Hematopoietic Stem Cell Transplantation ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; therapy ; Male ; Middle Aged ; Splenectomy ; adverse effects

OBJECTIVETo evaluate the effect of early application of high-volume hemofiltration treatment (HVHF) on the levels of lactic acid, pro-inflammatory cytokines and C-reactive protein (CRP) in plasma, as well as APACHE II score in patients suffering from severe sepsis.

METHODSThirty patients meeting the diagnosis of severe sepsis were enrolled in the trial within 24 hours of insults. The level of lactic acid, interleukin-6 (IL-6) and CRP in plasma were measured before HVHF and at 24, 48 or 72 h following HVHF treatment.

RESULTThe plasma levels of lactic acid and IL-6 decreased significantly at 24 h, 48 h, 72 h after HVHF (P <0.05), while, IL-10 did not differ significantly following HVHF (P>0.05), when compared with that before HVHF.

CONCLUSIONThe early application of HVHF could clear the plasma lactic acid and pro-inflammatory cytokines, and improve the tissue oxygenation in severe sepsis.

APACHE ; Adult ; C-Reactive Protein ; analysis ; Female ; Hemofiltration ; methods ; Humans ; Interleukin-10 ; blood ; Interleukin-6 ; blood ; Lactic Acid ; blood ; Male ; Middle Aged ; Sepsis ; blood ; therapy ; Treatment Outcome ; Young Adult

OBJECTIVETo achieve high expression of human renal cell carcinoma-associated antigen G250 in Escherichia coli.

METHODSThe gene fragments encoding the protein obtained by PCR was cloned into prokaryotic expression vector pET32a(+) and expressed in E. coli Rosseta. The immunogenicity of the recombinant protein was evaluated by Western blotting.

RESULTSThe plasmid pET32a(+)/G250 was constructed and expressed in E. coli Rosseta successfully. Western blot analysis showed that the recombinant protein could be specifically recognized by monoclonal antibody M75.

CONCLUSIONEfficient G250 expression is achieved in prokaryotic expression system, which may facilitate further functional study of the protein and its monoclonal antibody preparation.

Antibodies, Monoclonal ; immunology ; Antibody Specificity ; immunology ; Antigens, Neoplasm ; genetics ; immunology ; metabolism ; Biomarkers, Tumor ; genetics ; immunology ; metabolism ; Blotting, Western ; Carbonic Anhydrase IX ; Carbonic Anhydrases ; genetics ; immunology ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; Gene Expression ; Humans ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

The Pichia pastoris strain GS115-PreS could produce a high expression level of full-length PreS protein that secreted to the supernatant after methanol induction in the fermentation. The Western blot analysis showed a single band with expected molecular mass of 48kD and that the major component of the particles was the full-length PreS protein (PreS1 + PreS2 + S) and small envelope protein (S) of 48 and 28 kD, respectively. Electron microscopy image showed PreS particles with 30 nm in diameter. The supernatants of the fermentation were desalted and concentrated. Purified PreS protein was obtained by DEAE-SFF anion exchange column chromatography and the PreS particles were obtained by ultracentrifugation and sucrose density gradient. The ELISA assay results proved that both full-length PreS protein and particles showed high immunogenicity and specificity. P/N ratio further demonstrated that the immunogenicity of the particles is higher than the full-length PreS protein.
Hepatitis B Surface Antigens ; biosynthesis ; genetics ; Hepatitis B virus ; immunology ; Humans ; Pichia ; genetics ; metabolism ; Protein Precursors ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification ; Viral Envelope Proteins ; biosynthesis ; genetics

