Effect of simulated microgravity on human monocytic cell proliferation and tissue factor mRNA expression / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of simulated microgravity on the proliferation of human monocytic cells THP-1 and the expression of tissue factor (TF) mRNA.</p><p><b>METHODS</b>THP-1 cells were cultured under a simulated microgravity environment using the rotating cell culture system (RCCS). The changes in the cell proliferation after microgravity culture were assessed by cell counting and cell cycle analysis with flow cytometry. RT-PCR was used to detect the changes in the expression of TF mRNA in THP-1 cells.</p><p><b>RESULTS</b>Culture under simulated microgravity resulted in a significant decrease in the cell number of THP-1 cells in comparison with that of the control cells (P<0.01). After a 24-h culture under microgravity, the G0-Gl phase cells increased from the control level of (46.57∓1.64)% to (67.64∓2.71)% (P<0.05). The cells in both groups showed a low level of TF mRNA expression in the absence of LPS stimulation. A 4-h stimulation with LPS caused up-regulated expression of TF mRNA in both cells, but the microgravity group showed a significantly smaller increase in the expression (2.301∓0.179) than the control group (9.210∓1.328) (P<0.05).</p><p><b>CONCLUSION</b>Microgravity can inhibit the proliferation of THP-1 cells and suppress the cellular expression of TF mRNA.</p>

Urinary level of tissue factor and its procoagulant activity in patients with type 2 diabetes / 南方医科大学学报

<p><b>OBJECTIVE</b>To examine the urinary level of tissue factor (uTF) and its procoagulant activity (PCA) in patients with diabetes mellitus, and explore the relationship between uTF and renal damage in diabetes mellitus.</p><p><b>METHODS</b>Eighty-six patients with type 2 diabetes mellitus were divided into 3 groups according to urine albumin excretion (UACR), namely normal albuminuria group, microalbuminuria group and macroalbuminuria group. The levels of uTF, PCA, blood urea nitrogen (BUN), serum creatinine (CRE), serum cystatin C (CYSC), glycohemoglobin A1c (HbA1c), and high-sensitivity C-reactive protein (hs-CRP) were measured in all the patients and 21 healthy controls.</p><p><b>RESULTS</b>Compared with normal control, the diabetic patients showed significantly increased levels of uTF and PCA. The urinary TF-PCA was positively correlated to BUN, CYSC, CRE, UACR, fasting glucose and hs-CRP, but not to uTF; only hs-CRP, UACR were positively correlated to uTF.</p><p><b>CONCLUSION</b>uTF is probably implicated in the development and progression of diabetic nephropathy.</p>

Isolation and characterization of human rheumatoid arthritis fibroblast-like synoviocytes / 南方医科大学学报

<p><b>OBJECTIVE</b>To isolate and characterize human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs).</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>FLSs 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.</p>

Inhibition of tissue factor expression in endothelial cells by lentivirus mediated shRNA / 中南大学学报(医学版)

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.

Value of plasma tissue factor, tissue factor pathway inhibitor and factor VII assessments in patients with acute myocardial and cerebral infarction / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the clinical implications of changes in plasma tissue factor (TF), tissue factor pathway inhibitor (TFPI) and factor VII (FVII) after the onset of acute myocardial infarction (AMI) and acute cerebral infarction (ACI).</p><p><b>METHODS</b>Sixty-nine patients with AMI, 71 with ACI and 50 age-matched healthy volunteers were enrolled in this study. Blood samples were obtained from the healthy subjects and from the patients at the early stage of AMI and ACI onset for examination of plasma TF and TFPI activity using chromogenic assay, and the plasma TF and TFPI antigens were measured by enzyme-linked immunosorbent assay (ELISA). The plasma FVII coagulation activity (FVII:C) was also measured, and the plasma FVIIa determined using soluble TF assay.</p><p><b>RESULTS</b>Compared with the healthy control group, AMI patients had significantly enhanced plasma TF and TFPI activities and elevated TF and TFPI antigen levels (P<0.05), with also markedly increased FVIIa (P<0.05) but comparable FVII:C (P>0.05). In ACI patients, the plasma TF activity and antigen were obviously increased in comparison with the control group (P<0.05), but plasma TFPI activity and antigen were lowered (P<0.05), and both the FVII:C and FVIIa were markedly higher (P<0.05). Significant differences were noted in plasma TF and TFPI activities and their antigen levels as well as in FVII:C, but not in FVIIa between AMI and ACI patients.</p><p><b>CONCLUSION</b>V Following the onset of AMI and ACI, TF pathway is initiated and the risk of thrombogenesis increases, and the assessment of TF pathway is therefore of value for understanding the development of the condition.</p>

Construction and identification of RNA interference vector for human tissue factor gene / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a RNA interference vector for human tissue factor (TF) gene.</p><p><b>METHODS</b>Human TF short hairpin RNA (shRNA) sequence was designed using online design software (Invitrogen) and synthesized into double-strand oligonucleotide (ds oligo), which was cloned into the pENTRTM/U6 plasmid, followed by transformation of the product into competent Top10 E. coli cells. After expansion of the transformed bacteria, the plasmid was extracted and sequenced, which was subsequently transfected into human umbilical vein endothelial cells (HUVECs). The interference effect of the vector on the target gene expression was detected by RT-PCR and immunofluorescence assay.</p><p><b>RESULTS</b>The sequencing result indicated that the plasmid pENTRTM/U6-RelB-shRNA was constructed correctly, which resulted in effective inhibition of TF expression in HUVECs after transfection.</p><p><b>CONCLUSION</b>The RNA interference vector against human TF gene has been constructed successfully, which may provide a stable transfection vector for potential treatment of blood coagulation abnormalities.</p>