Abstract:Objective The aim of this study was to generate human cholesteryl ester transfer protein (CETP) transgenic rabbits and analyze their biological properties. Methods We generated human CETP transgenic rabbits by DNA microinjection, and detected the expression of human CETP by real-time PCR and Western blot assay. The activity of CETP was measured using an activity assay kit. Results Human CETP transgenic rabbits were successfully generated by DNA microinjection. Compared with wide type rabbits, the expression of human CETP was dramatically increased in the liver of the human CETP transgenic rabbits. The plasma CETP activity was also much higher in the liver of human CETP transgenic rabbits than that of control rabbits. Conclusions The model of human CETP transgenic rabbits is successfully established by DNA microinjection. It will provide a useful tool for the studies of CETP biological function and its involvement in the mechanisms of cardiovascular diseases.

Abstract:Objective The aim of this study was to investigate how different concentrations of dextran sulfate sodium (DSS) influence the establishment of mouse model of inflammatory bowel disease (IBD) and the effect of DSS on the expression of colitis-associated immune factors. Methods The DSS solution in different concentrations (3%, 5%, 7%) were given to male C57BL/6J mice to generate mouse inflammatory bowel disease model. The IBD mice were observed by defecation characteristics, body weight, and survival time. The animals were sacrificed at 6 days after the start of DSS drinking. The general appearance of colons was observed and scored. Moreover, the pathological changes of the colon were examined and analyzed by routine histology. The expression of immune factors in the spleen was detected by real-time PCR.Results The mice in the 3%, 5%, 7% DSS groups developed murine colitis. In addition, the incidence of IBD and mouse mortality rate was directly proportional to the increase of DSS concentration. Furthermore, the higher concentration of DSS induced the expression of proinflammatory factors including TNF-α, IFN-γ and IL-17A, but cause a decrease of anti-inflammatory factors such as IL-4, IL-10 and Treg-related transcription factor Foxp3. Conclusions Our data suggest that giving 5% DSS solution to C57BL/6J mouse is appropriate to efficiently establish a murine IBD model. This laid an important foundation for further studies of the pathogenesis of IBD, biological characteristics, and intervention factors.

Abstract:Object To investigate the effect of PPARα activation on PPARγ-induced fatty liver in the mouse. Methods Wild type mice (C57BL/6) aged 4 to 5 weeks were used as animal models. All mice were divided into four groups. The mice in the first group were fed with chow diet. The mice in the second group were fed with a diet containing 0.125% Wy-14,643, an agonist of PPARa, for 8 days. The mice in the third group were injected with Ad/PPARγ via tail vein for 5 day. The mice in the fourth group were firstly fed with Wy-14,643 diet for 3 days and then injected with Ad/PPARγ via tail vein for another 5 day. Mouse livers were collected and photographed. The effect of PPARα activation on PPARγ-induced fatty liver was observed by H&E and Oil red O staining. Results Compared with the controls, wild-type mice treated with Wy-14,643 for 8 days exhibited marked hypertrophy of hepatocytes with increased cytoplasmic eosinophilia and proliferation of peroxisomes. The liver size was significantly increased in the wild-type mice treated with Ad/PPARγ for 5 days, and over-expression of PPARγ strongly induced hepatic steatosis. Importantly, the wild-type mice pretreated with Wy-14,643 for 3 days and then given Ad/PPARγ injection exhibited dramatically the increase of liver size, which might be due to the dual function of PPARa and PPARγ. Compared with the Ad/PPARγ group, the Wy-14,643 pretreatment group showed a reduced hepatic steatosis. Conclusions Activation of PPARα by Wy-14,643 effectively improves PPARγ-stimulated hepatic steatosis, which provides a novel target for prevention and therapy of fatty liver.

Abstract:Objective To establish a non-traumatic mouse model of acid aspiration-induced lung injury which allows longitudinal studies. Method C57BL/6 mice were anesthetized and orotracheally intubated with a 20 gauge angiocatheter guided by optical fiber. The mice were subsequently placed in the right lateral decubitus position and external compression to the left lung was manually applied. A polyethylene catheter was advanced into the right lung and used to instill either hydrochloric acid (2.5 μL/g, 0.1 mol/L, pH 1.5) or saline as control. Then the mice were recovered with supplemental oxygen for 4 hours. The pulmonary physiological function and survival of mice within 2 weeks after surgery were assessed. Results Methylene blue instillation showed that the staining fluid went into the right lung of the non-traumatically intubated mice. The survival rate of the mice with non-traumatic instillation was 80%, statistically significantly higher than those with tracheostomy instillation. Histological examination and lung function (wet/dry ratio, elastance and arterial oxygen saturation) assay demonstrated that acid instillation caused a profound pathological changes and functional impairment of the lung. Besides, acid aspiration into the mouse lung caused a significant increase in neutrophil infiltration in mouse pulmonary alveoli and high concentrations of inflammatory factors (TNF-α, IL-6, CXCL1 and CXCL2) in the bronchoalveolar lavage fluid. Conclusions We successfully established a mouse model of acid aspiration-induced lung injury, which may serve as a reliable model for longitudinally studying pulmonary immune-inflammatory mechanism in humans.

