SPI1 exacerbates iron accumulation and promotes osteoclast formation through inhibiting the expression of Hepcidin.

BACKGROUND: Osteoporosis (OP) can be caused by an overactive osteoclastic function. Anti-osteoporosis considerable therapeutic effects in tissue repair and regeneration because bone resorption is a unique osteoclast function. In this study, we mainly explored the underlying mechanisms of osteoclasts' effects on osteoporosis. METHODS: RAW264.7 cells were used and induced toward osteoclast and iron accumulation by M-CSF and RANKL administration. We investigated Hepcidin and divalent metal transporter 1 (DMT1) on iron accumulation and osteoclast formation in an ovariectomy (OVX)-induced osteoporosis. Osteoporosis was induced in mice by OVX, and treated with Hepcidin (10, 20, 40, 80 mg/kg, respectively) and overexpression of DMT1 by tail vein injection. Hepcidin, SPI1, and DMT1 were detected by immunohistochemical staining, western blot and RT-PCR. The bioinformatics assays, luciferase assays, and Chromatin Immunoprecipitation (ChIP) verified that Hepcidin was a direct SPI1 transcriptional target. Iron accumulation was detected by laser scanning confocal microscopy, Perl's iron staining and iron content assay. The formation of osteoclasts was assessed using tartrate-resistant acid phosphatase (TRAP) staining. RESULTS: We found that RAW264.7 cells differentiated into osteoclasts when exposed to M-CSF and RANKL, which increased the protein levels of osteoclastogenesis-related genes, including c-Fos, MMP9, and Acp5. We also observed higher concentration of iron accumulation when M-CSF and RANKL were administered. However, Hepcidin inhibited the osteoclast differentiation cells and decreased intracellular iron concentration primary osteoclasts derived from RAW264.7. Spi-1 proto-oncogene (SPI1) transcriptionally repressed the expression of Hepcidin, increased DMT1, facilitated the differentiation and iron accumulation of mouse osteoclasts. Overexpression of SPI1 significantly declined luciferase activity of HAMP promoter and increased the enrichment of HAMP promoter. Furthermore, our results showed that Hepcidin inhibited osteoclast differentiation and iron accumulation in mouse osteoclasts and OVX mice. CONCLUSION: Therefore, the study revealed that SPI1 could inhibit Hepcidin expression contribute to iron accumulation and osteoclast formation via DMT1 signaling activation in mouse with OVX.

Iron accumulation induced by hepcidin1 knockout accelerates the progression of aging osteoporosis.

OBJECTIVE: Iron accumulation is associated with osteoporosis. This study aims to explore the effect of chronic iron accumulation induced by hepcidin1 deficiency on aging osteoporosis. METHODS: Iron accumulation in hepcidin1 knockout aging mice was assessed by atomic absorption spectroscopy and Perl's staining. Bone microarchitecture was observed using Micro-CT. Hepcidin, ferritin, oxidative stress, and markers of bone turnover in serum were detected by enzyme-linked immunosorbent assay. Bone formation and resorption markers were measured by real-time quantitative PCR. Cell aging was induced by D-galactose treatment. CCK-8, flow cytometry, EdU assays, and Alizarin red staining were performed to reveal the role of hepcidin1 knockout in cell model. Iron Colorimetric Assay Kit and western blot were applied to detect iron and ferritin levels in cells, respectively. RESULTS: In hepcidin1-knockout mice, the ferritin and iron contents in liver and tibia were significantly increased. Iron accumulation induced by hepcidin1 knockout caused a phenotype of low bone mass and deteriorated bone microarchitecture. Osteogenic marker was decreased and osteoclast marker was increased in mice, accompanied by increased oxidative stress level. The mRNA expression levels of osteoclast differentiation markers (RANKL, Mmp9, OPG, Trap, and CTSK) were up-regulated, while bone formation markers (OCN, ALP, Runx2, SP7, and Col-1) were down-regulated in model group, compared to wild type mice. In vitro, hepcidin1 knockdown inhibited proliferation and osteogenic differentiation, while promoted apoptosis, with increased levels of iron and ferritin. CONCLUSION: Iron accumulation induced by hepcidin1 deficiency aggravates the progression of aging osteoporosis via inhibiting osteogenesis and promoting osteoclast genesis.