OBJECTIVE: To construct the recombinant lentivirus RNAi vector, and to determine whether the lentivirus mediated short hairpin RNA (shRNA) can inhibit the tissue factor (TF) expression in endothelial cells. METHODS: Two short hairpin RNAs targeting to human TF were cloned into pENTRTM/U6 plasmid to obtain an entry clone, and the positive clones were verified by sequencing. A recombination reaction was performed between the pENTR/U6 entry construction and pLenti6/BLOCKiTTM-DEST vector, and then the positive clones were confirmed by sequencing. The 293FT cell line was transfected by the above recombined plasmid and lentivirus packing materials, the culture supernatant was harvested, and the virus titer was determined. RT-PCR and ELISA were used to observe the inhibition of TF gene expression after the lentivirus transduction in human umbilical vein endothelial cells. RESULTS: The shRNA sequences targeting to human TF were cloned into the vectors, and an entry clone and an expression clone were constructed successfully, which were proved by sequence determination. Viral particles were packaged in the 293FT cell line, all virus stocks were collected, and the transfection titer was 5*10(5)/transduced unit. RT-PCR and enzyme linked immunosorbent assay demonstrated that the lentivirus stocks could suppress the TF expression in endothelial cells remarkably. CONCLUSION Lentivirus RNAi vectors containing human TF gene are successfully constructed, and lentivirus mediated shRNA can inhibit the TF expression in endothelial cells, which may provide a highly effective method for the prevention and treatment of thrombo-embolic diseases.
Base Sequence ; Down-Regulation ; Endothelial Cells ; cytology ; metabolism ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; Molecular Sequence Data ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Thromboplastin ; biosynthesis ; genetics ; Umbilical Veins ; cytology

BACKGROUNDAll-trans retinoic acid (ATRA) can influence the tumor cell proliferation cycle, and some chemotherapeutic drugs are cycle specific. In this study, we hypothesize that ATRA can enhance chemotherapeutic drug sensitivity by affecting the cell cycle of tumor cells.

METHODSThe cell cycle of LoVo cells was evaluated using flow cytometry (FCM). Cell viability was analyzed using the MTT assay. The morphologic changes in the treated LoVo cells were measured with acridine orange (AO)/ethidium bromide (EB) staining. Expression of survivin in LoVo cells was analyzed by immunofluorescence assay.

RESULTSAfter LoVo cells were treated with ATRA, the G0/G1 ratio of the tumor cells increased and the cell ratio of S- and G2/M-phase decreased. Viability of the cells decreased significantly after combined treatment with ATRA and 5-fluorouracil (5-FU) or mitomycin c (MMC) and was evaluated by fluorescence microscopy. Expression level of survivin in the tumor cells decreased after ATRA combination treatment.

CONCLUSIONSATRA enhances drug sensitivity of the LoVo cell line to cell cycle-specific agents and inhibits the expression of survivin in LoVo cells. The combination of ATRA and 5-FU or MMC promoted cell apoptosis, and the mechanism involved in apoptosis may be related to inhibition of survivin gene expression.

Cell Line, Tumor ; Colonic Neoplasms ; drug therapy ; metabolism ; Drug Resistance, Neoplasm ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; Neoplasm Proteins ; genetics ; Tretinoin ; pharmacology

OBJECTIVETo identify protein markers for the early diagnosis of pancreatic cancer by a comparative proteomic method.

METHODSComparative analysis on the pancreatic peripheral blood protein profiling from 20 pancreatic cancer patients, 10 chronic pancreatitis patients and 20 cancer-free controls from May 2007 to September 2008 was carried out by two-dimensional fluorescence electrophoresis (2D-DIGE). Differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The significance difference proteins were confirmed by Western-blot.

RESULTSA differentially expressed proteins: complement 3 (C3) was identified. The gray level of C3 in pancreatic cancer tissue, chronic pancreatitis, and normal control group were 1.63 ± 0.28, 0.65 ± 0.13 (t = 11.81, P = 0.00) and 0.88 ± 0.19 (t = 9.93, P = 0.00), respectively. C3 was high expression in pancreatic cancer group compared with normal control group. The expression of C3 was higher in pancreatic cancer group than in chronic pancreatitis group. The high expression of C3 in pancreatic carcinoma was confirmed by Western blot.

CONCLUSIONS2D-DIGE and MALDI-TOF-MS technology is a quick, easy and practical method to screen for specific biomarkers in serum of patients with pancreatic carcinoma. The identified protein C3 in this study may be as specific serum biomarkers of pancreatic carcinoma.

Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor ; blood ; Case-Control Studies ; Complement C3 ; analysis ; Early Diagnosis ; Female ; Humans ; Male ; Middle Aged ; Pancreatic Neoplasms ; blood ; diagnosis ; Pancreatitis, Chronic ; blood ; Proteomics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Two-Dimensional Difference Gel Electrophoresis

OBJECTIVETo study the difference of gene expression profile among colorectal cancer tissue, pericancerous mucosa and normal mucosa, and to screen associated novel genes in colorectal carcinogenesis by DNA microarray.

METHODScDNA chip containing approximate 8000 genes was used to detect differentially expressed genes in colorectal cancer tissues, pericancerous mucosa and normal mucosa, and to screen associated novel genes in colorectal carcinogenesis by DNA microarray.

RESULTSAs compared with normal mucosa, 769 genes differentially expressed in cancerous tissue were identified, which included 363 up-regulated and 406 down-regulated genes. In pericancerous mucosa 3 cm away from cancerous tissues, 155 genes differentially expressed were identified, of whom 52 genes were up-regulated and 103 were down-regulated. In pericancerous mucosa 5 cm away from cancerous tissues, 230 genes differentially expressed were identified, of whom 46 genes were up-regulated and 184 genes were down-regulated. The genes expressed differentially were associated with several functional types. According to the primary results, the differentially expressed genes with prominent functions included tumor-related genes, genes regulating cell proliferation and apoptosis, transcriptional control genes, and construction and degradation of extracellular matrix-associated genes. The cancerous mucosa was obviously different from the normal mucosa(about 20%, 769/3944). The differences between the normal mucosa and pericancerous mucosa were relatively small (3.9%,5.8%).

CONCLUSIONSDifferent tissues have their own biological property. Several genes play roles in the development of colorectal carcinogenesis. Genes in adjacent non-cancerous tissues are also expressed differentially, leading to a malignant change.

Colorectal Neoplasms ; genetics ; pathology ; DNA, Complementary ; genetics ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Oligonucleotide Array Sequence Analysis ; methods

The automatic cleaning machine we have developed, adopts a SCM system in automatic cleaning. The machine has five functions: ultrasonic cleaning, cold or hot water spraying, drying and greasing. The clinical applications show that the machine with a good effectiveness is suitable for the cleaning of many surgical instruments. It also raises working efficiency, cuts down on the cost of repair and maintenance and reduces the injury and infection to nurses caused by manual cleaning, satisfying the needs of clinical applications.
Automation ; instrumentation ; Disinfection ; instrumentation ; Equipment Design ; Surgical Instruments ; standards ; Ultrasonics ; instrumentation

OBJECTIVETo isolate and characterize human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs).

METHODSThe synovial membrane tissues were obtained from 4 RA patients, 1 chondroma patient and 1 healthy subject and FLS were isolated by means of tissue culture. The cell morphology was observed by phase-contrast microscope and the cell surface markers were detected by flow cytometry.

RESULTSThe FLSs were successfully cultured from the synovial membrane tissues with good cell homogeneity after the third passage. The FLSs of the 3rd to 7th passages were stable and proliferated actively, followed by slow proliferation and aging since the 8th passage. Flow cytometry showed that the 4th-passage FLSs from the RA patients contained 99.04% CD90(+) cells, 2.73% CD3(+) cells, 0.29% CD3(-)CD19(+) cells, 2.81% CD3(-)CD16(+)CD56(+) cells, 5.89% CD14(+) cells, and 54.17% CD55(+) cells. The presence of interleukin-1 receptor type I (IL-1RI, 158.63-/+20.32 pg/ml) and IL-1beta (4.67-/+0.82 pg/ml) were detected in the cell culture supernatant of the 4th-passage FLSs from the RA patients by enzyme-linked immunosorbent assay ELISA.

CONCLUSIONFLSs from RA patients can be effectively culture by means of tissue culture, and the cultured FLSs show high expressions of CD90, IL-1RI and IL-1beta.

Adult ; Aged ; Arthritis, Rheumatoid ; pathology ; Cell Proliferation ; Cell Separation ; Cells, Cultured ; Female ; Fibroblasts ; pathology ; Humans ; Interleukin-1beta ; metabolism ; Male ; Middle Aged ; Receptors, Interleukin-1 Type I ; metabolism ; Synovial Membrane ; cytology ; pathology ; Thy-1 Antigens ; metabolism