Abstract:Objective To clone partial env genes of porcine endogenous retrovirus (PERV) isolated from Guangxi Bama minipigs and study their expression differences in different organ tissues.Method The PERV-env genes from peripheral blood leukocytes of Bama minipigs were amplified by RT-PCR, and their homology and phylogenetic analysis were compared with those of some other PERV-env genes published home and abroad. The PERV-env genes obtained by amplification from Bama minipig were used as templates to design primers for analysis on the expressions of PERV mRNA in different tissues of the Bama minipig with a semi-quantitative RT-PCR analysis.Result Differences in the expression levels of PERV mRNA were detected in nine organ tissues of Bama minipig, in which the abundance of PERV mRNA in kidney and peripheral blood lymphocytes was the highest, followed by that in lung, heart, liver, spleen, thymus and ovary, but the expression differences among the six organs were not significant (P>0.05), while the expression abundance of PERV mRNA in pancreas was the lowest.Conclusions The potential risk of PERV infection in using the islet cells of Bama mini pig for xenotransplantation is probably lower than using the other organs. However, further research is still needed.

Abstract:Objective To investigate the mechanism of trace element strontium on alleviating non-alcoholic fatty liver disease (NAFLD) in rats.Methods Fifty SD rats were randomly divided into five groups. The control group was fed with ordinary diet and the other four groups were fed with a high fat diet. From the 6th week, rats in the strontium 18 mg/L and 36 mg/L groups were fed with water with strontium in concentration of 18 mg/L and 36 mg/L respectively. These two groups were separately given strontium water (3 mL/kg b.w.) by gavage from the 11th week, while the Simvastatin group was given simvastatin (10 mg/kg b.w.) by gavage from the 11th week. Rats in other groups were given matching normal saline by gavage at the same period. The rats were killed and the TG, TC, LDL-C levels in their serum and the TG, TC levels in the liver were detected at the end of the 14th week. The lipid accumulation in the liver tissue was observed using oil red O staining. The protein expression levels of GRP78, SREBP2, HMGCR and LDLr in the liver tissue were assessed with immunohistochemical staining.Results The levels of serum TC, LDL-C and liver TC, TG in the NAFLD group were significantly higher than that of the control group (P<0.05). The levels of serum TC, LDL-C and liver TC, TG in the strontium 36 mg/L group were significantly lower than that of the NAFLD group (P<0.05). ORO staining showed that lipid accumulation in liver increased abnormally in the NAFLD group compared with the control group, while the lipids accumulation in the liver decreased obviously in the strontium 18 mg/L group, 36 mg/L group and simvastatin group compared with the NAFLD group to a different degree. Immunohistochemical staining showed that the protein expression levels of GRP78, SREBP2, HMGCR and LDLr in the NAFLD group, those of GRP78, SREBP2 and LDLr in the strontium 18 mg/L group, those of LDLr in the strontium 36 mg/L group and those of SREBP2 and LDLr in the Simvastation group significantly increased compared with those of the control group (P<0.05). The protein expression levels of GRP78, SREBP2 and HMGCR in the strontium 36 mg/L group decreased obviously compared with those of the NAFLD group, while those of LDLr in the strontium 36 mg/L group increased compared with those of the NAFLD group (P<0.05). The protein expression levels of GRP78 and HMGCR in the simvastatin group decreased compared with the NAFLD group(P<0.05). The protein expression levels of GRP78, SREBP2, HMGCR and LDLr in the strontium 18 mg/L group had no significant difference compared with the NAFLD group (P>0.05).Conclusions Intake of trace element strontium at high concentration for long time can alleviate the disorder of lipid metabolism and non-alcoholic fatty acid liver disease (NAFLD) in rats. Its mechanism is probably related to its adjusting of endoplasmic reticulum stress, the activity of HMGCR and the function of LDLr.

