Objective To establish a non-irradiated precondition strategy for successful donor specific transplantation tolerance induced by bone marrow transplantation which may be introduced to clinical application by a murine model.Methods Male C57BL/6 and female BALB/c mice were used as skin transplant donors and recipients respectively. In all, 11 groups were studied: group 1, control mice with skin graft and without any other treatment; group 2, mice receiving only donor bone marrow transplantation (DBMT) before skin transplantation; groups 3～5, mice administrated by only high dose FK506, CsA or CTX respectively and then skin transplantation; groups 6～8, mice receiving DBMT preconditioned by high dose FK506, CsA or CTX respectively and followed by skin transplantation. Both skin and bone marrow donors were male C57BL/6 mice in above 8 groups. Mice in groups 9～11 were treated almost equally to groups 6～8 except that skin grafts were from the third party ICR donors to prove specificity of tolerance. Every group included 6 recipients. Survival time of skin graft was recorded. Macrochimerism were examined by PCR method.Results Neither standard dose DBMT nor high dose immnodepressants alone could prolong skin graft survival, and macrochimerism was not detected, either. But skin graft survival time was significantly prolonged and macrochimerism was also detected in mice of 6～8 groups. Survival time of skin graft from the third party mice was not prolonged. Conclusions Bone marrow transplantation preconditioned with high dose immunosuppressants and without irradiation can induce donor specific transplantation tolerance and prolong murine skin graft survival. It may work through the mechanism of establishment of macrochimerism.

Objective To evaluate the value of ultrasound-guided mammotome in the diagnosis and therapy of breast tumor.Methods From May 2006 to February 2009,a total of 205 cases diagnosed with breast tumor by physical examination or ultrasonography underwent ultrasound-guided mammotome in our department.In the patients,142 cases had single tumor and 63 cases showed multiple tumors.Among the 205 cases,impalpable lesions were detected by ultrasonography in 58 patients.Mammotome biopsy system with 11 G/8 G needles were used in the operation.After the surgery,pathological examination was carried out,and short-and medium-term follow-up results of the patients were analyzed.Results In the 205 cases,totally 356 tumors were detected.The size of the tumors ranged from 0.3 to 3.5 cm in diameter.The operation time of the patients was 10.2 min in average(1-50 min).After the surgery,22 patients showed bruise in the surgical area,and 19 patients developed hematoma(three of them received puncture of the hematoma).Post-operative pathological examination showed fibroadenoma in 98 cases,breast hyperplasia in 246 cases(154 of them were complicated with fibroadenoma),7 had intraductal papilloma,4 were diagnosed with breast cyst,and 1 showed tubular adenoma.Follow-up was achieved in 132 patients for 2 to 36 months,during the period,residual tumor was detected in 4 patients(3.0%).All the patients were satisfied with the cosmetic results of the surgery.Conclusions Ultrasound-guided mammotome is a minimally invasive procedure that can safely and reliably diagnose and remove breast tumor,particularly multiple tumors and impalpable lesions.The cosmetic results of the operation are satisfying.

This study examined the adipocytokine-vascular interactions and link between epicardial adipose tissue and coronary artery atherosclerosis. Thirty-four patients undergoing open heart surgery were chosen randomly, and divided into group I (non-coronary artery disease group) and group II (coronary artery disease group). Blood samples were taken through peripheral vein prior to surgery. Plasma levels of a panel of proteins (adiponectin, IL-10, TNF-α) were detected by using ELISA. Epicardial adipose tissue was taken near the proximal tract of the right coronary artery and subcutaneous adipose was taken from the leg before cardiopulmonary bypassing, adiponectin and CD68 + were detected by using RT-PCR and immunohistochemistry. Our results showed that plasma adiponectin level was significantly lower in the group II as compared with group I (P<0.05). There were no differences in plasma concentration (IL-10, TNF-α, tatal-chol, HDL-chol, LDL-chol) between group I and group II. The number of CD68+ cells in epicardial adipose tissue of group II was significantly higher than that in subcutaneous adipose tissue. Adiponectin mRNA expression was 6 fold higher in subcutaneous adipose tissue than in epicardial adipose tissue of group II (P<0.01). Furthermore, the level of adiponectin mRNA in the epicardial adipose tissue in group II was also significantly lower than in group I (P<0.05). We are led to conclude that inflammation that occurs locally in epicardial adipose tissue of CAD contributes to the pathogenesis of coronary artery disease.