Opening a door to the spatiotemporal history of plants from the tropical Indochina Peninsula to subtropical China.

The complexity of global biodiversity in the tropical Indochina Peninsula and subtropical China bioregions has fascinated biologists for decades, but little is known about the spatiotemporal patterns in these regions. Accordingly, the aims of present study were to investigate the evolutionary and distribution patterns of Engelhardia in these regions and establish a model for examining biogeographic patterns and geological events throughout the tropical Indochina Peninsula and subtropical China. The effects of geological events occurring in the area between the Indochina Peninsula and subtropical China bioregions on the two trees species (i.e., E. roxburghiana and E. fenzelii) were evaluated. A robust phylogenetic framework of 884 individuals from 79 populations was used to generate time-calibrated cytoplasmic and nuclear phylogenetic frameworks based on cpDNA, nrDNA, and nSSR data, respectively. When considered along with ancestral area reconstructions, the genetic data were also used to assess and reconstruct the species' population genetic structure and diversity. These analyses yielded important information about the (1) historical distribution relationships between the tropical and subtropical flora of China; (2) effects of the East Asian summer monsoon (EASM) on the evolutionary history of Asia's plants; and (3) importance of biogeography in conservation planning. Although cytoplasmic-nuclear discordance indicated cpDNA and nrDNA were subject to distinct evolutionary mechanisms that reflected respective evolutionary histories of the plastid and nuclear genomes of prior demographic and biogeographic events. The tropical elements of Engelhardia occupied the Indochina Peninsula during the early Eocene, whereas the subtropical elements were transformed from the tropical elements during Miocene cooling and the onset of the EASM at the Oligocene-Miocene boundary, intensified during the late Miocene and Pliocene, facilitating the transformation of Engelhardia from the tropical Indochina Peninsula to subtropical China. Demographic history provided insights into prominent planning frameworks in conservation biology, namely that subtropical China functioned as a refugium during past climate oscillations and will continue to serve in this capacity in the future.

Changes in tumor suppressors and inflammatory responses during hydrogen peroxide-induced senescence in rat fibroblasts.

Cell proliferation and senescence are processes induced by oxidative stress. In this study, we aimed to establish a cellular model of rapid proliferation and senescence of rat tail-tip fibroblasts by hydrogen peroxide (H2O2), a well-known oxidant. On this basis, changes in oxidative stress, inflammatory response and cell cycle of fibroblasts were studied. After H2O2 treatment, cell counting and flow cytometry results showed that 50 µM of H2O2 for 12 h and 100 µM for 8 h effectively promoted fibroblast proliferation, while 500 µM rapidly led to cell cycle arrest. In addition, stimulation with H2O2 at a concentration of 50 µM also promoted the inflammatory effects of the cells. At a concentration of 100 µM H2O2, the cellular antioxidant system began to collapse at 8 h and began to affect cellular activity. 500 µM of H2O2 at 4 h the levels of senescence-associated ß-galactosidase, a marker of senescence and oxidative stress, were almost positive in fibroblasts. In addition, we found that the risk of fibroblasts carcinogenesis increased with increased H2O2 stimulation. The results of this study indicate that H2O2 can cause rapid proliferation and senescence of fibroblasts and that its mechanism of action may be mainly through influencing cellular antioxidant systems, cellular inflammatory responses and cell cycle.

Miocene diversification of a golden-thread nanmu tree species (Phoebe zhennan, Lauraceae) around the Sichuan Basin shaped by the East Asian monsoon.