Abstract:Objective To improve the gene targeting efficiency with C57BL/6 embryonic stem (ES) cells. Methods Three different genetically modified C57BL/6 ES cell lines, named TLX3, Ai3K and SL, were microinjected into ICR, B6(Cg)-Tyr^c-2J and BALB/c mouse blastocysts, respectively. The efficiency was statistically evaluated according to three aspects: blastocyst collection, chimera production and germline transmission. Results None of the three ES cell lines was germline transmitted with B6(Cg)-Tyr^c-2J mice as blastocyst donors, while it was achieved with both BALB/c and ICR mouse blastocysts. Compared in the aspect of blastocysts collection, ICR mouse was much better than BALB/c mouse (P<0.05), and the chimera production efficiency of ICR mouse was comparable to that of BALB/c mouse (P=0.115). As to the germline transmission efficiency, that of BALB/c mice is significantly higher than that of the ICR mice (P< 0.01). Conclusions The germline transmission efficiency of BALB/c mouse is highest among these three mouse strains. However, it has the disadvantages of blastocyst collection, developmental delay and zona pellucida fragility, compared with ICR mouse. Therefore, ICR mouse is also a good candidate as blastocyst donor for embryonic stem cell microinjection.

Abstract:Objective To investigate the regulation of blood-testis barrier by Rab13-PKA pathway in rats. Method First, shRNA vector targeting at Rab13 was constructed and then the Rab13 shRNA was transfected into the rat testis by injection. Western blot was used to detect the knock-down effect of Rab13 and the expression of blood-testis barrier (BTB) constituent proteins. PKA activity was detected by autoradiography and scintillation counting. Further, immunofluorescence analysis and phalloidin staining were applied to observe the distribution of occludin and F-actin, respectively. Results The expression level of Rab13 in the testis was reduced by approximately 70% after transfection of Rab13 shRNA as compared with the non-targeted control group (P<0.01), while the expression of BTB constituent proteins remained unchanged. PKA activity was significantly increased after Rab13 RNAi transfection (P<0.01). The distribution of occludin at BTB was remarkably increased after Rab13 RNAi silencing around stage VIII but not at other stages of the seminiferous epithelial cycle. The assembly of F-actin at BTB was also intensified in Rab13-silenced testis. Both the changes of distribution of occludin and F-actin induced by Rab13 shRNA were found to be antagonized by the PKA specific inhibitor H89. Conclusions Rab13 can modulate the distribution of occludin and F-actin at the blood-testis barrier in rats by regulating PKA activity, which may participate in the regulation of BTB function.

Abstract:Objective To isolate and identify viruses from fecal samples of tree shrew with diarrhea. Methods Fecal sample supernatant of tree shrew with diarrhea was inoculated to three cell lines (Vero, LLC-MK2 and KMB17), and the cytopathic effects on the cells were observed. The infectious particles in the culture supernatant were further analyzed by transmission electron microscopy (TEM), genomic RNA-PAGE, rotavirus detection kit, amplification of S1 complete segment and bioinformatics analysis. Results Constant cytopathic effects were induced in Vero, LLC-MK2 and KMB17 cell lines after three passages of culture. The results from TEM, RNA-PAGE and rotavirus analysis indicated that they belong to reoviruses. Analysis of the S1 segments revealed that the S1 sequence from KMB17 cell culture had the highest homology with that of prototype isolate T1L (85% nucleotide homology and 90% amino acid homology), therefore this isolate was named as type I reovirus. The other two S1 sequences from LLC-MK2 and Vero cell culture were identical to have 85% nucleotide homology and 92% amino acid homology with the prototype isolate T3D, named as type III reovirus. Phylogenetic analysis indicated that the isolates in this study are evolutionally adapted to tree shrews.Conclusions It is the first report here that 2 genotypes of Tupaia orthoreovirus are isolated and identified from one fecal sample via three cell lines and viral S1-specific primers, which provides useful guidelines for the isolation and identification of other reoviruses from tree shrew or other hosts.