Objective: To investigate the expression of voltage-dependent potassium channel 1.3(Kv1.3) mRNA and protein during human monocyte-derived macrophage differentiation into foam cells and its function in foam cell formation. Methods: Human peripheral blood monocytes were isolated from healthy male volunteers by density gradient centrifugation and then by adherent method. The obtained monocytes were cultured for 5 days to differentiate into macrophages. Based on establishment of the human macrophage-derived foam cell model, the expression of Kv1.3 channel was investigated by immunocytochemical staining, reverse transcription-polymerase chain raction (RT-PCR) and Western blot. Furthermore, the effects of rMargatoxin, a Kv 1.3 channel-specific inhibitor, on cholesterol metabolism in macrophages incepting oxidized low density lipoprotein (OxLDL) were studied. Results: After the macrophages co-incubated with 30 mg/L OxLDL at 37 ℃ for 60 hours, the cellular volume obviously enlarged and many red lipid granules were deposited in cytoplasm. The total amount of cholesterol (TC),free cholesterol ( FC ) and cholesterol ester ( CE ) in cells markedly increased and the ratio of CE/TC rose from ( 14.4±6.8) % to (57.9±3.5) % (n=7,P＜0.05). However, the expression of Kv1.3 channel had no significant change. r Margatoxin (0.1 nmol/L and 10 nmol/L) markedly reduced the contents of TC, FC and CE in macrophages and the ratios of CE/TC decreased to (42.8±11.6) % and (22.6±8.0)% , respectively (n=7, P＜0.05). Meanwhile, the red lipid granules deposited in the cytoplasm of macrophages also decreased. Conclusion: These data clearly show that the expression of Ky1.3 channel does not change obviously during human monocyte-derived macrophage differentiation into foam cells and the blocking of it would prevent foam cell formation.

Objective To discuss the effect of body posture change on the catheter tip position of totally implantable venous access port (TIVAP).Methods Under ultrasound guidance,implantation of TIVAP was carried out through bedside puncturing of internal jugular vein or subclavian vein.After the implantation of TIVAP,X-ray chest films of both erect position and supine position were taken to check the catheter tip position.The distance from the upper edge of the first thoracic vertebra to the catheter tip was separately measured on the erect position and supine position chest films.The shift of the catheter tip position was judged by the difference in the distance measured on chest films as well as by the comparison with the bony anatomic marks.Results Successful implantation of TIVAP was accomplished in 86 patients.When the patients changed from erect position to supine position,the catheter tip of TIVAP moved caudally in 71 patients,with the mean displace distance being (12.29±7.48) mm;the catheter tip of TIVAP moved cephalad in 31 patients,with the mean displace distance being (5.00±3.79) mm;and the catheter tip of TIVAP remained in the same position in 2 patients.When the patients changed from erect position to supine position,the catheter tip of TIVAP had a tendency to move toward the foot side,the average displace distance was (-9.32±9.36) mm,the difference in catheter tip location between two photographic positions was statistically significant (P＜0.000 1).No statistically significant correlation existed between the changes of catheter tip position and the sex,age,height,weight as well as body mass index (P＞0.05).Conclusion After the implantation of TIVAP,the position of catheter tip will change with patient's body posture.When patient's posture changes from erect position to supine position the tip of the catheter tends to shift towards the atrium.

