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.

AIM:To study the high-level HBV replication transgenic mice for evaluation of drugs treating hepatitis B virus.METHODS:The HBV transgenic mice were treated respectively with lamivudine,large dose recombinant hepatitis B protein vaccine,?-1b interferon,siRNA to evaluate their pharmacodynamics and mechanism of action.RESULTS:HBV DNA titre was reduced significantly in transgenic mice which were treated with lamivudine(100 mg?kg-1?d-1),recombinant hepatitis B protein vaccine(HBsAg 6 ?g/mouse),?-1b interferon(50 ?g /mouse),respectively.Recombinant hepatitis B protein vaccine and ?-1b interferon promoted the level of IL-2 and IFN-? and increased the Elispot number of spleen cells secreting IFN-? in the treated transgenic mice.HBV transgenic mice were treated with RNAi expression vector pU6-siHBV against HBV through vena caudalis by hydrodynamics technique.Five days later,the level of serum HBsAg was reduced by 56.7% and the inhibition lasted at least 14 days.The HbcAg(+)cells were decreased obviously by immunohistochemistry detection in liver tissue,but the RNAi did not reduce the serum HBV DNA titre.CONCLUSION:These inbreeding high-level HBV replication transgenic mice are reliable and feasible for evaluating the anti-HBV drugs and have its economical and convenient superiority.

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 investigate the effects of gene transfection with human growth/differentiation factor 5(GDF5)on the growth and difierentiation of bone nlarrow stromal stem cells (BMSCs).Methods GDF5 gene was trans fected into BMSCs by liposome method. Then cell proliferation and cycles were examined by MTT and flow cytometry respectively. Cell morphology was observed under light microscope and electron microscope (EM).GDF5 and Collagen Ⅱ were detected at the level of mRNA and protein by RT-PCR and immunocytochemistry. Alkaline phosphate activity was examined by lead citrate method. Osteocalcin mRNA expression was determined bv RT-PCR. ResulIs GDF5 gene was transfected into BMSCs successfully and the transfected cells still maintained their natural growth and proliferation features. Stable expression of GDF5 gene in BMSCs was obtained. The trans fected ceils had basically the same proliferation ability and cell cycles as the untransfccted ones. After transfection comparatively more polygonal cells could be seen in light microscope, showing irregular arrangement mode. Plenty organells were observed and cell nucleus showed irregular shape under EM. The expressions of Collagen Ⅱ mRNA and protein were positive. But osteocalcin mRNA expression was negative. Conclusion Since BMSCs can be induced by GDF5 to differentiate into chondrogenic cells. GDF5 gene-modified BMSCs may be used as candidate seed cells of cartilage tissue engineering.

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.

Klebsiella pneumoniae (KP) is a common conditional pathogen, and also one of the common pathogens causing infection in immunocompromised patients, with its infection rate increasing year by year. Carbapenem antibiotics are effective drugs to control KP infection. But with the widespread use of carbapenem antibiotics, carbapenemresistant Klebsiella pneumoniae (CRKP) appears and increases year by year. Organ transplant recipients are at high risk of CRKP infection due to the suppressed immune system. Once drug-resistant bacteria infection occurs, it is often difficult to control and the survival rate of transplant organs is reduced, which brings great challenges to clinical treatment. In this article, the current status and treatment progress of CRKP infection in organ transplantation are summarized.

OBJECTIVETo observe the pathological changes in the myocardial and pulmonary tissues in miniature pigs with chronic pulmonary hypertension induced by monocrotaline (MCT).

METHODSTwelve male miniature pigs (weigh 15.0-18.0 kg, aged 4.0-4.5 months) were examined for baseline mean pulmonary artery pressure (mPAP), followed by intraperitoneal injection of 10.0 mg/kg MCT in 10 randomly selected pigs. The mean pulmonary artery pressure at 4 and 8 weeks were determined, and the pathological changes in the myocardial and pulmonary tissues were observed.

RESULTSThe baseline mPAP of normal miniature pigs was 15.19∓0.70 mmHg. At 4 and 8 weeks after MCT injection, the sPAP and dPAP were 19.69∓2.47 mmHg and 25.62∓4.88 mmHg, respectively, and the mPAP increased significantly compared with that of the normal control group (P<0.01). Obvious pathological changes such as pulmonary hypertension and right ventricular hypertrophy were found in the pigs 4 weeks after MCT injection, and at 8 weeks, significant pathological changes occurred including right ventricular fibrosis and thickening of the tunica media of the pulmonary artery.

CONCLUSIONMCT can cause pulmonary hypertension in miniature pigs 8 weeks after drug administration, shown as increased pulmonary artery pressure and pulmonary vascular remodeling.

Animals ; Hypertension, Pulmonary ; chemically induced ; pathology ; Lung ; pathology ; Male ; Monocrotaline ; adverse effects ; Myocardium ; pathology ; Swine ; Swine, Miniature

OBJECTIVETo compare the efficiency of three different serotypes of adeno-associated virus (AAV) in mediating the transfection of enhanced green fluorescent protein (EGFP) in Tibet minipig fetal fibroblasts (PFFs).

METHODSThree recombinant AAV of different serotypes encoding EGFP were constructed and transfected into primary cultured PFFs at the multiplicity of infection (MOI) ranging from 10(3) to 10(5). The expression rates of EGFP in the PFFs were assessed 72 h after the infection by flow cytometry, and the transfected PFFs were observed under inverted fluorescence microscope. The toxicity of AAVs to PFFs was analyzed using MTT assay.

RESULTSThe transfection efficiency of AAV2-EGFP increased with MOI. At the MOI of 10(3), the transfection efficiency of AAV2-EGFP was (33.68∓1.18)%, which increased to (50.80∓2.59)% at the MOI of 10(4) but without obvious further increase at the MOI of 10(5). The other two serotypes of the virus (AAV8 and AAV9) showed no obvious changes in the infection efficiency at any MOIs. The transfection efficiency of AAV8 was (8.3∓0.02)% and that of AAV9 was (2.20∓1.02)% at the MOI of 10(5). Transfection with the 3 viruses caused no adverse effects on the normal cell growth of the PFFs.

CONCLUSIONSAAV2 has a significantly higher infection rate in cultured PFFs than AAV8 and AAV9, and the latter two have a rather low infection efficiency. All the three AAVs have no cell toxicity to the PFFs.

Animals ; Animals, Genetically Modified ; Cell Line ; Dependovirus ; classification ; genetics ; Fibroblasts ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Swine ; Swine, Miniature ; Transfection