Abstract:Objective To investigate the modulatory effects of mouse spleen-derived mesenchymal stem cells (Sp-MSCs) on the proliferation and activation of T lymphocytes.Methods The mouse Sp-MSCs were isolated from mouse spleens by using explant tissue culture protocol and the cells were induced to differentiate into osteoblasts and adipocytes. The effects of Sp-MSCs on proliferation of activated T lymphocytes were determined by carboxyfluorescein succinimidyl ester (CFSE) staining assay. Furthermore, the effects of Sp-MSCs on lymphocyte transformation test (LTT) and mixed lymphocytes reaction (MLR) were observed, respectively. In addition, the mRNA expression of T cell derived proinflammatory cytokines by Sp-MSCs was detected by quantitative PCR.Results The results of CFSE assay demonstrated that Sp-MSCs suppressed ConA and allogeneic lymphocyte-induced T cell proliferation. Moreover, the Sp-MSCs were capable of suppressing LLT and MLR. Additionally, the results of quantitative PCR revealed that Sp-MSCs significantly suppressed the T cells expressing interferon-γ, tumor necrosis factor-α and interleukine-17A.Conclusions Sp-MSCs are capable of suppressing the proliferation and activation of T lymphocytes as well as the expression of T cell-derived proinflammatory cytokines.

Abstract:Objective To establish a new orthotopic gastric cancer model in nude mice.Methods The BALB/c nude mice were anesthesized by Matrx VIP 3000 type gas anesthesia systems. Orthotopic gastric cancer models were established by embedding method, thread-ligation method or gastric bursa method in the nude mice. The tumor growth after transplantation in living body was detected by a Caliper IVIS Kinetic small animal imaging system. Samples of the transplanted tumor tissues for histological examination were taken at 28 days after transplantation.Results The orthotopic gastric cancer model was successfully established by embedding method. Compared with the models prepared by other commonly used methods, it showed obvious advantages such as a high success rate of transplantation, simple operation, short time, less difficulty, and less direct contact of the implanted tumor tissue with other organs, etc.Conclusions Large amount of mouse models of orthotopic gastric cancer can be prepared rapidly by the embedding method, thus, provide a convenient tool for related research of gastric cancer in future.

Abstract:Objective To conduct an epidemiological investigation of several pathogens in zebrafish, and to provide the basis for subsequent development of relevant standards.Methods This study mainly used traditional methods based on morphological, physiological and biochemical characteristics of bacteria, combined with molecular biology methods, to isolate bacteria and artificially infect healthy zebrafish with the bacteria. Through investigating the incidence and mortality in the experimental group, we distinguished the virulent effect of the pathogens, and further understanding the pathogens affecting the health of zebrafish.Results There were three kinds of pathogens identified in all sampling institutions, including pathogenic Aeromonas hydrophila, Aeromonas sobria and Pseudomonas aeruginosa. Aeromonas hydrophila was detected in most sampling institutions, and showed to possess strong virulence. Aeromonas sobria and Pseudomonas aeruginosa were detected in some sampling institutions, and the virulence of Aeromonas sobria was revealed to be weaker than that of Aeromonas hydrophila. The virulence of Pseudomonas aeruginosa was the weakest among these three bacteria, did not induce typical clinical symptoms and death in the zebrafish.Conclusions Aeromonas hydrophila may cause contamination of fish experimental facilities, and it has been shown to be potentially able to infect humans. Thus regarding the fish infection routes, enough attention should be payed to Aeromonas hydrophila, and it should be the focus in the pathogens monitoring.

Abstract:Objective To explore the feasibility of catheter implantation in a rat model of uterine horn fistula.Methods The catheter was placed into the uterine horns at one side in the rats, then histopathology and pregnancy function were studied.Results The endometrial thickness of the fistula side in the fistula model group (497.68±81.64 μm) was not significantly different compared with the control group (537.90±77.29 μm) and the opposite side (503.56±76.34 μm) (P>0.05). The number of embryo breeding in the fistula model group (9.67±1.95) was significantly decreased compared with the control group (12.15±2.06). The number of embryo breeding of the fistula side (5.83±3.54) was not significantly different compared with the opposite side (6.17±3.67) (P>0.05).Conclusions The effect of catheter on uterine tissue and pregnancy function is minimal, and the model of uterine horn fistula applies to the requirement of long term injection of pathogenic microorganisms or drugs.

