Abstract:Objective During zebrafish gastrulation, dramatic movements rearrange cells into three germ layers and contribute to the formation of the shield organizer, which acts as a dorsal signal center to pattern the body axis. Global identification of shield organizer-specific genes in early gastrulas will be valuable for studying the regulatory cascades during organizer formation and body axis establishment. Methods Tg(gsc:GFP) transgenic embryos express GFP in the shield organizer, which is controlled by a 1.8 kb gsc promoter. Flow cytometry technology and RNA deep sequencing analysis were applied to isolate GFP positive cells from the Tg(gsc:GFP) transgenic embryos and systematically uncover the genes highly expressed in the dorsal organizer. Subsequently, the expression of shield organizer-specific genes was further confirmed by quantitative real-time PCR and whole mount in situ hybridization method during zebrafish embryonic development. Results GFP-positive cells exceeding 96% purity were isolated from shield-stage Tg(gsc:GFP) transgenic embryos and 657 organizer highly expressed genes were identified through RNA deep sequencing analysis. The results of in situ hybridization experiments revealed that a number of genes including KIAA1324, ripply1,twist2, isthmin1, nme4, zgc174153 and rrbp1b were expressed in shield organizer during zebrafish gastrulation. Conclusions The identification of these shield organizer-specific genes in the current study provides useful clues to explore the zebrafish developmental functions in further studies.

Abstract:Objective To investigate whether TG2 plays an important role in the osteoblast differentiation and mineralization. Methods TG2 mRNA of SaOS-2 cells was knocked down using a lentivirus stably expressing short-hairpin(sh) RNA targeting TG2.Then the cells were cultured in osteo-inductive medium for 14 d to measure mineralization and for 7 d to measure the levels of osteoblastic differentiation markers including ALP activity and mRNA of collagen I, osteocalcin(OCN) and BMP-2.The wild-type SaOS-2 cells and scrambled shRNA-transducted SaOS-2 cells served as the controls. Results The controls displayed an increasing trend of the level of ALP activity and mRNA of collagen I, osteocalcin and BMP-2,and notable mineralization at 14 d.When TG2 was knocked down, ALP activity, mRNA of collagen I, osteocalcin and BMP-2 at 7d,and mineralization at 14 d were all significantly lower in comparison with the corresponding values in the controls.Conclusion TG2 is involved in the differentiation and mineralization of osteoblasts in vitro.

Abstract:Objective To compare the differences between the cell swelling of cultured astrocytes (AST) from Wistar and Sprague-Dawley (SD) rats after incubation with glutamate. Methods Primary cultured AST derived from the cerebral cortex of one-day-old Wistar or SD rats were prepared. The cultured AST received 1 or 10 mmol/L glutamate treatment for 48 h on the tenth day after subculture. The viability of AST was determined by lactate dehydrogenase (LDH) kit to assess the cell injury, and the perimeter of AST was measured using Image Pro Plus software after glial fibrillary acidic protein immunofluorescence staining to evaluate the astrocyte swelling. Then, the expression of aquaporin 4 (AQP4) in cultured AST was detected by quantitative reverse transcription polymerase chain reaction. Results No significant difference was found in the LDH release after the glutamate treatment in cultured AST from these two strains (P>0.05). The perimeter of AST from normal Wistar rats was shorter than that from SD rats, but was longer after the treatment of glutamate (P<0.05). Meanwhile, AQP4 expression in the Wistar rats was significantly higher than that from SD rats after incubation with 1 mmol/L glutamate (P<0.05). Conclusions These results suggeste that cultured AST from Wistar rats are more susceptible to glutamate-induced swelling than that from SD rats, and there are differences between the effects of glutamate on AQP4 expression in astrocytes of Wistar and SD rats.