Understanding the role of climate changes and geography as drivers of population divergence and speciation is a long-standing goal of evolutionary biology and can inform conservation. In this study, we used restriction site-associated DNA sequencing (RAD-seq) to evaluate genetic diversity, population structure, and infer demographic history of the endangered tree, Phoebe zhennan which is distributed around the Sichuan Basin. Genomic patterns revealed two distinct clusters, each largely confined to the West and East. Despite sympatry of the two genomic clusters at some sites, individuals show little or no evidence of genomic introgression. Demographic modeling supported an initial divergence time between the West and East lineages at ~15.08 Ma with further diversification within the West lineage at ~7.12 Ma. These times largely coincide with the two independent intensifications of the East Asian monsoon that were initiated during the middle (Langhian) and late Miocene (Messinian), respectively. These results suggest that the Miocene intensification phases of the East Asian monsoon played a pivotal role in shaping the current landscape-level patterns of genetic diversity within P. zhennan, as has been found for the interspecific divergence of other subtropical Chinese plants. Based on isolation-by-distance and species distribution modeling, we hypothesize that P. zhennan followed a ring diversification which was facilitated by the Sichuan Basin acting as barrier to gene flow. In situ and ex situ conservation management plans should consider the results obtained in this study to help secure the future of this beautiful and culturally significant endangered tree.

IGF-1 induces cellular senescence in rat articular chondrocytes via Akt pathway activation.

Cellular senescence decreases cell proliferation over time and is characterized by typical markers, including larger cell volume, a flattened morphology, irreversible cell cycle arrest, augmentation of senescence-associated ß-galactosidase (SA-ß-gal) activity and senescence-associated secretory phenotype. A variety of factors are implicated in the process of cellular aging, which mediates an organisms' lifespan. Insulin-like growth factor-1 (IGF-1) serves an essential role in regulating cell growth, division, proliferation and senescence. In the present study, the role of IGF-1 and the downstream Akt signaling pathway in rat articular chondrocyte senescence was assessed. The results of the current study demonstrated that IGF-1 promoted cellular senescence in rat articular chondrocytes via activation of SA-ß-gal and the upregulation of p53 and p21 mRNA and protein levels. IGF-1 enhanced Akt phosphorylation and treatment with Akt inhibitor, MK-2206, significantly suppressed the induction of these markers. Overall, the results indicated the involvement of IGF-1 and Akt in senescence exhibited by rat articular chondrocytes.

Comparison of Gut Microbiota Diversity and Predicted Functions Between Healthy and Diseased Captive Rana dybowskii.

The gut microbiota plays a key role in host health, and disruptions to gut bacterial homeostasis can cause disease. However, the effect of disease on gut microbiota assembly remains unclear and gut microbiota-based predictions of health status is a promising yet poorly established field. Using Illumina high-throughput sequencing technology, we compared the gut microbiota between healthy (HA and HB) and diarrhoeic (DS) Rana dybowskii groups and analyzed the functional profiles through a phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis. In addition, we estimated the correlation between gut microbiota structures and predicted the functional compositions. The results showed significant differences in the phylogenetic diversity (Pd), Shannon, and observed richness (Sobs) indices between the DS and HB groups, with significant differences observed in the gut microbiota composition between the DS group and the HA and HB groups. Linear discriminant analysis (LDA) effect size (LEfSe) results revealed that Proteobacteria were significantly enriched in the DS group; Bacteroidetes were significantly enriched in the HA and HB groups; and Aeromonas, Citrobacter, Enterococcus, Hafnia-Obesumbacterium, Morganella, Lactococcus, Providencia, Vagococcus, and Staphylococcus were significantly enriched in the DS group. Venn diagrams revealed that there were many more unique genera in the DS group than the HA and HB groups. Among 102 sensitive species selected using the indicator method, 33 indicated a healthy status and 69 (e.g., Acinetobacter, Aeromonas, Legionella, Morganella, Proteus, Providencia, Staphylococcus, and Vagococcus) indicated a diseased status. There was a significant and positive association between the composition and functional composition of the gut microbiota, thus indicating low functional redundancy of the frog gut bacterial community. Rana dybowskii disease was associated with changes in the gut microbiota, which subsequently disrupted bacterial-mediated functions. The results of this study can aid in revealing the effect of the R. dybowskii gut microbiota on host health and provide a basis for elucidating the mechanism of the occurrence of R. dybowskii disease.