Abstract:Objective To establish and evaluate a reliable and highly reproducible neonatal rat model of hyperbilirubinemia and to provide an experimental basis for research of kernicterus and related mechanism of neuroinjury. Methods Sixty 7-day old SD rats (28 male and 32 female) were used in this study. Three doses of phenylhydrazine hydrochloride (25, 50, and 75 mg/kg) were intraperitoneally injected respectively to the neonatal rats to establish models of hyperbilirubinemia induced by hemolysis. The control group was set up at the same time. 48 hours after the experimental treatment, the bilirubin in blood and brain tissue, neuron-specific enolase (NSE) of brain tissue, and hemoglobin were detected to evaluate the models. Results Compared with the control group, the bilirubin in the blood and brain tissue and the brain tissue NSE in the three experimental groups were significantly higher than that in the control group (P<0.05), while hemoglobin content was significantly lower than that in the control group (P<0.05). The bilirubin of blood and brain tissue and brain tissue NSE in the 50 mg/kg and 75 mg/kg dose phenylhydrazine hydrochloride groups were significantly higher than that of the 25 mg/kg dose group (P<0.05), while hemoglobin content was significantly lower than that of the 25 mg/kg dose group (P<0.05). There were no significant differences between the 50 mg and 75 mg dose groups (P>0.05). Conclusions Intraperitoneal injection of phenylhydrazine hydrochloride can be used to produce neonatal rat models of hyperbilirubinemia, mimicking the clinical features of this disease, and 50 mg/kg of phenylhydrazine hydrochloride is the best concentration. It is an ideal method to establish newborn rat models of hyperbilirubinemia.

Abstract:Objective To investigate the expression of AIF, CYT C, PAF-1, caspase-3, and XIAP in Sprague-Dawley rats with spontaneous mammary neoplasms. Methods One-hundred and thirty 3-4-week old SPF Spargue-Dawley rats (♀:♂=1:1) were fed in a specific pathogen free (SPF) breeding barrier for 60 weeks. The occurrence of spontaneous breast tumors was recorded and histopathology was performed to identify the types of tumors. The rats were divided into 3 groups: rats with normal breast tissue (group I), with benign tumors (group II) and with malignant tumors (group III). The expression of AIF, CYT C, APAF-1, caspase-3 and XIAP proteins and mRNAs were detected by immunhistochemistry (IHC) and RT-PCR assay. Results Among these 130 SD rats, 14 rats were observed having spontaneous mammary neoplasms with the incidence rate of 10.77% (14/130). In these neoplasm cases, 7 cases were mammary fibroadenomas, 7 cases of breast carcinoma, both with an incidence rate of 5.38%. Immunohistochemistry showed that, compared with the group I, the positive expressions of AIF, APAF-1, caspase-3 were decreased significantly (P<0.01), and the CYT C and XIAP expressions were significantly increased in the group II. The positive expression of all genes except XIAP was decreased in the group III(P<0.01). Compared with the group II, APAF-1 and XIAP were significantly higher in the group III (P<0.01), and the positive expression of AIF, Cyt C, and caspases-3 were significantly decreased (P<0.01). In the results of RT-PCR assay, except APAF-1 which showed significant correlation with the results of immunohistochemistry (P<0.05), all the others showed an extremely significant correlation with immunohistochemical results (P<0.01). Conclusions Mammary tumors are most common spontaneous neoplasms in SD rats. Abnormal expression of mitochondrial apoptotic pathway-related factors AIF, CytC, APAF-1, caspase-3, and XIAP are correlated with the carcinogenesis and development of breast tumors.

Abstract:Objective To establish a rat model of persistent intestinal mucosal injury. Methods The rat model of persistent intestinal mucosal injury was induced by intraperitoneal injection of methotrexate. The food intake and body weight of all the rats were recorded. Pathological changes were observed using HE staining, the level of D-lactate and diamine oxidase in plasma, and myeloperoxidase and malondiadehyde in the intestinal tissue were measured by biochemistry. Results After modeling, the rat body weight and food intake were decreased. On day 4, the scores of mucosal damage, the levels of plasma D-lactate, DAO, MPO and MDA were significantly increased (P<0.05). On day 5, the intestinal damages of rats began to restore, and there was no significant difference among groups on day 6. The symptoms after the secondary injection were similar to those after the first injection, and the rats recovered gradually at day 12. Conclusions Intestinal mucosal injury in rats induced by 20 mg/kg MTX is an acute injury process, the course only lasts for 4-5 days. Intermittent injections twice of 10 mg/kg MTX can cause persistent intestinal mucosal injury in rats. This persistent injury model is more suitable for nutritional therapy evaluation in medium-and long-term studies of nutritional therapy.