Abstract:Objective To study the effect of paeoniflorin on inflammatory chemokines and their receptor in a rat model of ovalbumin-induced asthma.Methods Sixty 6-week old SPF female BALB/c mice were used in this study. To establish a mouse model of asthma by sensitizing and challenging with ovalbumin.ELISA was used to analyze the serum IL-6 and TNF-α, and the specific IgE against ovalbumin (OVA-IgE), CCL19 and CCL21 in bronchoalveolar lavage fluid (BALF). RT-PCR was performed to determine CCR7mRNA and protein expression of chemokine receptor CCR7, and the level of NF-κB was tested by Western blot.ResultsIn In the paeoniflorin groups, the serum IL-6 and TNF-α levels were significantly lower, and the OVA-IgE, CCL19 and CCL21 levels in BALF were significantly reduced, compared with that in the control group.CCR7mRNA and protein expression of chemokine receptor CCR7 and NF-κB in the lung were significantly reduced by paeoniflorin.Conclusions Paeoniflorin has a remarkably inhibitory effect on the airway inflammatory chemokines CCL19/CCL21 and the receptor CCR7 in the mouse model of asthma.

Abstract:Object To study the effect of puerarin on the expression of inflammatory factors and miR-155-3p in human umbilical vein endothelial cells (HUVEC) induced by visfatin. Methods The HUVEC cell injury model was established with visfatin. Cell proliferation was measured by MTT assay. Cell apoptosis was detected by flow cytometry. The level of CRP and NF-κB was detected by ELISA, and the expression of miR-155-3p was detected by RT-PCR. The expression of myeloid differentiation factor 88 (MyD88) was identified by western blotting. Results Visfatin induced cell proliferation and inhibited apoptosis in HUVEC, meanwhile the expressions of both CRP and NF-κB were significantly increased, compared with that of the control group (P<0.01). Puerarin at moderate and high concentrations obviously reduced the HUVEC injury induced by visfatin, mainly through down-regulating the expression of CRP and NF-κB, as well as up-regulating the level of miR-155-3p in the HUVEC. MiR-155-3p mimic markedly decreased the level of MyD88, CRP and NF-κB in the HUVEC induced by visfatin (P<0.05). Conclusions Pueprarin obviously alleviates HUVEC injury induced by visfatin, probably related to down-regulating the level of MyD88, CRP, NF-κB, and up-regulating the expression of miR-155-3p in HUVEC.

Abstract:Objective In this study, the glucose and energy metabolism-related genes (PGC-1α, Glut-4, ERRα, NRF-1, TFAM and mtDNA gene) were detected in type 2 diabetes mellitus (T2DM) and non-T2DM minipigs, and the gene function was explored for T2DM pathogenesis. Methods The longissimus muscle of T2DM and non-T2DM Guangxi Bama mini-pigs was used as experiment material. The expression of glucose and energy metabolism-related genes was detected by QRT-PCR. Results The expressions of PGC-1α, Glut-4, ERRα and NRF-1 genes were significantly higher than that of non-T2DM group, the expressions of TFAM and mtDNA gene were lower than that of non-T2DM group. Conclusions The upregulated expression of PGC-1α gene and its downstream genes Glut-4, ERRα, NRF-1 may improve the glucose metabolic functions in skeletal muscle in the Bama minipigs, whereas insufficient mitochondrial synthesis may induce decreasing ATP synthesis, and results in skeletal muscle insulin resistance, finally leading to the T2DM occurrence.

Abstract:Objective To analyze the effect of Noggin silencing on the BMP and Wnt signaling pathways in hair follicle development. Methods The expression of BMP-2, BMP-4, BMPR-IA, BMP-6, BMP-7, LEF-1 and β-catenin in Noggin silencing MC3T3-E1 stable cell line was detected by RT-PCR and western blot. Results RT-PCR results showed that the expressions of five genes in BMP signaling pathway were all significantly influenced by Noggin silencing, the expressions of BMP-2 (P<0.001), BMP-4 (P<0.01), BMP-6 (P<0.001) and BMP-7 (P<0.001) were all increased and the expression of BMPR-IA (P<0.01) was decreased. While the expressions of the two genes LEF-1 (P<0.001) and β-catenin (P<0.001) in Wnt signaling pathway were significantly decreased. Western blot results showed that the expressions of these proteins in the two signaling pathways were also affected. The expressions of BMP-2 (P<0.05), BMP-4 (P<0.05), BMP-6 (P<0.05) and BMP-7 (P<0.05) were all increased, while the expressions of BMPR-IA (P<0.05), LEF-1 (P<0.01) and β-catenin (P<0.001) were decreased. Conclusions There may be a negative feedback regulation of Noggin on the BMP signaling pathway in vitro, but a positive feedback regulation on the Wnt signaling pathway in vitro. It provides certain evidence for studies on the effect of Noggin gene on BMP and Wnt signaling pathways in vivo. There may be an interaction between hair follicle development-related signaling pathways, which still needs further experiments to prove.