Comparison of the gut microbiota of Rana amurensis and Rana dybowskii under natural winter fasting conditions.

Rana amurensis and R. dybowskii occupy similar habitats. As temperatures decrease with the onset of winter, both species migrate to ponds for hibernation. Our goal was to determine whether different species possess different intestinal microbiota under natural winter fasting conditions. We used high-throughput Illumina sequencing of 16S rRNA gene sequences to analyse the diversity of intestinal microbes in the two species. The dominant gut bacterial phyla in both species were Bacteroidetes, Proteobacteria and Firmicutes. Linear discriminant analysis (LDA) effect size revealed significant enrichment of Proteobacteria in R. amurensis and Firmicutes in R. dybowskii. There were significant differences in the gut microbiota composition between the species. The core operational taxonomic unit numbers in R. amurensis and R. dybowskii shared by the two species were 106, 100 and 36. This study indicates that the intestinal bacterial communities of the two frog species are clearly different. Phylum-level analysis showed that R. amurensis was more abundant in Proteobacteria and Verrucomicrobia than R. dybowskii was This is the first study of the composition and diversity of the gut microbiota of these two species, providing important insights for future research on the gut microbiota and the role of these bacterial communities in frogs.

Effects of Tiletamine-xylazine-tramadol Combination and its Specific Antagonist on AMPK in the Brain of Rats.

INTRODUCTION: Tiletamine-xylazine-tramadol (XFM) has few side effects and can provide good sedation and analgesia. Adenosine 5'-monophosphate-activated protein kinase (AMPK) can attenuate trigeminal neuralgia. The study aimed to investigate the effects of XFM and its specific antagonist on AMPK in different regions of the brain. MATERIAL AND METHODS: A model of XFM in the rat was established. A total of 72 Sprague Dawley (SD) rats were randomly divided into three equally sized groups: XFM anaesthesia (M group), antagonist (W group), and XFM with antagonist interactive groups (MW group). Eighteen SD rats were in the control group and were injected intraperitoneally with saline (C group). The rats were sacrificed and the cerebral cortex, cerebellum, hippocampus, thalamus, and brain stem were immediately separated, in order to detect AMPKα mRNA expression by quantitative PCR. RESULTS: XFM was able to increase the mRNA expression of AMPKα1 and AMPKα2 in all brain regions, and the antagonist caused the opposite effect, although the effects of XFM could not be completely reversed in some areas. CONCLUSION: XFM can influence the expression of AMPK in the central nervous system of the rat, which can provide a reference for the future development of anaesthetics for animals.

[Application of area percent of positive neuron and relative intensity of staining grey level in the image quantitative analysis of rat brain tissues immunohistochemistry].

