wistar rats were selected to study their respective characteristics of oral microbial mmunities and compared with normal data of humans.Methods Total DNA was extracted from the specimens of oral microbial communities of Tibet minipigs, beagle dogs, rhesus monkey, New Zealand white rabbits and Wistar rats, and used to amplify 16S rRNA V4 fragments with labeled universal primers.The diversity and structure of microbial communities from those animals were compared with that of humans using BIPES and QIIME analysis after Illumina sequencing of 16S rRNA V4 fragments.Results The richness of the oral microbial communities of humans and the five species of laboratory animals was significantly different (P <0.05).Different species of animals have their own unique oral flora, among which the oral flora of the monkey is the most similar to that of humans.Conclusions Among the five species of laboratory animals, the oral microbial communities of rhesus monkeys and humans have highest similarity. Specifically, the Fusobacterium and Porphyromonas levels of rhesus monkeys is most similar to those of humans.Our findings indicate that rhesus monkeys may be suitable animal model for studies of human oral microbial communities.Tibet minipigs may be suitable animal model for Proteobacteria studies, while beagle dogs may be appropriate for modeling of diseases related to Spirochaetes.

Objective To analyze the Cyt b gene sequences in Tibet mini-pigs and clarify the differences and genetic relationship with other Chinese pigs.Method The sequence of Cyt b gene was amplified from genome DNA of Tibet mini-pig,Bama miniature pigs,Guizhou xiang pigs and Wuzhishan (WZS) pigs.After sequencing,the base sequences were compared and analysed.The blood relationship tree and evolution position of Tibet mini-pig were established.Result There were 14 mutation sites between domestic pigs in China and pigs from Europe.Besides there was a significant differenee in two nucleotide site:a T→C switch in site 420 and the G→A switch in site 883 at the same time.Conclusion Chinese pigs include Bama miniature pigs,Guizhou xiang pigs and WZS pigs,have a very close blood relationship with some of Tibet mini-pigs.It has been confirmed that there is a certain genetic differentiation in the Tibet mini-pig.

Objective To transfect EGFP gene to porcine embryonic fibroblasts ( PEFs) of Tibetan miniature pigs by Lonza Nucleofector II machine and compare the tansfection efficiency between this method and the lipofection method. Method A plasmid carrying green fluorescent protein ( GFP) was transfected into PEFs of Tibetan miniature pigs via the Lonza Nucleofector II machine ( program U020) and by Lipofectamine 2000.Results 5 hours after nucleofection, green fluorescence was observed, indicating 80%transfecting efficiency in the nucleofection group, which is significantly higher than the lipofection group. Conclusion Nucleofector II machine can efficiently transfect PEFs, provides a reliable method for efficiently generate transgenic Tibetan minipigs.

Objective To evaluate the efficacy, safety and compatibility of a new type of atrial septal defect ( ASD) occluder in atrial spetal defect mini-pigs model.Methods Five Tibet mini-pigs were selected as the ASD models which were established by the combination of atrial septal puncture and balloon dilation.Then the new type occluder was implanted for the therapy of ASD.Transthoracic echocardiography with color Doppler was used in all animals during closure and in follow up examinations.The animals were killed at 3 months after occlusion for electron microscopical observation and microscopic examination.Result The ASD models had been created in five piglets successfully without complication, all of whom were implanted successfully with the new device without shunts.Postmortem and microscopic examination of the 5 specimens 3 months after placement showed complete.Conclusion Transcatheter ASD occlusion with new type ASD occluder is safe,feasible and effective.This occlusion can repair the atrial septal defect successfully.

Objective To amplify the encoding full-length sequence of Tibet mini-pigs myostatin ( MSTN) gene and analysis the sequence by bioinformatics software.Method The RNA of liver tissues from Tibet mini-pig was extracted, and reversely transcribed into cDNA.The gene coding region sequence of myostatin gene was amplified through RT-PCR, and then the purified product of PCR was ligated with a pMD19-T and transferred into the bacterium DH5αfor replication.The positive clones were screened and sequenced. The sequence characters were analyzed by using bioinformatics method and phylogeny evolution tree was constructed with other twelve species.Results The coding redion of MSTN gene was 1128bp, and coded 375 amino acids.The amino acid homology analysis showed that the homology rate of amino acid sequence was 99%.Conclusions Molecular phylogeny evolution indicated that it had a close relation with human, dog, banna minipig, sheep, goat, cattle, horse, chimpanzee, rat, mouse except chick and zebrafish, and the most closely related with banna minipig.