Abstract:Objective To study the effect of once-weekly injection of recombinant human parathyroid hormone rhPTH(1-34) on the healing of bone fracture in rats.Methods Fifty male 3-month old SD rats were used in this study to produce unilateral tibial fracture and received internal fixation with a Kirschner needle. Based on the dose and frequency of rhPTH (1-34) injection, the rats were randomly divided into five groups (n=10 each) as follows: subcutaneous injections of saline, and rhPTH in a dose of 10, 20, 10, and 20 μg/kg/d.After 4 weeks of treatment, the rats were euthanatized and the fractured tibia were assessed by X-ray, dual energy X-ray absorptiometry (DXA),micro-computed tomography (microCT) and three-point bending test.Results The fracture healing in the 20 μg/kg/w group was better than the saline group.The fracture healing in the 20 μg/kg/w group was as well as the 10 μg/kg/dgroup. The BMD of 20 μg/kg/w group was 26.2% higher than the saline group. The mineralized callus volume in the 20 μg/kg/w group was 51.4% higher than the saline group. The total callus volume in the 20 μg/kg/w group was 21.6% higher than the saline group. The ultimate load of the 20 μg/kg/w group was 29.3% higher than the saline group. There was no significant difference between the 20 μg/kg/w group and 10 μg/kg/d group in BMD, bone micro-architecture,and biomechanical strength (P>0.05). Conclusions Once weekly injection of rhPTH (1-34)can promote the bone fracture healing.

Abstract:Objective The aim of this study was to observe the morphological changes of the retina at early stage of diabetes in SD rat induced by multiple injections of low-dose (multi-low-dose) streptozotocin (STZ). Methods Seventy-five male SD rats were randomly divided into control (CON)(n=30) and diabetes mellitus (DM)(n=45) groups. Rats in the DM group received multiple low-dose STZ i.v. injection for 5 consecutive days (30 mg/kg). The blood glucose, body weight, and other parameters were observed once a week after injection. In the 4, 8 and 12 w, the morphological changes in the retina were observed using HE and immunohistochemical (IHC) staining, vasculature digest preparation and transmission electron microscopy (TEM). Results The body weight of animals in the DM group was decreased from 2 w. The average body weight of the DM group was significantly lower than that of CON group (P<0.01). The average blood glucose of DM group was significantly higher than that of the CON group (P<0.01). TEM examination revealed thickened capillary basement membrane and dilatation of capillary at 4 w. trypsin digest preparation showed endothelial cell hyperplasia, mitochondrial swelling, cristae disruption, and vacuolar degeneration in capillary endothelial cells at 8 w. Retina HE staining showed retinal capillary dilatation and interstitial edema, capillary intumescence, apoptosis in endothelial cells in the DM group at 12 w. The control group had no obvious abnormalities. Staining of GAFP in the retina indicated decreased expression in the ganglion cell layer and nerve fiber layer, bipolar cells and ganglion cells, mitochondrial swelling and cristae disruption in pericytes, decreased membranous disc, and widened gap between membranous discs at 12 w. Conclusions Our findings indicate that an early stage diabetic retinopathy (DR) can be induced in rats by multiple low-dose streptozotocin injection. This may serve as a valuable preclinical model for studying the pathogenesis, pharmacodynamics and potential therapies for DR and its complications.