Abstract:Objective To explore the effect of yeast and adenine administration in different doses and different duration on the establishment of rat models of gouty nephropathy.Methods Fifty-seven healthy male 2 months old Dawley rats (220-250 g) were used in this study. The rats of model groups were gavaged with 15 g/kg yeast and 50 mg/kg or 100 mg/kg adenine, for different number of days. The kidney hypertrophy index, kidney function, urine indexes, liver xanthine oxidase activity, N-acetyl-β-D-glucosaminidase (NAG) content in the renal cortex, and the renal histology were detected. The pathological changes of renal tissue were examined along with detection of the expression of NGAL, OAT1 and TIMP-1 by immunohistochemical staining.Results The renal pathological changes were more obvious with the increasing yeast and adenine doses and time of modeling. Compared with the normal group, the renal histological changes in the group administered with 100 mg/kg adenine and 15 g/kg yeast for 8 days showed apparent inflammatory cell infiltration and swelling of tubular epithelial cells, but there was not destruction of renal tubules and glomerular structures, showing a significant difference between the model and normal groups (P<0.01). Compared with that of the normal control group, kidney function was significantly decreased in the model groups A, B, E, and F (P<0.01). Compared with the normal control group, the 24 h urine volume and content of urinary protein were significantly increased in the model groups A, B, E, and F (P<0.01), and the expressions of protein OAT1 were significantly reduced in the renal tubules, while expressions of NGA, TIMP-1 were increased in the model group E (P<0.01).Conclusions Administration of adenine at a dose of 100 mg/kg and yeast at 15 g/kg for 8 days is an ideal procedure for the establishment of a rat model of gouty nephropathy. The renal alterations of this rat model of gouty nephropathy are affected by the changes of expressions of NGAL, TIMP-1 and OAT1 protein expressions.

Abstract:Objective To establish an HPLC-MS/MS method for determination of the plasma concentration of granisetron hydrochloride administered by transdermal patch in Beagle dogs and evaluate its pharmacokinetics.Methods Six beagle dogs were given topically applied transdermal patches (72 h) for 6 times with an interval of 24 h. HPLC-MS/MS was applied to determine the concentration of granisetron hydrochloride in blood samples, thereafter the pharmacokinetic parameters were calculated.Results The trough concentration of Cmin was 0.0459 ng/mL and 0.2593 ng/mL after the first-dosage and sixth-dosage of granisetron transdermal patches, respectively. The peak concentration of Cmax was 0.4637 ng/mL and 0.4637 ng/mL, respectively. The AUC_0~96 was 18.92 h·ng/mL and 40.21 h·ng/mL, respectively. The Tmax was 48.3 hours and 45.3 hours, respectively, and the MRT_0-96 was 48.9 hours and 45.84 hours, respectively.Conclusions The concentration of granisetron has accumulation effect after repeated administration of granisetron hydrochloride transdermal patches, and it reaches a steady state on the sixteenth day by multiple administrations.

Abstract:Objective Studies have suggested that human amniotic mesenchymal stem cells (hAMSCs) have multiple differentiation and proliferation functions, and can be induced to differentiate into hepatocyte-like cells to repair the liver injury.Methods Human amniotic mesenchymal stem cell swere prepared by resuscitation of frozen cells. Sixty-six healthy female Wistar rats were divided randomly into normal control group (n=22) without any treatment,and rat models of chronic alcoholic liver injury(n=44) treated by gastric gavage of alcohol for consecutive 30 days. After modeling,22 of those 44 rats were randomly divided into model group (tail vein injection of 1 mL PBS), ad another 22 rats as hAMSCs group which received tail vein injection of 1 mL (2×10⁶) hAMSCs.Four weeks after the transplantation, the rat serum alanine aminotransferase (ALT),aspartate aminotransferase(AST), total bilirubin (TBIL), albumin (ALB), total protein (TP)were detected by a full automatic biochemical analyzer, and the catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase(GSH PX), malondialdehyde (MDA) and IL-4 content in the liver tissue were detected,the distribution of PKH-26 labeled hAMSCs was observed by immunofluorescence microscopy, and TUNEL staining was used to detect apoptosis in liver cells.Results The rat blood biochemical test showed that compared with normal control group, the serum content of ALT, AST, Tp and TBIL in the model group was significantly higher,compared with those of the model group; the serum contents of ALT, AST, TBIL and Tp in the hAMSCs transplantation group were significantly decreased(P<0.05), compared with hose of the normal control group. The content of ALB in the model group was significantly decreased, and the content of ALB in hAMSCs transplantation group was significantly higher (P<0.05)compared with those of the model group. The contents of SOD GSH- PX and CAT in the liver tissues were significantly increased in the hAMSCs transplantation group. The contents of MDA and IL-4 were decreased significantly, compared with those of the normal control group. The contents of SOD GSH- PX, CAT in the liver tissues were decreased in the hAMSCs transplantation group, and the content of MDA and IL-4 in the liver tissues were significantly increased(P<0.05). PKH-26-labeled positive hAMSCs cells were observed to be distributed in the liver tissue of the hAMSCs transplantation group but not in the liver tissue of other groups (P<0.05).TUNEL assay detected apoptotic cells in the liver of all model groups, with the highest number in the model group (34.27±571) and less in the hAMSCs transplantation group (18.42±3.95), but not found in the normal control group.Conclusions Human amniotic mesenchymal stem cell transplantation can improve the blood biochemical indexes of rats with cirrhosis, and hAMSCs can reduce the apoptosis in liver cells in rats with chronic alcoholic liver injury.

