Isolation and differentiation of embryonic stem cells from BALB/c mouse

Objective To invest the efficient method which can culture and induce embryonic stem cells to neuroeyte in vitro. Methods Isolate the blastula o f 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass （inner cell mass, ICM） which were isolated by mechanical method on the mouse embryonic fibroblaste cell （MEF） feeder layer or 0.1% gelatin coated dishes. The stem ceils were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunoehemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 （ stage specific embryonic antigen 1 ）. Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated ceils presented the characters of ESCs. Then the isolated cells were able to differentiate into neuroeytes in vitro. Conclusion Mouse embryonic stem ceils isolation, culture and differentiation system has been established.

Insights into sensitizing and eliciting capacity of gastric and gastrointestinal digestion products of shrimp(Penaeus vannamei)proteins in BALB/c mice

Shrimp(Penaeus vannamei)proteins have been shown an allergenic potential;however,little information is available on the sensitizing and eliciting capacity of shrimp protein digestion products.In this study,a BALB/c mice model was used to explore the allergenicity of shrimp protein sample(SPS)and their gastric and gastrointestinal digestion products(GDS/GIDS).As compared with the SPS groups,the GDS/GIDS groups caused lower specific immunoglobulins(Ig E/Ig G1)levels(P<0.05),but higher than the control groups,indicating that the digestion products sensitized the mice.Meanwhile,spleen index,mouse mast cell protease-1(m MCP-1)concentration and proportion of degranulated mast cells were significantly reduced in the GDS/GIDS groups(P<0.05);simultaneously,allergic symptoms,vascular permeability and histopathological changes of tissues were alleviated.Nevertheless,the allergenicity of digestion products cannot be eliminated and still cause systemic allergic reactions in mice.The study showed that the digestion products of shrimp still had high sensitizing and eliciting capacity.

Establishment of an orthotopic pancreatic cancer mouse model: Cells suspended and injected in Matrigel

AIM: To establish an orthotopic mouse model of pancreatic cancer that mimics the pathological features of exocrine pancreatic adenocarcinoma.

Study on the Gene Transcription Level of Hippocampus NGF in C_(57) Mouse Development

β-Nerve growth factor (β NGF) mRNA levels in hippocampa from C57BL/6J mice of different ages were compared by semi-quantitative RT-PCR method, taking β-actin gene as an internal control. The levels of 6-and 12-month-old C57 mice were much lower compared with that of the l-month-old mouse. However, there was no significant difference between these two groups. This result suggests that gene transcription level of hippocampus β NGF in a C57, mouse descends evidently as it grows up. Base T at Site 294 of β NGF cDNA was substituted by C in the C57, mouse, as revealed by DNA sequencing for the first time. Nevertheless, this polymorphism of nucleotide did not make any difference in amino acid composition.

SOCS3 Expression Correlates with Severity of Inflammation in Mouse Hepatitis Virus Strain 3-induced Acute Liver Failure and HBV-ACLF

Summary： Recently, suppressor of cytokine signaling-3 （SOCS3） has been shown to be an inducible endogenous negative regulator of Janus kinase/signal transducers and activators of transcription （JAK/STAT） pathway which is relevant in inflammatory response, while its functions in acute liver failure and HBV-induced acute-on-chronic liver failure （HBV-ACLF） have not been fully elucidated. In this study, we explored the role of SOCS3 in the development of mouse hepatitis virus strain 3 （MHV-3）-induced acute liver failure and its expression in liver and peripheral blood mononuclear cells （PBMCs） of patients with HBV-ACLF. Inflammation-related gene expression was detected by real-time PCR, immtmohistochemistry and Western blotting. The correlation between SOCS3 level and liver injury was studied. Our results showed that the SOCS3 expression was significantly elevated in both the liver tissue and PBMCs from patients with HBV-ACLF compared to mild chronic hepatitis B （CHB）. Moreover, a time course study showed that SOCS3 level was increased remarkably in the liver of BALB/cJ mice at 72 h post-infection. Pro-inflammatory cytokines, interleukin （IL）-1 β, IL-6, and tumor necrosis factor （TNF）-α, were also increased significantly at 72 h post-infection. There was a close correlation between hepatic SOCS3 level and IL-6, and the severity of liver injury defined by alanine aminotransferase （ALT） and aspartate aminotransferase （AST） levels, respectively. These data suggested that SOCS3 may play a pivotal role in the pathogenesis of MHV-3-induced acute liver failure and HBV-ACLF.