Abstract:Objective To explore the regulatory effect of astragalus polysaccharide (APS) on immune function in immunosuppressed mice, and to establish a set of feasible indexes of immunological regulation and evaluation system of polysaccharides. Methods Both Cytoxan (CTX)-induced immunosuppressed mice and normal mice were treated with astragalus polysaccharide (APS). Spleen index, thymus index, phagocytosis rate and phagocytic index of peritoneal macrophages were assessed, and the spleen and thymus tissues were examined by histology. The percentage of CD3⁺, CD4⁺, and CD8⁺ cells in peripheral blood were determined by flow cytometry. Results APS significantly increased the thymus and spleen indexes in both immunosuppressed mice and normal mice, promoted the histological development of spleen and thymus in normal mice, enhanced the recovery of histological structures in the spleen and thymus in the CTX-induced immunosuppressed mice. APS also enhanced the phagocytosis rate and phagocytic index of peritoneal macrophages in both immunosuppressed mice and normal mice. In addition, the percentage of CD3⁺ and CD4⁺ cells,the ratio of CD4⁺/CD8⁺ in peripheral blood of both CTX-induced immunosuppressed mice and normal mice were increased while CD8⁺ cells were decreased. Conclusions The results of this study suggest that astragalus polysaccharide can improve the non-specific immunity and cellular immunity in mice, and indicate that we established comprehensive evaluation indexes of immunoregulatory effects of polysaccharides.

Abstract:Objective The aim of this study was to establish rat models of immunosuppression and immune hyperfunction. Methods Sixty-four SPF Sprague-Dawley rats were randomly divided into groups A, B, and C. All rats were immunized with intraperitoneal injection of 100 μg ovalbumin (OVA). The group A was used as control. At 6 hours after immunization, the rats of group B were injected with different doses of cyclophosphamide (Cy) at different time points. The rats of group C were injected with Cy in different ways at 3 days before immunization. Results Immunosuppressed rats were successfully induced by Cy (125 mg/kg or 100 mg/kg) at 6 h after immunization and also by injection of 225 mg/kg Cy at 3 days before immunization with ovalbumin. Small dose (20 mg/kg) of Cy injected once or a smaller dose (5 mg/kg/d) injected once a day for consecutive 3 days can also result in immune hyperfunction. Conclusions Rat models of immunosuppression and immune hyperfunction are successfully established, which provide methodological and data support for establishment of such animal models and useful reference for related research.

Abstract:Objective To prepare liposomes of glycyrrhizic acid, and evaluate its liver targeting property in mice. Methods The liposomes were prepared with conventional rotary-evaporated film-ultrasonication method. The liposomes were injected into the mouse tail vein, and the concentration of glycyrrhizic acid was detected by RP-HPLC. The glycyrrhizic acid injection was taken as control. The targeted indicators, including the relative tissue exposure (r_e), targeting efficiency (t_e), and index of peak concentration ratio (C_e), were used to evaluate the liver targeting property. Results The r_e was 1.4, T_e of the liposomes was 0.092, and t_e of the injection was 0.059. The C_e of the liver was 1.59, and the C_e of the blood was 0.99.Conclusion Compared with glycyrrhizic acid injection, the glycyrrhizic acid liposomes show good liver-targeting property.

Abstract:Objective HGF and its receptor (c-met) are principal mediators of mesenchymal-epithelial interaction in several different systems. Moreover, hair follicle is a model of mesenchymal-epithelial interaction. The aim of this study was to explore whether HGF/c-met signal plays a role in the control of hair follicle growth cycle. Methods To examine the localization of HGF/c-met in anagen, catagen and telogen in the hair follicle of ICR mice. Results HGF was mainly located in the dermal papilla and sebaceous gland, and c-Met in the hair matrix, root sheath and epidermis. Both HGF and c-met expressions peaked during anagen, not found in catagen, and increased during telogen-anagen phase. Conclusions Our results demonstrate a regulatory role of HGF and c-met in the control of hair follicle growth in ICR mice.

Abstract:Objective To compare three methods for culture of fetal cortical neurons of SD rats and find out the suitable culture conditions of fetal cortical neurons in vitro. Methods The cortex of 16-18-day embryonic rat was used for culture in this study. Mechanical dissociation, trypsin digestion and papain digestion were applied respectively to the neuron culture. The morphological characteristics of neuronal cells at different time points were observed and neuron purity was identified by immunofluorescence staining assay. Results High purity of the fetal rat cortical neurons was successfully achieved by all the three culture methods, and each had distinct morphological characteristics at different time points. The purity of neurons was 96.28%, 95.63% and 97.34%, respectively, with no significant differences among the three groups (P>0.05).Conclusions The three culture methods are improved in our study. Stable neurons with high purity can be obtained by all the three methods respectively, and each of these methods has distinct characteristics.

