The research progress of tissue factor pathway inhibitor / 中华检验医学杂志

Tissue factor pathway inhibitor (TFPI) is a major inhibitor of tissue factor-mediated extrinsic coagulation pathway, mainly derived from microvascular endothelial cells. Recent studies have found that TFPI plays a role in hemophilia, sepsis, antiphospholipid syndrome, venous thromboembolism and other diseases, and participates in the occurrence and development of diseases through anticoagulation mechanism. At present, there are many methods to detect the source, content and function of TFPI, which are helpful for the diagnosis and treatment of clinical diseases.

Qilin pills prevent ovarian hyperstimulation syndrome of patients after IVF-ET by regulating plasma-related indicators / 中国临床药理学与治疗学

AIM: To study the preventive effect of Qilin pill on ovarian hyperstimulation syndrome (OHSS) after in vitro fertilization and embryo transfer (IVF-ET) and its effects on vascular endothelial growth factor (VEGF), tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in plasma. METHODS: Sixty-four patients undergoing IVF-ET treated in our hospital from January 2016 to January 2019 were selected. On the day of ovulation induction injection of human chorionic gonadotropin (HCG), 32 patients with high risk factors of OHSS were randomly divided into two groups. The control group received western medicine therapy, while the observation group received extra Qilin pill. The incidence of mild to moderate OHSS, fresh cycle transplant cancellation rate, plasma VEGF, TF, TFPI levels, and clinical outcomes of patients undergoing IVF-ET (HCG positive rate, biochemical pregnancy rate, clinical pregnancy rate) were compared between the two groups.RESULTS:There was no severe OHSS occurred in the two groups, the incidence of OHSS in the observation group (12.50%) and the cancellation rate of fresh cycle transplantation (15.63%) were lower than those in the control group (50.00%, 43.75%)(χ2=6.063,P=0.014); The levels of VEGF and TF in the observation group on the day of egg retrieval and embryo transfer were [(368±103) pg/mL, (392±91) pg/mL],[(24±4)pg/ mL,(29±4) pg/mL], which were lower than the control group [(436±117) pg/mL, (448±108) pg/mL],[(26±4) pg/mL, (31±4) pg/mL] (t=2.450,2.237,4.093,5.204,P=0.017,0.029,<0.001,<0.001); The plasma TFPI levels in the observation group on the day of egg retrieval and embryo transfer were [(73±18) ng/mL,(66±12) ng/mL], higher than the control group [(62±16)ng/mL, (58±10) ng/mL](t=2.550,3.032,P=0.014,0.004); The biochemical pregnancy rate in the observation group (8.70%) was lower than that in the control group (42.86%) (χ2=4.147, P=0.042),the clinical pregnancy rate (91.30%) was higher than that of the control group (57.14%) (χ2=4.147,P=0.042).CONCLUSION:Qilin pill can prevent the occurrence of severe OHSS after IVF-ET, reduce the occurrence of mild to moderate OHSS, decrease the cancellation rate of fresh cycle transplantation and improve the pregnancy outcome after IVF-ET; Its mechanism may be related to the regulation of the expression of VEGF, TF and TFPI.

RG108 affects the proliferation and apoptosis of NSCLC cells by regulating TFPI-2 methylation and TMPRSS4 expression / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effect of RG108 on the proliferation and apoptosis of human non-small cell lung cancer (NSCLC) cell lines (A549, H1299) and explore its molecular mechanism. Methods: A549 and H1299 cells were cultured in vitro and treated with different concentrations of RG108. The cell proliferation, cell cycle and apoptosis were detected by MTT assay and Flow cytometry, respectively. qPCR and Western blotting (WB) were used to detect the TFPI-2 mRNA and protein expressions as well as the expression of TMPRSS4 in cells. Meanwhile, the methylation status and degree of TFPI-2 promoter in cells were detected with Methylation-specific PCR (MSP) and colorimetry. Finally, siRNA-TFPI-2 and pcDNA3.0-TMPRSS4 plasmids were used to silence TFPI-2 or overexpress TMPRSS4, and then the changes in cell proliferation and apoptosis were detected. Results: After treatment with RG108, the proliferation rate of A549 and H1299 cells were significantly decreased (all P<0.05), while the apoptosis rate were significantly increased(P<0.05), the cell cycle were arrested in G1/S phase (P<0.05), and the intracellular mRNA and protein expressions of TFPI-2 were significantly increased (P<0.01 or P<0.05). Meanwhile, the methylase degree in TFPI-2 promoter region and the expression of TMPRSS4 in cells were all significanly decreased ( all P<0.05). After TFPI-2 silence, the proliferation levels of A549 and H1299 cells were significantly increased(all P<0.05); however, the apoptosis rate of A549 and H1299 cells were significantly reduced after transfection with pcDNA3.0-TMPRSS4(all P<0.05). Conclusion: RG108 can inhibit proliferation of A549 and H1299 cells and promote apoptosis by inhibiting the methylation of TFPI-2 and negatively regulates the expression of TMPRSS4.