Abstract:Objective To evaluate the disease course of acute kidney injury (AKI) induced by single intravenous injection of cisplatin in rabbits.Methods Forty male New Zealand rabbits were divided into 5 groups, 8 rabbits in each group. Acute kidney injury models were made by a single injection of cisplatin in the ear vein of rabbits, and five groups were defined as T₆, T₁₂, T₂₄, T₃₆ and T₄₈ according to the detection time of the blood urea nitrogen (BUN) and serum creatinine (SCr) in the rabbits. Blood BUN and SCr were detected before and immediately after cisplatin injection. The data were analyzed with SPSS 22.0 software, and P<0.05 was considered as statistically significant. The rabbits were sacrificed and kidney samples were taken for routine pathological examination.Results Compared with the data pre-injection, the blood BUN and SCr were increased significantly, and the experimental rabbits were consistent with the criteria of AKI according to the criteria of "Kidney Disease:Improving Global Outcomes Organization (KDIGO standard)" at 24 h after injection of cisplatin. The acute kidney injury of the rabbits was progressively worsening in 48 hours. More severe pathological changes were observed in the 48 hour group than in the other groups.Conclusions Progressive renal injury of rabbit kidney can be induced by single intravenous injection of cisplatin.

Abstract:Objective To analyze the Rxra expression level in 6 tissues of diabetic gerbils and explore the molecular mechanism of diabetes in Mongolian gerbils.Methods We collected 6 tissues including skeletal muscle, liver, adipose tissue, kidney, heart and brain from 6 diabetic gerbils and 6 control animals, respectively. Rxra mRNA and protein expression level in the 6 tissues were separately detected using real-time PCR and western blotting.Results The real-time PCR showed that compared with control animal, mRNA expression of Rxra in skeletal muscle and adipose tissue was significantly decreased in the diabetic group. However, there were no significant differences in the liver between the two groups. The relative expression level in diabetic group exhibited uptrend compared with the control in kidney, heart and brain. According to the results of western blotting, RXRA protein in skeletal muscle was significantly decreased in the diabetes group. RXRA protein was almost undetectable in adipose tissue. Varying expression was observed in other tissues, but there was no significant difference.Conclusions Lower expression of Rxra is observed in the skeletal muscle and adipose tissue of diabetic gerbils. There is no significant difference in other four tissues between the two groups. The results illustrate that the effect of Rxra mainly occurres in skeletal muscle and adipose tissue in this diabetic model.