Abstract:Objective To observe the ultrastructure of blood-brain barrier in the nude mouse model of brain metastases from lung cancer by transmission electron microscopy using lanthanum nitrate tracing. Methods PC-9 cells (1×10⁶/0.1 mL) in logarithmic phase were respectively injected into six nude mice (model group) selected from eight nude mice randomly via the left ventricle, the other two mice without any treatment as the control group. The general status of the mice was observed after implantation. In the fourth week all the mice were sacrificed and brain tissue samples were taken and prepared for transmission electron microscopic observation using lanthanum nitrate tracing. besides, the lung and brain were removed and stained with HE to detect the presence of tumor metastasis. Results Mice in the model group began to lose weight almost simultaneously in the third week and became moribund slowly, and were all sacrificed at the fourth week when showing clear signs of cachexia. At autopsy, the thoraxes were clear, with normal lungs. Histology showed evidence of brain metastasis in all the six mice. The electron microscopy showed that lathanum nitrate tracer was escaped from the capillaries and diffusely or sparsely distributed in the brain tissues of the model group mice, however lathanum nitrate tracer was still confined in the capillary lumen in the mice of control group. Conclusions The diffuse lathanum nitrate tracer in the brain parenchymal tissue indicates the impairment of blood-brain barrier in the nude mouse model of lung cancer brain metastasis and the formation of these metastases is accompanied with the destruction of blood brain barrier.

Abstract:Objective In order to lay the foundation for establishment of a clean grade Meriones meridianus population, and provide reference for the formulation of Meriones meridianus parasite detection standard. Methods A survey of parasite infection in Meriones meridianus was carried out, and three kinds of antiparasitic drugs, tetramethrin/permethrin, praziquantel, and tinidazole were used in high-, medium- and low-doses for eradication of the target parasites. Results Three types of parasites were detected among the animals. The infection rate of surface parasite Myobia musculi was 82.1% (23/28), that of intestinal parasite Hymenolepis nana was 78.1% (25/32) and Tritrichomonas muris was 90.6% (29/32). Significant results of parasite eradication were achieved with the antiparasitic drugs. Medicated bath using 1% tetramethrin/permethrin for 30 s every 4 days for 5 times and 1.5% tetramethrin/permethrin for 30 s every 4 days for 3 times completely eradicated the surface Myobia musculi infection. Praziquantel in a dose of 31 mg/kg by daily gastric gavage for consecutive 7 days was the optimal dosage to completely eradicate the intestinal Hymenolepis nana. Tinidazole in a dose of 42 mg/kg administered by gastric gavage daily for consecutive 7 days was the optimal dosage for eradication of intestinal Tritrichomonas muris infection. Conclusions Three types of parasites are detected in Meriones meridianus in our laboratory, i.e. surface infection with Myobia musculi, and intestinal infection with Tritrichomonas muris and Hymenolepis nana. Tetramethrin/permethrin, praziquantel and tinidazole can be used to effectively eradicate these three types of parasites, respectively.

Abstract:Objective To comparatively investigate the characteristics of bone metabolism in forelimb malformation WHBE rabbits based on the OPG/RANK/RANKL system. Methods Healthy male 2-2.5-month old WHBE rabbit (HWR), Healthy Japanese rabbit (HJR) and forelimb malformation WHBE rabbit (FMWR) were used in this study and divided into 3 groups, HWR, HJR and FMWR groups, with 10 rabbits in each group. The shape of forelimb and mean gray value observed from X-ray film were examined, and the bone tissue micro-morphology was analyzed using HE staining. Real-time fluorescence quantitative PCR was used to determine the expression of osteoprotegerin (OPG) and receptor activator of NF-KB ligand (RANKL) mRNA. The expressions of OPG, receptor activator of NF-KB (RANK) and RANKL protein in serum and bone tissue were assayed by enzyme-linked immunoassay (ELISA) and immunohistochemistry, respectively. Results Compared with the HWR and HJR groups, rabbits in the FMWR group appeared abnormal in toutuous forelimb and thinner bone cortex. The mean gray values of X-ray in the FMWR group were lower than that in the HWR group (P<0.05). There were significant differences between the FMWR and healthy rabbits (HWR and HJR) in the following parameters: RANKL mRNA expression and RANKL/OPG mRNA ratio in the liver(P<0.05, P<0.01), serum protein expression of RANK and RANKL and RANKL/OPG ratio (P<0.05, P<0.01), and positive index of expression of RANKL protein and RANKL/OPG ratio in the bone tissue(P<0.05, P<0.01). Furthermore, the gene expression levels of OPG and RANKL of HWR were significantly higher than that of HJR(P<0.05, P<0.01). Conclusions The FMWR show some abnormal symptoms in bone metabolism as well as decrease of bone quality and histological changes of bone micro-structure, due to the significant increase of RANKL/OPG ratio of FMWR. The breed differences between WHBE and Japanese rabbits in RANKL mRNA expression level may be one of factors inducing limb malformation in the WHBE rabbits.