Objective To undertake an updated genetic spectrum analysis in patients with hereditary spinocerebellar ataxia (SCA) in mainland China. Methods SCA 1, 2, 3, 6, 7, 8, 10, 12, 17 and dentatorubral-pallidoluysian atrophy (DRPLA) nucleotide repeat mutations were detected in 430 families with autosomal dominant SCA (ADCA) and 237 patients with sporadic ataxias by PCR and DNA sequencing. Subsequently, point and Indel (Insertion/deletion) mutation analyses of SCA5, SCA11, SCA13, SCA14, SCA15/16/29, SCA27, SCA31 and SCA35 were detected in 91 families with ADCA and 196 patients with sporadic ataxias excluded from SCA1, 2, 3, 6, 7, 8, 10, 12, 17 and DRPLA genotypes via PCR and Denaturing High Performance Liquid Chromatography (PCR-DHPLC), Multiplex ligation-dependent probe amplification and DNA direct sequencing analysis. Results Among the 430 ADCA families, there were 25 SCA1 (5.81%), 27 SCA2 (6.28%), 267 SCA3/MJD (62.09%), 8 SCA6 (1.86%), 8 SCA7 (1.86%), 1 SCA12 (0.23%), 1 SCA17 (0.23%) and 2 SCA35 (0.47%), and the remaining 91 families (21.16%) were genetically unidentified. Among the 237 sporadic SCA patients, there were 6 SCA1 (2.53%), 9 SCA2 (3.80%), 23 SCA3/MJD (9.70%) and 3 SCA6 (1.27%), and the remaining 196 (82.7%) were genetically unidentified. No pathogenic point mutation causing SCA5, SCA11, SCA13, SCA14, SCA27 or SCA31 subtypes was found. Conclusion SCA3/MJD is substantially the most common subtype in patients with ADCA and sporadic forms in mainland China, followed by SCA2, SCA1, SCA6 and SCA7. While SCA12, SCA17 and SCA35 are seldom found, SCA5, SCA8, SCA10, SCA11, SCA13, SCA27, SCA31 and DRPLA are very rare. The high proportion of genetically unidentified cases further verify that SCAs are of highly genetic heterogeneity, suggesting that other disease-causing genes might be involved in the negative ADCA pedigrees, and other etiological factors may involve in those sporadic cases other than genetics.

Ebolavirus can cause hemorrhagic fever in humans with a mortality rate of 50%-90%. Currently, no approved vaccines and antiviral therapies are available. Human TIM1 is considered as an attachment factor for EBOV, enhancing viral infection through interaction with PS located on the viral envelope. However, reasons underlying the preferable usage of hTIM-1, but not other PS binding receptors by filovirus, remain unknown. We firstly demonstrated a direct interaction between hTIM-1 and EBOV GP in vitro and determined the crystal structures of the Ig V domains of hTIM-1 and hTIM-4. The binding region in hTIM-1 to EBOV GP was mapped by chimeras and mutation assays, which were designed based on structural analysis. Pseudovirion infection assays performed using hTIM-1 and its homologs as well as point mutants verified the location of the GP binding site and the importance of EBOV GP-hTIM-1 interaction in EBOV cellular entry.
Ebolavirus ; metabolism ; Flow Cytometry ; Glycoproteins ; metabolism ; Hepatitis A Virus Cellular Receptor 1 ; Hepatitis A Virus Cellular Receptor 2 ; Humans ; Membrane Glycoproteins ; metabolism ; Membrane Proteins ; metabolism ; Protein Binding ; Receptors, Virus ; metabolism ; Surface Plasmon Resonance ; Viral Envelope Proteins ; metabolism ; Viral Proteins ; metabolism