Abstract:Objective To investigate the microbial and parasitic infection in laboratory mice and rats in Guangdong province from 2013 to 2015.Methods The samples of mice and rats were collected from 12 supervised and 32 entrusting institutions in Guangdong province. The test items and methods were determined according to the national standards, additionally, the test of Helicobacter and Murine Norovirus were added in some samples.Results There were major differences between the results of supervisory test and commission test. There were 4 pathogens detected in mice during the 3-year supervisory tests, including Pseudomonas aeruginosa (0.7%), Pasteurella pneumotropica (0.3%), Klebsiella pneumoniae (0.7%) and Flagellates (1.7%), and with on virus. Meanwhile, only Pasteurella pneumotropica (1.1%) was detected in rats. In contrast, there were 15 pathogens found in commission test of mice, and they were Pseudomonas aeruginosa (3.7%), Pasteurella pneumotropica (5.2%), Mycoplasma spp (1.9%), Staphylococcus aureus (0.7%), Klebsiella pneumoniae (0.8%), Helicobacter (45.3%), MHV (8.5%), TMEV (7.0%), MNV (16.2%), Ect. (0.3%), MVM (0.5%), SV (0.1%), flagellates (11.7%), helminths (1.0%) and ectoparasites (0.1%). At the same time, 8 pathogens were detected in rats, i.e. Pseudomonas aeruginosa (3.4%), Pasteurella pneumotropica (8.6%), Mycoplasma spp (0.9%), Staphylococcus aureus (2.9%), Tyzzer's organism (4.8%), Escherichia coli (1.0%), H-1 (3.0%) and RCV (1.0%). Some pathogens showed high detection ranges and rates, and had characteristics of regional distribution, which could be tested repeatedly in contaminated facilities, such as Pseudomonas aeruginosa, Pasteurella pneumotropica, Helicobacter, flagellates, MHV, TMEV and MNV in mice, as well as Staphylococcus aureus and Tyzzer's organism in rats.Conclusions This study investigated the prevalence and contamination of pathogens in mice and rats in Guangdong province from 2013 to 2015, which provides important references for production, administration, application and revision of standards of laboratory animals.

Abstract:Objective To report the results of medicated bath with ivermectin for eradication of external parasites in mice.Methods Nearly 800 genetically engineered mice which are self-used were treated with ivermectin medicated bath in concentration of 1% (L/L) to eradicate external parasites. Each mouse swims at least 1 minute, including immersing the head into solution for 3-4 times with a colander. Manual auxiliary medicated bath was needed for less than 10-day ones.All cages and materials were changed after the medicated bath, and environmental disinfection was performed at the same time. Medicated bath was used only once.Results Mortality rate of the mice was about 2%. The mice were followed-up for 18 months, and no reinfection with external parasites was observed. The mice were healthy and reproduction was normal.Conclusions Medicated bath with ivermectin is effective and feasible for eradication of external parasites in laboratory mice.

Abstract:Optogenetics is a technology invented by Boyden in 2005 that allows targeted, fast control of precisely defined events in neural system. It was widely used in Parkinson disease and emotional neural circuit studies. Optogenetics was selected by Nature Methods for method of the year 2010. This paper reviews on the research advances and applications of optogenetics.

Abstract:Adeno-associated virus(AAV)belongs to the family of Parvoviridae. It is a small virus which can infect humans and some other primate species with extensive host cell and low-immunogenicity. Besides, modified AAV can target the specific tissues and cells effectively. As a kind of gene therapy vector,Adeno-associated virus have been widely known of its biological characteristics. By modification of the adeno-associated virus serotype and structure of capsid protein, we can extend the application range of AAV vectors. This article introduces the targeting mechanism and results of some related researches, the genetic engineering modification of AAV capsid proteins, the transcription regulation modification of target gene expression, and gene modification and the covalent coupling of AAV capsid proteins.

Abstract:Exosomes have a size of 30-100 nm and membrane-bound nanovesicles, which is widely present in nearly all human body fluids, such as serum, saliva and urine, etc. Exosomes contain multiple proteins, nucleotides, and even viruses, and play an important role in the substance exchange and information transmission between cells. Recently, research found that genetic materials secreted from exosomes such as mRNA and miRNA were directly derived from their parent tumor cells, and they can directly effect on the receptor cells. Therefore, exosomes may become a new target for diagnosis and treatment of cancer. In this review, we described exosomes as a marker in early diagnosis of cancer, and its applications in cancer therapy.

Abstract:Deep brain stimulation is a therapy that stimulates the corresponding areas of the brain, records and analyzes neural signals, and to effectively treat a variety of neuropsychiatric disorders. Neural stimulator and electrical signal recording system is the key device of this therapy. This paper reviews the characteristics of neuroelectrical signals, stimulation and recording systems, introduces the architecture and basic module of the system, analyzes the application of deep brain electrical stimulation in neurological diseases, and summarizes the future trend.