TLR4-HMGB1-, MyD88- and TRIF-dependent signaling in mouse intestinal ischemia/reperfusion injury

AIM: To characterize high-mobility group protein 1-toll-like receptor 4(HMGB1-TLR4) and downstream signaling pathways in intestinal ischemia/reperfusion(I/R) injury.METHODS: Forty specific-pathogen-free male C57BL/6 mice were randomly divided into five groups(n = 8 per group): sham, control, anti-HMGB1, anti-myeloid differentiation gene 88(My D88), and anti-translocatingchain-associating membrane protein(TRIF) antibody groups. Vehicle with the control Ig G antibody, antiHMGB1, anti-My D88, or anti-TRIF antibodies(all 1 mg/kg, 0.025%) were injected via the caudal vein 30 min prior to ischemia. After anesthetization, the abdominal wall was opened and the superior mesenteric artery was exposed, followed by 60 min mesenteric ischemia and then 60 min reperfusion. For the sham group, the abdominal wall was opened for 120 min without I/R. Levels of serum nuclear factor(NF)-κB p65, interleukin(IL)-6, and tumor necrosis factor(TNF)-α were measured, along with myeloperoxidase activity in the lung and liver. Inaddition,morphologic changes that occurred in the lung and intestinal tissues were evaluated. Levels of m RNA transcripts encoding HMGB1 and NF-κB were measured by real-time quantitative PCR, and levels of HMGB1 and NF-κB protein were measured by Western blot. Results were analyzed using one-way analysis of variance.RESULTS: Blocking HMGB 1, MyD 8 8, and TRIF expression by injecting anti-HMGB1, anti-My D88, or anti-TRIF antibodies prior to ischemia reduced the levels of inflammatory cytokines in serum; NF-κB p65: 104.64 ± 11.89, 228.53 ± 24.85, 145.00 ± 33.63, 191.12 ± 13.22, and 183.73 ± 10.81(P < 0.05); IL-6: 50.02 ± 6.33, 104.91 ± 31.18, 62.28 ± 6.73, 85.90 ± 17.37, and 78.14 ± 7.38(P < 0.05); TNF-α, 43.79 ± 4.18, 70.81 ± 6.97, 52.76 ± 5.71, 63.19 ± 5.47, and 59.70 ± 4.63(P < 0.05) for the sham, control, anti-HMGB1, anti-My D88, and anti-TRIF groups, respectively(all in pg/m L).Antibodies also alleviated tissue injury in the lung and small intestine compared with the control group in the mouse intestinal I/R model. The administration of antiHMGB1, anti-My D88, and anti-TRIF antibodies markedly reduced damage caused by I/R, for which anti-HMGB1 antibody had the most obvious effect.CONCLUSION: HMGB1 and its downstream signaling pathway play important roles in the mouse intestinal I/R injury, and the effect of the TRIF-dependent pathway is slightly greater.

Transglutaminase 3 expression in C57BL/6J mouse embryo epidermis and the correlation with its differentiation

Epidermal-type transglutaminase 3 (TGM3) is involved in the cross-linking of structural proteins to form the cornifiedenvelope in the epidermis. In the present study, we detected the expression of TGM3 in the mouse embryo using RT-PCR.TGM3 mRNA is weakly presented from E11.5 to E14.5 and increases significantly from E15.5 to birth. Then wedetermined the spatial and temporal expression pattern of TGM3 in the skin and other organs by in situ hybridization. Wefound a deprivation of TGM3 in skin at E11.5, while a rich supply in periderm cells and a weak expression in basal cellsfrom E12.5 to E14.5. From the period of E15.5 to E16.5, after keratinization in the epidermis, TGM3 was expressed inthe granular and cornified layers. The electron microscopic observation of the C57BL/6J mouse limb bud skin develop-ment provided several morphological evidences for the epidermal differentiation. The above findings suggest that theexpression of TGM3 plays a important role in the epidermis differentiation in embryogenesis.

Hepatocellular carcinoma xenograft supports HCV replication:A mouse model for evaluating antivirals

AIM: To develop a hepatocellular carcinoma (HCC) xenograft model for studying hepatitis C virus (HCV) replication in a mice, and antiviral treatment.METHODS: We developed a stable S3-green fluorescence protein (GFP) cell line that replicated the GFP-tagged HCV sub-genomic RNA derived from a highly efficient JFH1 virus. S3-GFP replicon cell line was injected subcutaneously into γ-irradiated SCID mice. We showed that the S3-GFP replicon cell line formed human HCC xenografts in SCID mice. Cells were isolated from subcutaneous tumors and then serially passaged multiple times in SCID mice by culturing in growth medium supplemented with G-418. The mouse-adapted S3-GFP replicon cells were implanted subcutaneously and also into the liver of SCID mice via intrasplenic infusion to study the replication of HCV in the HCC xenografts. The tumor model was validated for antiviral testing after intraperitoneal injection of interferon-α (IFN-α). RESULTS: A highly tumorigenic S3-GFP replicon cell line was developed that formed subcutaneous tumors within 2 wk and diffuse liver metastasis within 4 wk in SCID mice. Replication of HCV in the subcutaneous and liver tumors was confirmed by cell colony assay, detection of the viral RNA by ribonuclease protection assay and real-time quantitative reverse transcription polymerase chain reaction. High-level replication of HCV sub-genomic RNA in the tumor could be visualized by GFP expression using fluorescence microscopy. IFN-α cleared HCV RNA replication in the subcutaneous tumors within 2 wk and 4 wk in the liver tumor model. CONCLUSION: A non-infectious mouse model allows us to study replication of HCV in subcutaneous and metastatic liver tumors. Clearance of HCV by IFN-α supports use of this model to test other anti-HCV drugs.

Axonal damage in myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis in a C57BL/6 mouse model may be not secondary to inflammatory demyelination

The present study established a chronic experimental autoimmune encephalomyelitis model in C57BL/6 mice induced by myelin oligodendrocyte glycoprotein peptides and complete Freund＇s adjuvant. Onset latency was 12 days, with an incidence rate of 100%. Neuropathological characteristics included perivascular inflammatory cell infiltration, demyelination, neuronal degeneration, and axonal damage within cerebral and myelic white matter. Electron microscopy revealed swollen mitochondria, complete organ disappearance, and fused or broken myelin sheath structure, which were accompanied by myelin sheath reconstruction. Moreover, axonal damage was not consistent with demyelination distribution, and severity of axonal damage did not correlate with demyelination. Results suggested that axonal damage in an experimental autoimmune encephalomyelitis model is not secondary to inflammatory demyelination.

Neurodevelopmental Timing of Ethanol Exposure May Contribute to Observed Heterogeneity of Behavioral Deficits in a Mouse Model of Fetal Alcohol Spectrum Disorder (FASD)

Maternal drinking during pregnancy can result in a wide spectrum of cognitive and behavioral abnormalities termed fetal alcohol spectrum disorders (FASD). The heterogeneity observed in FASD-related phenotypes can be attributed to a number of environmental and genetic factors;however, ethanol dose and timing of exposure may have significant influences. Here, we report the behavioral effects of acute, binge-like ethanol exposure at three neurodevelopmental times corresponding to the first, second, and third trimester of human development in C57BL/6J mice. Results show that developmental ethanol exposure consistently delays the development of basic motor skill reflexes and coordination as well as impairs spatial learning and memory. Observed changes in activity and anxiety-related behaviors, however, appear to be dependent on timing of alcohol exposure. The variability in behaviors between different treatment models suggests that these may be useful in evaluating the mechanisms disrupted by ethanol at specific neurodevelopmental times. The results provide further evidence that, regardless of developmental stage, the developing brain is acutely sensitive to alcohol exposure.

Contributions of transgenic mouse studies on the research of hepatitis B virus and hepatitis C virus-induced hepatocarcinogenesis

Transgenic mouse technology has enabled the investigation of the pathogenic effects, including those on development, immunological reactions and carcinogenesis, of viral genes directly in living organism in a real-time manner. Although viral hepatocarcinogenesis comprises multiple sequences of pathological events, that is, chronic necroinflammation and the subsequent regeneration of hepatocytes that induces the accumulation of genetic alterations and hepatocellular carcinoma(HCC), the direct action of viral proteins also play significant roles. The pathogenesis of hepatitis B virus X and hepatitis C virus(HCV) core genes has been extensively studied by virtue of their functions as a transactivator and a steatosis inducer, respectively. In particular, the mechanism of steatosis in HCV infection and its possible association with HCC has been well studied using HCV core gene transgenic mouse models. Although transgenic mouse models have remarkable advantages, they are intrinsically accompanied by some drawbacks when used to study human diseases. Therefore, the results obtained from transgenic mouse studies should be carefully interpreted in the context of whether or not they are well associated with human pathogenesis.