Abstract:Objective To observe the changes of mRNA expression related to kidney yang deficiency and the ultrastructure of hypothalamus in Lewis rats, and further study the constitution of kidney yang deficiency in Lewis rats. Methods Ten 7-8-week old SPF male Lewis rats and twenty 7-8-week old SPF male Wistar rats were used in this study. The rats received subcutaneous injection of hydrocortisone to establish kidney yang deficiency model. The expressions of TNF-α, IFN-γ, IL-10, CRH, MR and GR mRNA in the hypothalamus of the two groups were detected and the ultrastructural changes of hypothalamus and adrenal gland were observed to compare the differences between the two groups, and to explore the mechanism of kidney yang deficiency constitution in the Lewis rats. Results Compared with the normal Wistar rats, the expressions of GR and IL-2 mRNA in the hypothalamus were significantly increased (P<0.01), while the expressions of CRH, TNF-α, IFN-γ and IL-10 mRNA were significantly decreased in the Lewis rats (P<0.01). The zona fasciculata of the adrenal cortex was slightly thinner, the mitochondria were slightly swollen and the amount of mitochondria was decreased. The nuclei of hypothalamic neurons were larger, and the mitochondria, neuron synapses and secretory vesicles in the presynaptic neurons were decreased. Conclusions There are abnormal expression of immune-related cytokines and CRH mRNA, and ultrastructural changes in the hypothalamus and adrenal gland of Lewis rats with kidney yang deficiency. Such changes may be related to the functional imbalance of the hypothalamus-pituitury-adrenal axis.

Abstract:Objective To investigate the effects of different drugs and to explore the mechanism of pain in mice with adenomyosis (ADM). Methods The mouse model of adenomyosis was induced by oral administration of tamoxifen. The vaginal smear was examined by cytology, and serum levels of 5-HT, GnRH-R, NGF and NF were determined. Results Higher level of 5-HT was detected in the controls compared with the models. The expression of GnRH-R and NGF in normal endometrium and eutopic endometrium were significantly lower than those in ectopic endometrium. The expression on NF in normal endometrium was significantly lower than that in the eutopic and ectopic endometria. Conclusions Similar with regular dose of GnRH-a, half dose of GnRH-a can slow down the progress of ADM as well as reducing pain. Combination of regular dose of GnRH-a and Diane-35 could consolidate the therapeutic effect and even achieve pathological cure. 5-HT may take an active part in the mechanism of pain in ADM.

Abstract:Objective To evaluate the mutagenicity of hydrolysate of Meretrix meretrix Linnaeus soft tissue, so as to provide experimental basis for its exploitation. Methods Three mutagenicity tests were used to evaluate the mutagenic effects, including Ames test, CHL chromosome aberration assay and bone marrow micronucleus assay in mice. Results In Ames test, the revertant colonies numbers in each group were twice less than the numbers of spontaneous revertant colonies, five bacterial strains showed negative results with or without S9 activation, and the result of Ames test was negative. The CHL chromosome aberration assay and bone marrow micronucleus assay showed that the chromosome aberration rate and micronucleus rate of each dose group showed no significant difference compared with the negative control group, respectively (P>0.05). Conclusions Under this condition, the results show that all of the Ames test, chromosome aberration assay and bone marrow micronucleus assay are negative, and no mutagenicity is observed in the hydrolysate of Meretrix meretrix Linnaeus soft tissue.