Abstract:Objective To investigate the existence of pulmonary vascular remodeling after left pneumonectomy in rats and the role of hypoxia inducible factor-lα (HIF-1α) and vascular endothelial growth factor (VEGF) in pulmonary vascular remodeling. Methods Twenty-four healthy male Sprague-Dawley rats were randomly divided into experimental and control groups, 12 in each group. The rat models of pulmonary vascular remodeling were created by open-chest left pneumonectomy. After 12 weeks of feeding, the mean pulmonary artery pressure (mPAP) and partial pressure of arterial oxygen (PaO₂) of each rat were measured. The ultrastructure of small arteries in the lung specimens were examined by electron microscopy. Muscularized degree of three kinds of small pulmonary vessels (muscularized artery MA, partially muscularized artery PMA, and non-muscularized artery NMA) were observed by light microscopy, and the percentage of each kind of pulmonary arteries (MA%, PMA%, NMA%) were calculated. Arterial external diameter, media thickness of vessel (MTV), total vascular area, media area of vessel (MAV), MTV% and MAV% were calculated as indicators of pulmonary vascular remodeling. The expressions of HIF-1α and VEGF in artery were detected by immunohistochemistry. Results The values of mPAP, MA%, PMA%, MTV, MAV, MTV% and MAV% in the experimental group were significantly higher than those in the control group (P<0.01), but the value of PaO₂ and NMA% were significantly lower than those in the control group (P<0.01). The IOD value of HIF-1α and VEGF expressed in the pulmonary arterial wall of the experimental group were 26.47±4.16 and 42.04±3.79, respectively, significantly higher than those in the control group (6.12±2.14 and 11.53±2.29, P<0.01). Linear correlation analysis showed that the expression of HIF-1α and VEGF was positively correlated with MTV% and MAV%, negatively correlated with PaO₂, and the HIF-1α expression was positively correlated with VEGF expression. Conclusions A rat model of pulmonary vascular remodeling can be successfully established by left pneumonectomy. Hypoxia is a key factor in the development of pulmonary vascular remodeling, HIF-1α and VEGF may play an important role in its pathogenesis.

Abstract:Prostate cancer is one of the most common malignant tumors in men and related studies have achieved great breakthrough in recent years. But because of the lack of effective in vivo animal models, the process to translate basic research into clinical application has been severely hampered. Patient derived prostate tumor xenograft (PDPTX) model is an ideal animal model in which freshly isolated tumor tissues from patients were inoculated into immunodeficient mice. This model can duplicate the heterogeneity of primary tumor in a better way and keep the tumor complexity at molecular, genetic and pathological levels. Particularly, the PDPTX model, in which the isolated tumor tissue is inoculated under the renal capsule, is even better, because it solves the clrawbacks of traditional subcutaneous inoculation model. In traditional models, the success rate is low, it's not easy for lower grade tumor to form xenograft, and it's not easy to reconstruct metastasis, etc. PDPTX provides a more ideal in vivo model for prostate cancer studies. It has irreplaceable advantages, especially in target therapy, new drug screening and individualized tumor treatment.

Abstract:Because of the unique molecular structure and stable performance of yolk immunoglobulin (IgY), it has been a hot spot in the field of antibody research. As a new type of vaccine, nucleic acid vaccine has more advantages than most traditional vaccines, such as production cost, immunizing dose and immune protection. The approach combining the advantages of nucleic acid vaccine and IgY production in egg yolk and suck IgY has wide scope of application. In this review, we introduce a quick and inexpensive production method of IgY, namely, IgY produced by nucleic acid vaccine immunization.

Abstract:Systemic lupus erythematosus (SLE) is a chronic multisystem relapsing-remitting autoimmune disease, which affects human health seriously. There are numerous animal models that have long been employed in an effort to understand the mechanism and treatment of SLE. Animal models of SLE were reviewed and compared in this paper, to provide references for the researchers to choose appropriate models for studying specific pathogenic mechanism and diagnostic criteria, searching for targeted treatment interventions and developing potential therapeutic drugs.

Abstract:The neonatal maternal separation (NMS) model has been ubiquitously used in studies of irritable bowel syndrome (IBS),which mimics the disease characterized by visceral hypersensitivity, and GI dismotility and mental disorder. This minireview focuses on the features and certain pathological mechanism of the animal models.

Abstract: