Adenosine monophosphate-activated protein kinase activation enhances embryonic neural stem cell apoptosis in a mouse model of amyotrophic lateral sclerosis

Alterations in embryonic neural stem cells play crucial roles in the pathogenesis of amyotrophic lateral sclerosis. We hypothesized that embryonic neural stem cells from SOD1G93A individuals might be more susceptible to oxidative injury, resulting in a propensity for neurodegeneration at later stages. In this study, embryonic neural stem cells obtained from human superoxide dis- mutase 1 mutant （SOD1G93A） and wild-type （SOD1wv） mouse models were exposed to H202. We assayed cell viability with mitochondrial succinic dehydrogenase colorimetric reagent, and measured cell apoptosis by flow cytometry. Moreover, we evaluated the expression of the adenos- ine monophosphate-activated protein kinase （AMPK） ct-subunit, paired box 3 （Pax3） protein, and p53 in western blot analyses. Compared with SOD1wr cells, SOD1~93A embryonic neural stem cells were more likely to undergo H202-induced apoptosis. Phosphorylation of AMPKct in SOD1G93A cells was higher than that in SOD1wr cells. Pax3 expression was inversely correlated with the phosphorylation levels of AMPKct. p53 protein levels were also correlated with AMPKct phosphorylation levels. Compound C, an inhibitor of AMPKa, attenuated the effects of H20~. These results suggest that embryonic neural stem cells from SOD1C93A mice are more susceptible to apoptosis in the presence of oxidative stress compared with those from wild-type controls, and the effects are mainly mediated by Pax3 and p53 in the AMPKa pathway.

Pattern of expression of the CREG gene and CREG protein in the mouse embryo

Background The cellular repressor of ElA-stimulated genes(CREG) is a secreted glycoprotein that inhibits cell proliferation and/or enhances differentiation.CREG is widely expressed in adult tissues such as the brain,heart, lungs,liver,intestines and kidneys in mice.We investigated the level of CREG expression during mouse embryogenesis and its distribution at 18.5 days post coitus(dpc).Methods Immunohistochemical staining with diaminobenzidine,western blotting and reverse transcription-polymerase chain reaction were used.Results CREG expression was rst detected in mouse embryos at 4.5 dpc.It was expressed at almost all stages up to 18.5 dpc.The level of CREG was found to increase gradually and was highest at 18.5 dpc.Western blotting showed that the CREG protein was expressed at higher levels in the brain,heart,intestines and kidneys than in the lungs and liver at 18.5 dpc.In 9.5 dpc embryos,CREG was expressed only in the endothelial cells of blood vessels,after the vascular lumen had formed.With advanced differentiation, vascular smooth muscle cells developed in the embryonic vascular structures;the expression of smooth muscle a-actin protein and CREG were positive and increased gradually in 10.5 dpc embryonic vessels.CREG expression in the embryonic blood vessels peaked at 15.5 dpc and was reduced slightly at 18.5 dpc.Conclusions These results indicate that CREG is expressed during mouse embryogenesis and might participate in the differentiation of these organs during embryogenesis.

Effect of Panax notoginseng saponins on the expression of beta-amyloid protein in the cortex of the parietal lobe and hippocampus, and spatial learning and memory in a mouse model of senile dementia

BACKGROUND： The pharmacological actions of Panax notoginseng saponins （PNS） lie in removing free radicals, anti-inflammation and anti-oxygenation. It can also improve memory and behavior in rat models of Alzheimer＇s disease. OBJECTIVE： Using the Morris water maze, immunohistochemistry, real-time PCR and RT-PCR, this study aimed to measure improvement in spatial learning, memory, expression of amyloid precursor protein （App） and β -amyloid （A β ）, to investigate the mechanism of action of PNS in the treatment of AD in the senescence accelerated mouse-prone 8 （SAMP8） and compare the effects with huperzine A. DESIGN, TIME AND SETTING： A completely randomized grouping design, controlled animal experiment was performed in the Center for Research ＆ Development of New Drugs, Guangxi Traditional Chinese Medical University from July 2005 to April 2007. MATERIALS： Sixty male SAMP8 mice, aged 3 months, purchased from Tianjin Chinese Traditional Medical University of China, were divided into four groups： PNS high-dosage group, PNS low-dosage group, huperzine A group and control group. PNS was provided by Weihe Pharmaceutical Co., Ltd. （batch No.： Z53021485, Yuxi, Yunan Province, China）. Huperzine A was provided by Zhenyuan Pharmaceutical Co., Ltd. （batch No.： 20040801, Zhejiang, China）. METHODS： The high-dosage group and low-dosage group were treated with 93.50 and 23.38 mg/kg PNS respectively per day and the huperzine A group was treated with 0.038 6 mg/kg huperzine A per day, all by intragastric administration, for 8 consecutive weeks. The same volume of double distilled water was given to the control group. MAIN OUTCOME MEASURES： After drug administration, learning and memory abilities were assessed by place navigation and spatial probe tests. The recording indices consisted of escape latency （time-to-platform）, and the percentage of swimming time spent in each quadrant. The number of A β 1-40, A β 1-42 and App immunopositive neurons in the brains of SAMP8 mice was analyzed by immunohistochemistry. The mRNA content ofApp, tau, acetylcholinesterase, and synaptophysin （Syp） was tested by real time PCR and RT-PCR. RESULTS： The PCR results show that PNS can downregulate the expression of the App gene and upregulate the expression of the Syp gene in the parietal cortex and hippocampus of SAMP8 mice. The therapeutic effects of the PNS high-dosage group were greater than those of the PNS low-dosage group and the huperzine A group （P 〈 0.05）. The results of the Morris water maze and immunohistochemistry indicated that PNS can improve the capacity for spatial learning and memory in SAMP8 mice, and reduce the content of A β 1-40, A β 1-42 and expression of App in the brains of SAMP8 mice. The therapeutic effects of the PNS high-dosage group were greater than that of the PNS low-dosage group and the huperzine A group （P 〈 0.05）. CONCLUSION： These results support the hypothesis that PNS plays a therapeutic and protective role on the pathological lesions and learning dysfunction of Alzheimer＇s disease. The therapeutic effects of PNS for Alzheimer＇s disease are possibly achieved through downregulating the expression of the App gene and upregulating the expression of the Syp gene. The therapeutic effects of PNS are dose-dependent and are greater than the effect of huperzine A.

Lower Concentrations of Glucose or Insulin Decrease the Risk of Various Types of Cancer in the Long-Lived Ames Dwarf Mouse by Increasing the Expression of p27Kip1, a Cell-Cycle Repressor Protein

Introduction. The molecular biological mechanism of the increased incidence of the various types of cancer in obesity or type 2 diabetes in rodents or humans has largely been resolved in recent years. By contrast, the molecular biological mechanism of the decreased, not increased, incidence of the various types of cancer in the homozygous long-lived Ames dwarf mice still remains unresolved. Objective. The first objective of the present study was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the increase, not decrease, in the expression of p27Kip1, a cell cycle repressor protein. The second objective was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the decrease, not increase, in the levels of glucose or insulin. Methods. To achieve these objectives, we first performed western immunoblot analysis of the hepatic expression of p27Kip1 protein. We then performed, using a human breast cancer cell line in vitro, the luciferase reporter plasmid assay to determine whether the translation initiation activity of the p27Kip1 mRNA is increased when the concentrations of either glucose or insulin are decreased. Results and Conclusion. The results of the first objective indicated that the hepatic expression of p27Kip1 protein was up-regulated in the homozygous long-lived Ames dwarf mice as expected. We also found that the lower concentrations of glucose or insulin increased the translation initiation activity of the p27Kip1 mRNA.

Immunolocalization assessment of metastasis-associated protein I in human and mouse mature testes and its association with spermatogenesis

Aim： To investigate the stage-specific localization of metastasis-associated protein 1 （MTA1） during spermatogenesis in adult human and mouse testis. Methods： The immunolocalization of MTA1 was studied by immunohistochemistry and Western blot analysis. The distribution pattern of MTA1 in mouse testis was confirmed by using quantitative analysis of purified spermatogenic cells. Results： The specificity of polyclonal antibody was confirmed by Western blot analysis. MTA1 was found expressed in the nucleus of germ cells, except elongate spermatids, and in the cytoplasm of Sertoli cells; Leydig cells did not show any specific reactivity. MTA1 possessed different distribution patterns in the two species： in humans, the most intensive staining was found in the nucleus of round spermatids and of primary spermatocytes while in mice, the most intense MTA 1 staining was in the nucleus of leptotene, zygotene and pachytene spermatocytes. In both species the staining exhibited a cyclic pattern. Conclusion： The present communication initially provides new evidence for the potential role of MTA1 in mature testis. In addition, its distinctive expression in germ cells suggests a regulatory role of the peptide during spermatogenesis.

Chondrogenic Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells Induced by Cartilage-derived Morphogenetic Protein-2 In Vitro

To study the cartilage differentiation of mouse mesenchymal stem cells （MSCs） induced by cartilage-derived morphogenetic proteins-2 in vitro, the MSCs were isolated from mouse bone marrow and cultured in vitro. The cells in passage 3 were induced into chondrogenic differentiation with different concentrations of recombinant human cartilage-derived morphogenetic proteins-2 （0, 10, 20, 50 and 100 ng/mL）. After 14 days of induction, morphology of cells was observed under phase-contrast microscope. Collagen Ⅱ mRNA and protein were examined with RT-PCR, Western blotting and immunocytochemistry respectively and the sulfate glycosaminoglycan was measured by Alcian blue staining. RT-PCR showed that CDMP-2 could promote expression of collagen Ⅱ mRNA in an dose-dependant manner, especially at the concentration of 50 ng/mL and 100 ng/mL. Immunocytochemistry and Western blotting revealed a similar change. Alcian blue staining exhibited deposition of typical cartilage extracellular matrix. Our results suggest that mouse bone marrow mesencymal stem cells can differentiate into chondrogenic phonotype with the induction of CDMP-2 in vitro, which provides a basis for further research on the role of CDMP-2 in chondrogenesis.

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.

Construction of Prokaryotic Expression Vector of Mouse Nanog Gene and Its Expression

The aim of this study is to construct a prokaryotic expression vector of mouse Nanog gene and to express it in E. coli. A pair of primers was designed according to digestion sites in plasmid pGEX-KG and the Nanog gene sequence published by GenBank. The DNA fragment of 918 bp was amplified by polymerase chain reaction （PCR） from the pNA992 recombinant plasmid with Nanog gene, then cloned into pGEX-KG and transformed into the host E. coli strain TG Ⅰ. The sequence of the fragment was matched with the original sequence of pNA992. It indicated that fusion expression vector, pGEX-KG- Nanog, was constructed successfully. The pGEX-KG-Nanog plasmid was extracted from E. coli strain TG Ⅰ and was transformed into BL21（DE3） for expression. After induction by isopropyl-β-D-thiogalactoside （IPTG） at 37℃, the expression product of Nanog gene was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, and the expression condition was optimized. Nanog fusion protein was successfully expressed in the form of inclusion bodies. The molecular weight of the inclusion body was 63 kDa. Meanwhile, the optimum condition for the expression of Nanog fusion protein was induced with 0.8 mmol L^-1 IPTG for 5 h. The mouse Nanog gene was successfully expressed in E. coli, which laid a foundation for the purification of Nanog protein and for the preparation of polyclonal antibody.

High-yield expression of recombinant mouse coagulation factor Ⅶ in methylotrophic yeast Pichia pastoris

Objective: To explore high-yield secretory expression of recombinant mouse coagulation factor Ⅶ (rmF Ⅶ ) protein in Pichia pastoris (P. pastoris). Methods: The fragment of mF Ⅶ cDNA was amplified by PCR from a pcDNA3-mFⅦ plasmid. Then the cDNA fragment was subcloned into α-factor secretion signal open reading frame of pPIC9K secretory expression vector. The mutagenesis of mF Ⅶ was performed by Site-Direct Mutation and then verified by DNA sequencing. The yeast expression vector of rmF Ⅶ, named as pPIC9K-rmFⅦ, was linearized with Sac I and transferred into GS115 strains(his-Mut+)by electroporation. The recombinants were identified by direct PCR and selection on MM and MD plates. rmF Ⅶ was expressed in recombinant strains (his+Mut+) for 4 d. The expression level and activation of rmF Ⅶ in the BMMY medium were detected by SDS-PAGE and Western blot respectively. Results:pPIC9K-rmFⅦ was constructed and transferred to GS115 strains successfully. 48-hour post induction by methanol rmFⅦ protein was secreted into the culture supernatant. The molecular weight of the expressed products was shown to be about 46 kD by SDS-PAGE analysis. Western blot showed that the expressed rmF Ⅶ exhibited specificity and antigenicity. Conclusion: Since mFⅦ is considered as a tumor-targeting molecule , this study may provide a basis for further anti-tumor strategy on rmFⅦ.

PTPN22 and islet-specific autoimmunity:What have the mouse models taught us?

An allelic variant of the protein tyrosin phosphatase non-receptor 22(PTPN22) gene, PTPN22 R620 W, constitutes the strongest non-HLA genetic risk factor for the development of type 1 diabetes(T1D). A numberstudies using mouse models have addressed how PTPN22 predisposes to T1D. PTPN22 downmodulation, overexpression or expression of the variant gene in genetically manipulated mice has generated controversial results. These discrepancies probably derive from the fact that PTPN22 has differential effects on innate and adaptive immune responses. Moreover, the effects of PTPN22 are dependent on other genetic variables. Here we discuss these findings and try to explain the discrepancies. Exploring the mechanism by which PTPN22 contributes to islet-specific autoimmunity could help us understand its role in T1D pathogenesis and exploit it as a potential therapeutic target to prevent the disease.

Differential expression of glial cell line-derived neurotrophic factor splice variants in the mouse brain

Glial cell line-derived neurotrophic factor(GDNF) plays a critical role in neuronal survival and function. GDNF has two major splice variants in the brain,α-pro-GDNF and β-pro-GDNF, and both isoforms have strong neuroprotective effects on dopamine neurons. However, the expression of the GDNF splice variants in dopaminergic neurons in the brain remains unclear. Therefore, in this study, we investigated the mRNA and protein expression of α-and β-pro-GDNF in the mouse brain by real-time quantitative polymerase chain reaction, using splice variant-specific primers, and western blot analysis. At the mRNA level,β-pro-GDNF expression was significantly greater than that of α-pro-GDNF in the mouse brain. In contrast, at the protein level,α-pro-GDNF expression was markedly greater than that of β-pro-GDNF. To clarify the mechanism underlying this inverse relationship in mRNA and protein expression levels of the GDNF splice variants, we analyzed the expression of sorting protein-related receptor with A-type repeats(SorLA) by real-time quantitative polymerase chain reaction. At the mRNA level, SorLA was positively associated with β-pro-GDNF expression, but not with α-pro-GDNF expression. This suggests that the differential expression of α-and β-pro-GDNF in the mouse brain is related to SorLA expression. As a sorting protein, SorLA could contribute to the inverse relationship among the mRNA and protein levels of the GDNF isoforms. This study was approved by the Animal Ethics Committee of Xuzhou Medical University, China on July 14, 2016.

Lutein delays photoreceptor degeneration in a mouse model of retinitis pigmentosa

Retinitis pigmentosa is a retinal disease characterized by photoreceptor degeneration.There is currently no effective treatment for retinitis pigmentosa.Although a mixture of lutein and other antioxidant agents has shown promising effects in protecting the retina from degeneration,the role of lutein alone remains unclear.In this study,we administered intragastric lutein to Pde6brd10 model mice,which display degeneration of retinal photoreceptors,on postnatal days 17(P17)to P25,when rod apoptosis reaches peak.Lutein at the optimal protective dose of 200 mg/kg promoted the survival of photoreceptors compared with vehicle control.Lutein increased rhodopsin expression in rod cells and opsin expression in cone cells,in line with an increased survival rate of photoreceptors.Functionally,lutein improved visual behavior,visual acuity,and retinal electroretinogram responses in Pde6brd10 mice.Mechanistically,lutein reduced the expression of glial fibrillary acidic protein in Müller glial cells.The results of this study confirm the ability of lutein to postpone photoreceptor degeneration by reducing reactive gliosis of Müller cells in the retina and exerting anti-inflammatory effects.This study was approved by the Laboratory Animal Ethics Committee of Jinan University(approval No.LACUC-20181217-02)on December 17,2018.

The expression of Usp26 gene in mouse testis and brain

Deubiquitinating enzymes （DUBs） play an important role in ubiquitin-dependent processes as negative regulators of protein ubiquitination. Ubiquitin-specific protease 26 （USP26） is a member of this family. The expression of Usp26 in mammalian testis and in other tissues has yet to be fully elucidated. To study the expression of Usp26 mRNA and protein in various murine tissues, reverse transcription （RT）-PCR and immunohistochemistry analyses were carried out. The RT-PCR analysis showed that the Usp26 transcript was expressed in all of the tested tissues. USP26 protein localization was examined by immunohistochemistry, and it was shown that USP26 was not detectable at 20 days postpartum, with the expression restricted to the cytoplasm of condensing spermatids （steps 9-16）, Leydig cells and nerve fibers in the brain. In addition, the USP26 protein was detected at moderate levels in myocardial ceils, the corpus of epidydimis, epithelium of the renal tubules and the seminal gland of postnatal day 35 mice. Its spatial and temporal expression pattern suggests that Usp26 may play an important role in development or function of the testis and brain. Further research into these possibilities is in progress.

Expression of HSP72 in Mouse Preimplantation Embryos with Heat Shock

The objective of the paper was to detect HSP72 expression and HSP72 gene sequence in heat shocked mouse preimplantation embryos and the effects of different thermo conditions on hatching rates of embryos. The mouse blastocysts cultured in vitro were heat treated at 40℃ and 38℃ for 1 h, 2 h and 3 h and then recovered at 370C for 3 h, 2 h and 1 h, respectively, to detect their HSP72 gene expression by using RT-PCR after the total R.NA extraction. The hatching rate of the blastocysts for different treated groups was recorded and the expression of liSP72 in the blastocysts was determined by Western blot. The results showed that all the groups of blastocysts, including the control, had the expression of HSP72 gene. The expression of HSP72 protein had the highest level in the embryos stressed at 38℃ for 2 h, and it was significantly higher than that in the control group. The expression of HSP72 in the groups of blastocysts treated at 40℃ was not significantly different from that in the control group. The embryos with induction of mild heat shock at 38℃ for 2 h, then subjected to heat shock at 40℃ for 2 h, had a significant higher （P〈0.05） hatching rate of 54.74% compared to 47.85% in the embryos treated directly at 40℃ for 2 h. The above results indicated that the mouse blastocysts were sensitive to heat shock and a mild heat shock induced HSP72 gene expression. Induction of HSP72 expression with mild heat shock helped embryos to tolerate more severe heat shocks.

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.

Analysis of Proteotranscriptomics Landscape Reveals Differentially Regulated Pathways in Toxoplasma gondii Infected Mouse Liver

Toxoplasma gondii (T. gondii) an intracellular protozoan parasite, infects mammals including human population world-wide. Upon primary infection, the parasite contributes to mild flu like symptoms in immune competent host, but life threatening complication is seen in immune compromised patients and in pregnant women. Understanding the host-parasite interaction is critical for understanding the pathogenesis and biology parasite reactivation in the host. In this study, we used proteotrasncriptomics analyses by integrating the transcriptomics and proteomics data of T. gondii infected mouse liver to uncover the effector molecules responsible for disease pathogenesis that can be used as candidate markers for diagnosis and drug target. With this aim, we systematically integrated transcriptomicand proteomic data, representing the parasite infected mouse liver. Out of 2758 differentially expressed genes (DEGs) and 301 differentially expressed proteins (DEPs), 159 overlapping genes were identified. Among them, 86 genes were upregulated and 72 were downregulated in their respective mRNA and protein levels in the infected condition. Gene Ontology (GO) analysis revealed that the upregulated genes were mostly associated with immune system processes whereas the downregulated genes were involved in oxidation-reduction process and metabolism of lipid, and fatty acids. Protein-protein interaction (PPI) network analysis uncovered an interaction-hub including, Psmb8, Psmb9 and Tap1 for upregulated proteins and Cyp1A2, Cyp4A10 and Cyp3A11 for down-regulated proteins. Further studies are needed to validating these effector molecules. These molecules are likely to play a vital role in disease pathogenesis, as well as can be used as potential diagnostic marker and drug target candidates.

ICOS-Ig combined with CsA induces long term survival of cardiac allografts in mouse

Objective: To study the synergistic effect of ICOS-Ig combined with cyclosporine (CsA) on mouse heart transplantation and explore its therapeutic potential. Methods: ICOS-Ig fusion protein was generated by fusing the extracellular portion of human ICOS and Fc portion of human IgG. To investigate the effect of ICOS-Ig on T-cell proliferation in vitro, ICOS-Ig or IgG was added to the primary MLR cultures (BALB/c spleen T cells as responder cells and irradiated C57BL/6 spleen cells as stimulator cells). The cells responsiveness rates were detected by 3H-TdR methods. Then the T cells of each group in primary MLR were cultured as responder cells for secondary MLR, and irradiated C57BL/6 (donor) or C3H (third party) spleen cells as stimulator cells. To study the effect of ICOS-Ig on T-cell proliferation in vivo, CFSE-labeled C57BL/6 spleen cells were transferred to irradiated BALB/c mice. Mice were then treated with IgG, ICOS-Ig or CsA. Seventy two hours after transfer, the spleen cells of the mice were harvested for the detection of CD4+CFSE+ and CD8+CFSE+ by FACS. C57BL/6 mouse underwent transplantation of the hearts of BALB/c mouse and were then randomly divided into five equal groups: no treatment group, control IgG treated group (250 μg i.p. d2, 4, 6), ICOS-Ig treated group (250 μg i.p. d2, 4, 6), CsA treated group (10 mg/kg i.p. d0-6), ICOS-Ig combined with CsA group. The cardiac allograft survival was monitored by daily palpation. Results: In primary MLR, ICOS-Ig inhibited T-cell proliferation, (inhibition ratio 58±8.2% in 50 μg/ml). In secondary MLR, ICOS-Ig specifically inhibited donor spleen cells, which suggested ICOS-Ig could induce donor-specific hyporesponsiveness. In the CFSE dye assay, CD4+CFSE+ and CD8+CFSE+ in ICOS-Ig and CsA group was stronger than those in control group, which showed ICOS-Ig and CsA could inhibit the proliferation of allo-reactive T cells in vivo. In mouse heart transplantation model, survival was significantly prolonged in animals treated with ICOS-Ig or CsA as compared with controls. Moreover, ICOS-Ig combined with CsA group had even longer engraftment (>100 d) than ICOS-Ig or CsA used alone. In histological examination, it was found that there were congestions and edemas in no treatment and IgG treated recipients, together with a lot of inflammatory cells infiltrated. Allogeneic hearts from ICOS-Ig and/or CsA immunized recipients revealed milder histological changes. It was revealed in mechanical analysis that splenic T cells from recipients also exhibited depressed mixed leukocyte reactions (MLR) and cytotoxic lymphocyte reactions (CTL). Conclusion: These data suggest that ICOS-Ig combined with CsA induces a long-term survival of mouse cardiac allografts, whereas monotherapy is less effective in this regard. Thus, ICOS-Ig combined with CsA treatment may be a novel regimen to combat allograft rejection.

SIRT2、P65和Survivin在乳腺癌中的表达及其与临床病理特征的关系

目的通过检测SIRT2、P65和生存素(Survivin)蛋白在乳腺癌中的表达,探讨其在乳腺癌中的相互关系及与乳腺癌的临床病理特征的关系。方法384例乳腺浸润性癌患者作为乳腺癌组,31例乳腺纤维腺病患者作为对照组,采用免疫组化EnVision二步法检测两组乳腺病变组织内SIRT2、P65、Survivin的蛋白表达,同时收集乳腺癌组患者相关临床病理特征资料(年龄、分子分型、TNM分期、组织学分级、淋巴结转移及月经情况),分析SIRT2、P65及Survivin蛋白在乳腺癌组中的表达与临床病理特征的关系,分析SIRT2、P65及Survivin蛋白在乳腺癌组表达的相关性。结果乳腺癌组中SIRT2、Survivin蛋白高表达,表达率均高于对照组,差异具有统计学意义(P<0.05);SIRT2、P65及Survivin蛋白在不同分子分型、TNM分期乳腺癌患者的高表达,差异有统计学意义(P<0.05);SIRT2蛋白在LuminalB1型中的高表达率高于其他分型(P<0.05),P65蛋白在LuminalA型中的高表达率高于其他分型(P<0.05),Survivin蛋白在HER-2阳性型的高表达率高于其他分型(P<0.05);SIRT2、P65及Survivin蛋白在TNM分期中Ⅲ~Ⅳ期高表达率较Ⅰ~Ⅱ期高(P<0.05);SIRT2和P65蛋白在淋巴结转移与非转移乳腺癌患者间的高表达,差异有统计学意义(P<0.05),其中转移的高表达率较非转移高;P65和Survivin蛋白在不同组织学分级乳腺癌患者间高表达,差异有统计学意义(P<0.05),其中Ⅰ级的高表达率较Ⅱ、Ⅲ级高;乳腺癌组中SIRT2与P65和Survivin蛋白表达均呈正相关(r分别为0.371和0.310,P<0.001),P65与Survivin蛋白表达呈正相关(r=0.466,P<0.001)。结论乳腺癌组织存在SIRT2及Survivin蛋白表达上调,这可能是影响乳腺癌组织学分级及TNM分期、分子分型等临床病理特征的机制。

Dissociation of FK506 Binding Protein 12.6 from Ryanodine Receptor Type 2 Is Regulated by cADPR but not β-Adrenergic Stimulation in Mouse Cardiomyocytes

AIMS: β-adrenergic augmentation of Ca2+ sparks and cardiac contractility has been functionally linked to phosphorylation-dependent dissociation of FK506 binding protein 12.

Effects of Andrographolide on the Activation of Mitogen Activated Protein Kinases and Nuclear Factor-κ B in Mouse Peritoneal Macrophage-derived Foam Cells

Objective： To observe the effect of andrographolide on the activation of mitogen-activated protein kinases （MAPKs） and expression of nuclear factor- kB （NF-kB） in macrophage foam cells. Methods： The mouse peritoneal macrophages were cultured in the media in the presence of oxidized low-density lipoprotein （ox-LDL）, ox-LDL＋andrographolide, or neither （control）. The phosphorylation of MAPK molecules （p38MAPK, JNK, ERK1/2） and the expressions of NK- kB p65 were examined by Western blot. Results： As compared with cells in the control group, the expressions of phospho-p38 and NF- kB p65 were increased in the cells cultured with either ox-LDL or ox-LDL＋andrographolide （P〈0.01）, but attenuated significantly in the presence of ox-LDL＋ andrographolide when compared with ox-LDL （P〈0.05）. The phospho-JNK increased in the presence of either ox-LDL or ox-LDL＋andrographolide when compared with control cells （P〈0.01）, but no significant difference existed between ox-LDL and ox-LDL＋andrographolide （P〉0.05）. The expression of phospho-ERK1/2 was increased in the presence of ox-LDL compared with the control cells （P〈0.01）, but no significant differences existed between the cells cultured in the presence of ox-LDL＋andrographolide and the control medium （P〉0.05）. Conclusions： Andrographolide could inhibit the activation of ERK1/2, p38MAPK and NK-kB induced by ox-LDL in macrophage foam cells, which might be one of its mechanisms in preventing atherosclerosis.