Abstract:Objective To observe the changes in estrous cycle and vaginal smears in ovarectomized NOD/SCID mice.Methods To continuously observe the estrous cycle time by vaginal smears of NOD/SCID mice in consecutive nine days, twice daily. After ovariectomy, the changes of estrous cycle were observed by vaginal smears for 7 days.Results The estrous cycle in NOD/SCID mice was 4-6 days. Regular estrous mice accounted for 80%. There was no significant correlation between vaginal opening and estrous cycle status. After ovariectomy, the vaginal smears showed characteristics of metestrus or diestrus. Conclusions Vaginal smear cytology can be used to determine the estrous cycle and characteristics of NOD/SCID female mice.The ovariectomized operation of NOD/SCID female mice is effective.

Abstract:A member of miR-16 family, miR-424 has been found to be closely related with tumorigenesis, tumor progrssion, prognosis and therapy. This article reviews the expression changes, roles and possible regulating mechanisms of miR-424 in leukemia and various tumors such as breast, cervical, lung, liver and colorectal cancers. Recent studies have demonstrated that the expression of miR-424 is affected by many factors, and miR-424 could be a biomarker of diagnosis, staging and prognosis in cancers,to identify the area of tumor, and be a target of therapy.

Abstract:The established immunodeficient animal models could be used as valuable resource for mechanism research of related disease in humans, drug discovery and development, translational research and stem cell research. However, it is difficult and low-efficient to establish the genetic modified animal models using traditional technologies. The reports for immunodeficient animal models are few in middle-size and large animals. Recently, several effective gene-targeting tools, including ZFNs, TALENs, CRISPR/Cas9, develop quickly and provide technology basis for the establishment of immunodeficient animal models. In this paper, the technology principles and research progresses of ZFNs, TALENs, CRISPR/Cas9 are introduced. The significant progresses of these emerging technologies achieved in immunodeficient animal models are also elaborated, including KO Rag/1 Rag2 rabbit, KO Rag/1 Rag2 pig, KO IL2rg pig, KO Ppar-γ/ Rag1 monkey, and so on. In addition to being models for researching SCID-related diseases in humans, and evaluating the efficacy and safety of stem-cell engraftment, these models may be also useful to develop surgical procedures for placement of grafts before clinical trials in humans, to produce humanized animals and bridge the gap between laboratory animal and medicical research. The immunodeficient animal models described here represent a step toward the comprehensive evaluation of preclinical cellular regenerative strategies.

Abstract:Alzheimer's disease (AD) is one of the most common dementia of neurodegenerative disorders, which results from the deposition of amyloid-beta (Aβ) and there are no curative treatments for this disease at present. It had been proved that prion protein is the receptor for Aβ and it plays a key role in the progress of AD with dual-side effects. Prion protein can not only transfer neurotoxicity to neurons but also protect them from neurotoxicity of Aβ. The polymorphisms of prion protein encoding gene (PRNP) affect the AD incubation period and clinical symptoms in humans and other animals. The discovery of PRNP mutational mouse fills the gaps of existing AD mouse models in this research area, which is potential for the studies of pathogenesis, new drugs design and testing aspects. The role and effects of prion protein in AD pathogenesis were summarized in this paper, furthermore, the discovery and utility of PRNP gene mutational mouse in research on AD and/or amyloid diseases were reviewed, and in order to provide some guidance for AD animal model study.

Abstract:Germ-free (GF) pigs are a special and adaptable experimental animal model for biomedical studies and animal productions, which are negative for bacteria, viruses, yeast and fungi tested by current microbiological examination. GF pigs were initially used in cleanse of epidemic diseases in animal production and in a bid to study the relationship between animal disease and intestinal flora. Because of the similarities to humans in anatomy, physiology and hematology, and the clear microbiological background, GF pigs have been playing an important role in detecting the relationship between intestinal flora with growth and the development of diseases in medical biology, and also providing a special medical animal model for intestinal flora targeted prevention, diagnosis and treatment for update technology research in the clinic. This paper reviews the characteristics, advancements and research tendency of GF piglets.