Cow milkα_(s1)-casein induces allergic responses in a mouse model of atopy

α_(s1)-Casein is a potential allergen to induce hypersensitivity in cow milk.We had identifiedα_(s1)-casein and its epitopes in previous studies.The present study aimed to evaluate the allergic mechanism ofα_(s1)-casein in a BALB/c mouse model.The levels of specific IgE,mast cell proteinase,histamine and cytokines in sensitized mice were determined,and the clinical and pathological observation were evaluated.Results showed that the levels of specific IgE,mast cell proteinase,histamine,IL-4,IL-5,IL-10 increased significantly with a dose-dependent trend.The local alveolar septum collapsed or thickened,and lymphatic foci were produced in the spleen and thymus,and the inflammatory cells infiltrated in small intestinal mucosa mesenchyme.In conclusion,the levels of specific IgE,mast cell proteinase,histamine,IL-4,IL-5,IL-10 and some inflammatory factors could possibly serve as allergic biomarkers ofα_(s1)-casein,however,additional studies on signal transduction and gene expression are necessary in future.

Changes of the intestinal endocrine cells in the C57BL/6 mouse after implantation of murine lung carcinoma (3LL): An immunohistochemical quantitative study

AIM: To study the distributions and frequencies of intestinal endocrine cells in the C57BL/6 mouse with immunohistochemical method using seven types of specific antisera against chromogranin A (CGA), serotonin,somatostatin, glucagons, gastrin, cholecystokinin (CCK)-8 and human pancreatic polypeptide (hPP) after abdominal subcutaneous implantation of murine lung carcinoma (3LL).METHODS: The experimental animals were divided into two groups, one is non-implanted Sham and the other is 3LL-implanted group. Samples were collected from six regions of intestinal tract at 28th d after implantation of 3LL cells (1×105 cell/mouse).RESULTS: In this study, five types of immunoreactive (IR) cells were identified except for gastrin and hPP. The regional distributions of the intestinal endocrine cells in the 3LL-implanted group were similar to those of the non-implanted Sham. However, significant decreases of IR cells were detected in 3LL-implanted group compared to those of non-implanted Sham. CGA- and serotonin-IR cells significantly decreased in 3LL-implanted groups compared to that of non-implanted Sham. Somatostatin-IR cells in the jejunum and ileum and CCK-8-IR cells in the jejunum of 3LL-implanted groups significantly decreased compared to that of non-implanted Sham. In addition,glucagon-IR cells were restricted to the ileum and colon of non-implanted Sham.CONCLUSION: Implantation of tumor cell mass (3LL)induced severe quantifiable changes of intestinal endocrine cell density and the abnormality in density of intestinal endocrine cells may contribute to the development of gastrointestinal symptoms such as anorexia and indigestion, frequently encountered in patients with cancer.

Effects of DHRS3 in C2C12 Myoblast Differentiation and Mouse Skeletal Muscle Injury

Myoblast differentiation is an essential process during skeletal muscle development.C2C12 myoblast is a commonly used experimental model to study muscle cell differentiation in vitro.Dehydrogenase/reductase(SDR family)member 3(DHRS3)is a highly conserved member in short-chain alcohol dehydrogenase/reductase superfamily and has been shown to be involved in the metabolism of retinol.Previous experimental results showed that the expression of DHRS3 increased significantly during the differentiation of myoblasts differentiation.However,the effect of DHRS3 on mouse muscle cell differentiation was unclear.The objective of current study was to determine if DHRS3 affected muscle cell differentiation,and if DHRS3 was involved in muscle regeneration.Protein expression was determined by western blot and immunofluorescence analysis.The activation and inhibition of DHRS3 increased and decreased C2C12 myoblast differentiation respectively,which indicated that DHRS3 could affect C2C12 myoblast differentiation.DHRS3 expression was significantly changed during muscle regeneration,with the regeneration of muscle injury,the expression of DHRS3 tended to increase first and then decrease.It suggested that DHRS3 might be involved in muscle regeneration.In summary,this study confirmed the involvement of DHRS3 in C2C12 myoblast differentiation and mouse skeletal muscle regeneration and provided a theoretical basis for further elucidating the molecular mechanism of muscle development.

One extraction tool for in vitro-in vivo extrapolation?SPME-based metabolomics of in vitro 2D,3D,and in vivo mouse melanoma models

Solid phase microextraction(SPME)in combination with high-resolution mass spectrometry was employed for the determination of metabolomic profile of mouse melanoma growth within in vitro 2D,in vitro 3D,and in vivo models.Such multi-model approach had never been investigated before.Due to the low-invasiveness of SPME,it was possible to perform time-course analysis,which allowed building time profile of biochemical reactions in the studied material.Such approach does not require the multiplication of samples as subsequent analyses are performed from the very same cell culture or from the same individual.SPME already reduces the number of animals required for experiment;therefore,it is with good concordance with the 3Rs rule(replacement,reduction,and refinement).Among tested models,the largest number of compounds was found within the in vitro 2D cell culture model,while in vivo and in vitro 3D models had the lowest number of detected compounds.These results may be connected with a higher metabolic rate,as well as lower integrity of the in vitro 2D model compared to the in vitro 3D model resulting in a lower number of compounds released into medium in the latter model.In terms of in vitro-in vivo extrapolation,the in vitro 2D model performed more similar to in vivo model compared to in vitro 3D model;however,it might have been due to the fact that only compounds secreted to medium were investigated.Thus,in further experiments to obtain full metabolome information,the intraspheroidal assessment or spheroid dissociation would be necessary.