Objective: To assess the effect of viral macrophage inflammatory protein (vMIP) -Ⅱ on the expression of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) , and to explore the mechanisms. Methods: A recombinant plasmid pEGFP-N3-K4 (vMIP-Ⅱ plasmid group) and an empty plasmid pEGFP-N3 (empty plasmid group) were separately transfected into 293T cells, and quantitative PCR and Western blot analysis were performed to evaluate the effect of transfection with vMIP-Ⅱ gene on the APOBEC3G expression in 293T cells. Some 293T cells in the empty plasmid group and vMIP-Ⅱ plasmid group were treated with 1 000 IU/ml interferon (IFN) -α for 36 hours, and then Western blot analysis was conducted to determine the APOBEC3G expression in the empty plasmid group and vMIP-Ⅱ plasmid group with or without IFN-α treatment. Some 293T cells transfected with vMIP-Ⅱ plasmids were treated with 75 μmol/L AG490 (a JAK/STAT signaling pathway inhibitor) and 20 μmol/L U0126 (an ERK signaling pathway inhibitor) separately; after 24 hours, total protein was extracted from 293T cells, and Western blot analysis was conducted to determine the expression of APOBEC3G. A recombinant plasmid containing APOBEC3G promoter was constructed by using a luciferase reporter gene, and the promoter fragment included the full-length promoter sequence (POS) of APOBEC3G, sequences with the lengths of 1 560, 960, 720, 480, 420, 360, 330 and 240 bp, and the regulatory element-free region (NEG) of APOBEC3G, separately. Some 293T cells were co-transfected with the recombinant plasmid carrying luciferase reporter gene and vMIP-Ⅱ plasmid (experimental group), or the recombinant plasmid and empty plasmid (control group). Subsequently, the activity of the APOBEC3G promoter was evaluated, and the key promoter region through which the transcriptional activity of APOBEC3G was regulated by vMIP-Ⅱ was analyzed. Statistical analysis was carried out by using t test, one-way analysis of variance and least significant difference (LSD) -t test. Results: The mRNA and protein expression of APOBEC3G was significantly higher in the vMIP-Ⅱ plasmid group (2.500 ± 0.013, 1.472 ± 0.013 respectively) than in the control group (1, 0.364 ± 0.030 respectively; t = 6.22, 6.54 respectively, both P < 0.05) . The APOBEC3G expression significantly differed among the empty plasmid group, vMIP-Ⅱ plasmid group, empty plasmid + IFN-α group and vMIP-Ⅱ plasmid + IFN-α group (1, 2.030 ± 0.108, 2.700 ± 0.081 and 2.600 ± 0.099 respectively; F = 67.026, P < 0.001) , but there was no significant difference between the vMIP-Ⅱ plasmid group and empty plasmid + IFN-α group (t = 3.46, P > 0.05) . The APOBEC3G expression also significantly differed among the vMIP-Ⅱ plasmid group, vMIP-Ⅱ plasmid + AG490 group and vMIP-Ⅱ plasmid + U0126 group (0.617 ± 0.025, 0.179 ± 0.061, 0.359 ± 0.012 respectively; F = 70.019, P < 0.001) , and was significantly lower in the vMIP-Ⅱ plasmid + AG490 group and vMIP-Ⅱ plasmid + U0126 group than in the vMIP-Ⅱ plasmid group (t = 9.66, 11.836 respectively, both P < 0.01) . Luciferase activity assay showed that the promoter activity significantly differed among the vMIP-Ⅱ plasmid groups transfected with POS, 1 560-, 960-, 720-, 480-, 420-, 360-, 330-, 240-bp or NEG sequences (F = 81.092, P < 0.001) , and the APOBEC3G promoter activity decreased greatly from the 720-bp group to 480-bp group. Conclusion vMIP-Ⅱ upregulates the expression of APOBEC3G, likely through the JAK/STAT signaling pathway or the key promoter region regulating the transcriptional activity of APOBEC3G.