OBJECTIVE: The acetylcholine expression in hypothalamus arcuate nucleus is detected and then the images are processed and analyzed. The features of the image quantitative analysis of immunohistochemistry (IHC) with the method combining two parameters of area percent of positive neuron (APPN) and relative intensity of staining grey level (RISGL) were investigated. METHODS: Samples were the im-munohistochemical slices of acetylcholine(ACh)expression of hypothalamic arcuate nucleus cholinergic neurons in the process of exercise in-duced immunosuppression, which included twelve groups of "0 w, 2 w, 4 w, 6 w" and three groups of "control, immediately after exercise, 3 hours after exercise" in every week. IHC technology was used to detect the ACh expression. The image quantitative analysis of IHC was con-ducted in accordance with the parameters of ACh total area of positive neuron (TAPN), average intensity of staining grey level (AISGL), APPN, RISGL, APPN/RISGL. Then the differences among APPN, RISGL and traditional parameters in the quantitative analysis were com-pared and the advantages were found. RESULTS: The changes of TAPN and APPN showed almost the same variation. Namely the corresponding significant differences could be found through these two parameters(P < 0.05), but the sensitivity and anti-interference of APPN was higher. The results of AISGL and RISGL were not coincident completely. Furthermore, with the combination of APPN and RISGL, the positive expres-sion could be reflected better than any single parameter. CONCLUSIONS: The parameters of immunohistochemical image analysis, APPN and RIS-GL, can be reliable and accurate in image quantitative analysis of IHC. The combination of APPN and RISGL can not only reflect the expres-sion of positive neurons, but also help analyze its mechanism, which is better than traditional analysis parameters.

Hepatitis B virus induces expression of cholesterol metabolism-related genes via TLR2 in HepG2 cells.

AIM: To investigate whether hepatitis B virus (HBV) exacerbates hepatic cholesterol accumulation, and explore the underlying mechanisms. METHODS: HepG2 cells were infected with adenovirus (Ad) containing 1.3-fold overlength HBV genome. Real-time polymerase chain reaction and Western blotting were used to measure mRNA and protein expression of target genes. Cholesterol accumulation was measured by fluorescence microscopy. Cell toxicity due to Ad-HBV treatment was determined by the mitochondrial tetrazolium assay. The protein levels of toll-like receptors (TLRs) were determined by Western blotting. RESULTS: Ad-HBV increased hepatic cholesterol accumulation and enhanced the mRNA and protein levels of low-density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMGCoAr) mRNA and protein expression in HepG2 cells. In addition, these inductive effects were partly offset by suppressing TLR2 expression levels by small interfering RNA in HepG2 cells. CONCLUSION: Ad-HBV increases LDLR and HMGCoAr expression, resulting in exacerbated cholesterol accumulation in HepG2 cells, which was mediated via the TLR2 pathway.

Dynamic characteristics of MR diffusion-weighted imaging in a rabbit liver VX-2 tumor model.

PURPOSE: To investigate prospectively dynamic characteristics of the apparent diffusion coefficient (ADC) on MR diffusion-weighted imaging (DWI) in a rabbit VX-2 tumor model. MATERIALS AND METHODS: Forty New Zealand rabbits were included in the study, and 47 rabbit VX-2 tumor models were developed by direct and intrahepatic implantation after opening the abdominal cavities. DWI was carried out periodically and respectively on days 7, 14 and 21 after implantation. The VX-2 tumor samples were studied by pathology. The distinction of VX-2 tumors on DWI was assessed by their ADC values by analysis of variance (ANOVA) using SPSS12.0 software. RESULTS: The ADC values (mean ± SD) × 10(-3) mm(2)/s of 47 VX-2 tumors in the peripheral and central areas were 2.18 ± 0.29, 1.96 ± 0.33, 1.80 ± 0.35, 2.20 ± 0.29, 2.05 ± 0.30 and 1.96 ± 0.48, respectively, on days 7, 14 and 21 after implantation. ADC values of 47 VX-2 tumors in the area of the tumor periphery, center and normal parenchyma were higher when the b-value was 100 s/mm(2) than those when the b-value was 300 s/mm(2) (F = 17.964, p < 0.001; F = 13.986, p < 0.001; F = 128.681, p < 0.001). ADC values in the area of normal liver parenchyma were higher than those in the area of the VX-2 tumor periphery and center when the b-value was 100 or 300 s/mm(2). ADCs of viable tumor cells in VX-2 tumors were lower on DWI than those in the area of normal liver parenchyma around the tumor, and ADCs of dead tumor cells in VX-2 tumors were unequal, including high, equal and low values, but they were higher than in the area of normal liver parenchyma around tumors after dead tumor cells had been liquefied or had become cystic. CONCLUSION: ADC is correlated with the tumor histology and degree of malignancy, and DWI has potential value for dynamically monitoring tumors and evaluating the degree of malignancy and therapeutic effect.

[Effect of deletion mutant IPS-1 on IFN-beta induction and its antiviral to HSV-1].

AIM: The deletion mutant gene of IFN-beta promoter stimulator 1(DeltaIPS-1) was constructed by depleting the amino acid region from 300 to 444 loci and the recombinant plasmid pEF-BOS-FLAG/DeltaIPS-1 were constructed to study the effect of deletion mutant DeltaIPS-1 to IFN-beta induction and its antiviral to HSV-1. METHODS: The mutant DeltaIPS-1 was obtained by overlap extension PCR. The depleting mutant DeltaIPS-1 recombinant plasmid pEF-BOS-FLAG/DeltaIPS-1 was constructed and identified by Xba I/Cla I digestion and DNA sequencing. HEK293T was Transiently transfected using the calcium phosphate precipitation method. The expression of pEF-BOS-FLAG/IPS-1 and pEF-BOS-FLAG/DeltaIPS-1 in HEK293T cell line was detected by Western blot. After transfected with different plasmids, HEK293T cells were infected by HSV-1 at different multiplicity of infection (MOI). The cytopathic effect (CPE) of the different plasmids in transfected cell groups was determined by amido black stainning. The viral titer of the supernatant of the cell culture medium in different plasmids of transfected groups at different time points was tested by plaque assay. RESULTS: The eukaryotic expression vector of the depleting mutant IPS-1 gene was constructed successfully. The secretory volume of IFN-beta in pEF-BOS-FLAG/DeltaIPS-1 class was much lower compared with that in pEF-BOS-FLAG/IPS-1 class. The plaque assay showed the virus titre in pEF-BOS-FLAG/DeltaIPS-1 class was higher than that in pEF-BOS-FLAG/IPS-1 transfected class. CONCLUSION: DeltaIPS-1 could decrease the secretory volume of IFN-beta in HEK293T cell and could not completely suppress the CPE of the cell infected by HSV-1. The antiviral function of DeltaIPS-1 to HSV-1 is weakened to some extent.

Gene expression and MR diffusion-weighted imaging after chemoembolization in rabbit liver VX-2 tumor model.

AIM: To investigate the dynamic characteristics and the correlation between PCNA, Bax, nm23, E-cadherin expression and apparent diffusion coefficient (ADC) on MR diffusion-weighted imaging (DWI) after chemoembolization in rabbit liver VX-2 tumor model. METHODS: Forty New Zealand rabbit liver VX-2 tumor models were included in the study. DWI was carried out periodically after chemoembolization. All VX-2 tumor samples in each group were examined by histopathology and Strept Avidin-Biotin Complex (SABC) immunohistochemical staining. RESULTS: The PCNA expression index in VX-2 tumors was higher than in the normal parenchyma around the tumor (P < 0.001). Nm23, Bax or E-caderin expression index in VX-2 tumors were lower than in the normal parenchyma around the tumor (all P < 0.001). PCNA and nm23 expression in the VX-2 tumor periphery first increased and then decreased (P < 0.001 and P = 0.03, respectively), while the expression of Bax and E-cadherin before and after chemoembolization was insignificant. When b-value was 100 s/mm(2), there was a linear correlation between PCNA expression and ADC in the area of VX-2 tumor periphery (P < 0.001), and PCNA expression in VX-2 tumor periphery influenced the ADC. CONCLUSION: The potential of VX-2 tumor infiltrating and metastasizing decreases, while its ability to proliferate increases for a short time after chemoembolization. To some degree, the ADC value indirectly reflects the proliferation of VX-2 tumor cells.

Characteristics of liver on magnetic resonance diffusion-weighted imaging: dynamic and image pathological investigation in rabbit liver VX-2 tumor model.

AIM: To investigate dynamical and image pathological characteristics of the liver on magnetic resonance (MR) diffusion-weighted imaging (DWI) in the rabbit VX-2 tumor model. METHODS: Forty New Zealand rabbits were included in the study and VX-2 tumor piece was implanted intrahepatically. Fifteen animals received two intrahepatic implantations while 25 had one intrahepatical implantation. DWI, T1- and T2-weighted of magnetic resonance imaging (MRI) were carried out on the 7th and the 14th d after implantation and DWI was conducted, respectively on the 21st d. Ten VX-2 tumor samples were studied pathologically. RESULTS: The rate of lump detected by DWI, T1WI and T2WI was 78.7%, 10.7% and 53.5% (c2=32.61, P<0.001) on the 7th d after implantation and 95.8%, 54.3% and 82.9% (c2=21.50, P<0.001) on the 14th d. The signal of most VX-2 tumors on DWI was uniform and it was equal on the map of apparent diffusion coefficient (ADC). The signal of VX tumors did not decrease on the 7th d after implantation, most of them slowly growing during the week following implantation without significant cell dying within the tumor. VX-2 tumors grew increasingly within 14 d after implantation but the signal of most VX-2 tumors on DWI or on the map of ADC was uniform or uneven and ADC of VX tumors decreased obscurely or slightly because tumor necrosis was still not obvious. On the 21st d after implantation, the signal of most VX-2 tumors on DWI or on the map of ADC was uneven because tumor necrosis was evident and ADC of VX-2 tumor necrotic areas decreased. The areas of viable cells in VX-2 tumors manifested a high signal on DWI and a low signal on the map of ADC. The areas of dead cells or necrosis in VX-2 tumors manifested low signals on DWI and low, equal or high signals on the map of ADC but they manifested high signals on DWI and on the map of ADC at the same time when the areas of necrotic tumor became liquefied or cystic. The border of tumors on DWI appeared gradually distinct and internal signals of tumor became progressively uneven. CONCLUSION: The manifestations of viable, necrotic and liquefied or cystic areas in VX-2 tumors on DWI are typical and DWI is of significant and potential values in clinical application in both the early detection and diagnosis of liver tumors.

[Preparation and application of anti-serum against mouse Foxp3].

AIM: To prepare the high titer and specific anti-serum against mouse Foxp3 and then use them to identify Foxp3 expression in normal mouse tissues. METHODS: The Foxp3 fragment was amplified by polymerase chain reactions(PCR) and cloned into the prokaryotic expression vector pGEX-6p-2. The recombinant plasmid pGEX-6p-2/Foxp3 was transformed into E.coli BL21(DE3) to be expressed. The expressed fusion protein GST-Foxp3 was analyzed by SDS-PAGE and Western blot. The polyclonal clanti-serum was obtained by immunizing New Zealand rabbits with the purified fusion protein as antigen. The Foxp3 expression in normal mouse tissues was detected by Western blot with the anti-serum. RESULTS: The expressed products were analyzed by SDS-PAGE and Western blot. The results showed that the molecular weight of the expressed protein was about 45 000. The titer of the anti-serum was above 1:12 800. We observed that the anti-serum could recognize Foxp3 protein expressed in NIH3T3 cells by immunoblotting. Western blot results showed that the Foxp3 protein was expressed highly in spleen, thymus and lymph nodes, less expressed in stomach, but not expressed in skeletal muscle and adipose tissue. CONCLUSION: High titer antiserum against mouse Foxp3 is produced and can be used to identify the Foxp3 expression in normal mouse tissues.

Characteristics and pathological mechanism on magnetic resonance diffusion-weighted imaging after chemoembolization in rabbit liver VX-2 tumor model.

AIM: To investigate dynamic characteristics and pathological mechanism of signal in rabbit VX-2 tumor model on diffusion-weighted imaging (DWI) after chemoembolization. METHODS: Forty New Zealand rabbits were included in the study and forty-seven rabbit VX-2 tumor models were raised by implanting directly and intrahepatically after abdominal cavity opened. Forty VX-2 tumor models from them were divided into four groups. DWI was performed periodically and respectively for each group after chemoembolization. All VX-2 tumor samples of each group were studied by pathology. The distinction of VX-2 tumors on DWI was assessed by their apparent diffusion coefficient (ADC) values. The statistical significance between different time groups, different area groups or different b-value groups was calculated by using SPSS12.0 software. RESULTS: Under b-value of 100 s/mm(2), ADC values were lowest at 16 h after chemoembolization in area of VX-2 tumor periphery, central, and normal liver parenchyma around tumor, but turned to increase with further elongation of chemoembolization treatment. The distinction of ADC between different time groups was significant respectively (F = 7.325, P < 0.001; F = 2.496, P < 0.048; F = 6.856, P < 0.001). Cellular edema in the area of VX-2 tumor periphery or normal liver parenchyma around tumor, increased quickly in sixteen h after chemoembolization but, from the 16th h to the 48th h, cellular edema in the area of normal liver parenchyma around tumor decreased gradually and that in the area of VX-2 tumor periphery decreased lightly at, and then increased continually. After chemoembolization, Cellular necrosis in the area of VX-2 tumor periphery was more significantly high than that before chemoembolization. The areas of dead cells in VX-2 tumors manifested low signal and high ADC value, while the areas of viable cells manifested high signal and low ADC value. CONCLUSION: DWI is able to detect and differentiate tumor necrotic areas from viable cellular areas before and after chemoembolization. ADC of normal liver parenchyma and VX-2 tumor are influenced by intracellular edema, tissue cellular death and microcirculation disturbance after chemoembolization.

[Induction of protective immunity against Schistosoma japonicum by pcDNA3.1/Sj20.8 nucleic acid vaccine in BALB/c mice].

Sj20.8 gene was amplified by PCR and inserted into eukaryotic expression plasmid pcDNA3.1 to construct recombinant plasmid pcDNA3.1/Sj20.8, which was then injected into the quadriceps femoris of the BALB/c mice. Results showed that the Sj20.8 antigen was low expressed in the local tissue of the mice, and was not able to significantly reduce eggs in the liver than in the control mice.

[Construction of Sjcb2 DNA vaccine and its expression in HeLa cells].

A recombinant plasmid containing cathepsin B endopeptidase of Schistosoma japonicum (Sjcb2) was constructed, indentified by PCR, restrictive enzyme, digestion and DNA sequencing, and expressed into mammalian cells. Immunochemistry examination showed that the Sjcb2 gene can be expressed in the eukaryotic system, providing a basis for the development of schistosome DNA vaccine.

[Acquisition and analysis of 149 ESTs and 18 novel genes of Schistosoma japonicum].

OBJECTIVE: To acquire and analyze adult stage Schistosoma japonicum (Chinese strain) expressed sequence tags and new genes from an adult S. japonicum cDNA library, and to search new vaccine candidates and drug targets. METHODS: A cDNA library was constructed from adult stage S. japonicum. Clones were selected randomly from the cDNA library and were sequenced. ESTs and new genes were acquired after analysis in GenBank databases by BLAST and other programs. All ESTs and new genes were submitted to GenBank and received accession numbers. RESULTS: 149 ESTs were acquired from a total 382 clones that were randomly selected from the adult S. japonicum cDNA library. All ESTs were successfully submitted to the dbEST at Genbank. Some of them were homologous with sequences of male, female, egg, schistosomula, cercaria and miracidia of S. japonicum. 18 new genes of adult S. japonicum were acquired. Some genes were housekeeping genes and some genes might be interesting as vaccine candidates or drugs targets. CONCLUSIONS: The EST strategy is a rapid, efficient and economical method to acquire ESTs and to discover new genes of adult stage S. japonicum from cDNA libraries.