Expression and characterization of mouse lactate dehydrogenase subunit C in Escherichia coli.

Objective: To construct a prokaryotic recombinant vector for mouse lactate dehydrogenase-C and to detect its expression in BL21. Methods: The coding sequence of mouse lactate dehydrogenase subunit C was amplified from mouse testis RNA with specific primers and cloned into pGEX-2T after restriction digestion with BamH I and EcoR I. GST fusion protein was expressed after induction with IPTG. Results: Sequencing and restriction digestion of the recombinant plasmid revealed the coding sequence for mouse lactate dehydrogenase subunit C. A protein band of about 60 000 could be induced by IPTG in the recombinant plasmid. Conclusion: The coding sequence of mouse lactate dehydrogenase subunit C was introduced into the pGEX-2T plasmid and a GST-fused protein could be induced at a high level.

Study on the Gene Transcription Level of Hippocampus NGF in C57 Mouse Development

β-Nerve growth factor (β NGF) mRNA levels in hippocampa from C57BL/6J mice of different ages were compared by semi-quantitative RT-PCR method, taking β-actin gene as an internal control. The levels of 6-and 12-month-old C57 mice were much lower compared with that of the l-month-old mouse. However, there was no significant difference between these two groups. This result suggests that gene transcription level of hippocampus β NGF in a C57, mouse descends evidently as it grows up. Base T at Site 294 of β NGF cDNA was substituted by C in the C57, mouse, as revealed by DNA sequencing for the first time. Nevertheless, this polymorphism of nucleotide did not make any difference in amino acid composition.

Construction of Cox7a2 fluorescent vector and its effect on cytochrome C oxidase activity in mouse Sertoli cell line TM4

Objective To construct Cox7a2 fluorescent vector and study its effect on cytochrome C oxidase ( COX) activity in mouse Sertoli cell line TM4. Methods The coding region of CoxTa2 was amplified from mouse Sertoli cell line TM4 by RT-PCR. PCR product was

Assessment of immune responses and intestinal flora in BALB/c mice model of wheat food allergy via different sensitization methods

Increasing incidences showed that food allergies have attracted more and more attention from researchers.BALB/c mice were sensitized with wheat gluten combined with aluminum hydroxide adjuvant via intraperitoneal injection,transdermal sensitization,and oral gavage sensitization route.Results showed that all the three sensitization methods could induce allergic symptoms;increase the serum antibody(total immunoglobulin E(IgE),specific IgE,IgG,IgA)and histamine content;promote the secretion of Th2 cytokines(interleukin(IL)-4,IL-5,IL-13)and inflammatory factors(IL-6,IL-17 A,IL-10);and inhibit the production of Th1 cytokines(IFN-γ,IL-2).However,the allergic symptoms of mice sensitized by intraperitoneal injection were the most obvious among the three models.The level of serum antibodies in intraperitoneal injection group was significantly higher than control.Subsequently,16 S rRNA sequencing technology was used to analyze the intestinal flora of mice.The results showed that the abundance of Firmicutes in the wheat protein sensitized group was lower than that in the normal group,while the abundance of Bacteroidetes was higher,and Lactobacillus was the difference marker in normal group.Bacterial species diversity analysis showed that the species richness and diversity of intestinal flora in mice were decreased,the difference between mice induced by intraperitoneal injection and normal control group mice was the most significant.Taken together,these results show that among three sensitization methods used to build a mouse model with aluminum hydroxide as adjuvant,intraperitoneal injection is the comparably best way to build a mouse sensitization mode.

Influence of whole peptidoglycan of bifidobacterium on cytotoxic effectors produced by mouse peritoneal macrophages

INTRODUCTIONBifidobacteria are physiologically beneficial bacteria which are perdominant in human intestine ,and possess the most important functions .They play an important role in maintaining microbial balance of the intestine .Furthermore , their presence is thought to be an important indication of health of the body [1-4].Whole peptidoglycan ( WPG) is the major component in the cell wall of bifidobacterium ,which is also a biological responsemodifier with nontoxic side dffcets.