This study examined the adipocytokine-vascular interactions and link between epicardial adipose tissue and coronary artery atherosclerosis. Thirty-four patients undergoing open heart surgery were chosen randomly, and divided into group Ⅰ (non-coronary artery disease group) and group Ⅱ (coronary artery disease group). Blood samples were taken through peripheral vein prior to surgery.Plasma levels of a panel of proteins (adiponectin, 1L-10, TNF-α) were detected by using ELISA.Epicardial adipose tissue was taken near the proximal tract of the right coronary artery and subcutaneous adipose was taken from the leg before cardiopulmonary bypassing, adiponectin and CD68 + were detected by using RT-PCR and immunohistochemistry. Our results showed that plasma adiponectin level was significantly lower in the group Ⅱ as compared with group Ⅰ (P＜0.05). There were no differences in plasma concentration (IL-10, TNF-a, tatal-chol, HDL-chol, LDL-chol) between group Ⅰ and group Ⅱ. The number of CD68 + cells in epicardial adipose tissue of group Ⅱ was significantly higher than that in subcutaneous adipose tissue. Adiponectin mRNA expression was 6 fold higher in subcutaneous adipose tissue than in epicardial adipose tissue of group Ⅱ (P＜0.01). Furthermore, the level of adiponectin mRNA in the epicardial adipose tissue in group Ⅱ was also significantly lower than in group Ⅰ (P＜0.05). We are led to conclude that inflammation that occurs locally in epicardial adipose tissue of CAD contributes to the pathogenesis of coronary artery disease.

OBJECTIVE: To screen differentially expressed genes and gene pathways in L02 cell line stably expressing hepatitis C virus (HCV) Ib type nonstructural protein 4B (NS4B) mediated by lentiviral system, and to provide a basis for further research of molecular biological mechanism of NS4B gene in chronic hepatitis C and hepatocarcinogenesis. METHODS: NS4B stably overexpressed L02 cell line and negative control stable L02 cell line, designated as L02-NS4B and L02-mkate2 respectively, were resurrected and amplified in vitro. The differentially expressed genes between L02-NS4B and L02-mkate2 were determined by gene expression microarray from Human Gene 1.0ST. The significant pathways of the differential genes were selected by the Fisher's exact test and χ2 test according to kyoto encyclopedia of genes and genomes (KEGG) database. The differential expression levels of 5 selected genes including protein kinase C delta binding protein (PRKCDBP), tumor protein p53 (TP53), v-akt murine thymoma viral oncogene homolog 1 (AKT1), baculoviral IAP repeat containing 3 (BIRC3) and B-cell lymphoma 2-like1 (BCL2L1) from cDNA microarray data were further verified by real-time quantitative polymerase chain reaction (real-time QPCR). RESULTS: Between L02-NS4B and L02-mkate2, the genes with flurescence intensity ratio >1.2 or <0.8 were considered as differentially expressed genes. A total of 2 682 differentially expressed genes in the known 28 869 human genes were detected in L02-NS4B, 1 446 genes were upregulated and 1 236 genes were downregulated. A total of 41 involved pathways of up-regulated differential genes were identified by KEGG database, mainly including apoptosis, extracellular matrix receptor interaction, cell cycle, pathways in cancer and Toll-like receptor signaling pathway; and 20 involved pathways of down-regulated differential genes were identified, mainly including pathways in cancer, Wnt signaling pathway and cell cycle pathway. Of the 5 upregulated genes selected from cDNA microarray data, 3 genes showed the same differential expression pattern by real-time QPCR as that shown in cDNA microarray data, namely AKT1, BIRC3 and BCL2L1. The confirmation rate of real-time QPCR was 60%. CONCLUSION HCVNS4B can up-regulate or down-regulate the expression of many genes in L02 cells, thus affecting multiple signaling pathways relevant to cell apoptosis, cell cycle and carcinogenesis.
Cell Cycle ; Cell Line ; Gene Expression Profiling ; Hepacivirus ; genetics ; Humans ; Oligonucleotide Array Sequence Analysis ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Transcriptional Activation ; Viral Nonstructural Proteins ; genetics