Correlation of interaction between genetic polymorphisms with thrombin activity in plasma and pathological stages of esophageal carcinoma / 西安交通大学学报(医学版)

Objective To investigate the correlation of interaction between polymorphisms of prothrombin gene G20210A in 3' untranslated region and tissue factor pathway inhibitor (TFPI) gene C399T in 5' untranslated region with thrombin activity in plasma and the pathological stages of esophageal carcinoma.Methods Based on TNM method,we selected 198 patients with stage Ⅰ esophageal carcinoma,198 with stage Ⅱ,198 with stage Ⅲ,and 198 with stage Ⅳ from the First Affiliated Hospital of Xinxiang Medical College from May 2011 to August 2015 for this study;198 patients with esophageal carcinoma of stage 0 served as the control group.The thrombin activity in plasma were determined by chromogenic substrate assay.The genetic polymorphisms of prothrombin gene G20210A in 3' untranslated region and TFPI gene C399T in 5' untranslated region in peripheral blood leukocytes of the above-mentioned patients were analyzed by PCR-RFLP technique.Unconditional logistic regression model and single factor analysis were performed to calculate the adjusted odds ratios (OR) and 95％ confidence intervals (95％ CI) of polymorphisms prothrombin gene G20210A and TFPI gene C399T polymorphisms and to analyze the interaction of nucleotide polymorphisms with thrombin activity in plasma and the pathological stages of esophageal carcinoma.Results The frequencies of G20210A (GA),G20210A (AA),C399T (CT) and C399T (TT) were 24.24％,26.77％,24.24％ and 25.76％ in stage Ⅰ group;34.34％,37.37％,34.85％ and 36.36％ in stage Ⅱ group;39.90％,42.93％,40.41％ and 41.92％ in stage Ⅲ group;45.45％,46.97％,45.35％ and 46.46 in stage Ⅳ group;and 13.64％,14.14％,13.13％ and 13.64％ in stage 0 group,respectively.Statistical tests showed significant difference in the frequencies among each group (all P＜0.01).The risks of invasion and metastasis of esophageal carcinoma significantly increased in the subjects with G20210A,in those with G20210A(AA) genotype,in those with C399T (CT) genotype and in those with C399T (TT) genotype.Combined analysis of the polymorphisms showed that percentage of G20210A (AA)/C399T (TT) in stage Ⅰ group,stage Ⅱ group,stage Ⅲ group,stage Ⅳ group and stage 0 group was 7.07％,14.14％,18.18％,21.71％ and 1.52％,respectively,and statistical tests showed significant difference in the frequency among each group (all P＜0.01).People who carried G20210A(AA)/C399T(TT) had higher risks of invasion and metastasis of esophageal carcinoma,and statistical analysis suggested a positive interaction between G20210A (AA) and C399T (TT) in increasing the risks of invasion and metastasis of esophageal carcinoma (All γ＞ 1).Likewise,there were also positive interactions in the pathogenesis of invasion and metastasis of esophageal carcinoma between G20210A (GA) and C399T (TT),G20210A (GA) and C399T(CT),G20210A (AA) and C399T (CT) (All γ＞1).The thrombin activities in plasma in stage Ⅰ,Ⅱ,Ⅲ and Ⅳ groups were all significantly higher than those in stage 0 group,and there were significant differences among stage Ⅰ,stage Ⅱ,stage Ⅲ and stage Ⅳ in thrombin activities (all P＜0.01).Patients with mutation genotype had significantly higher thrombin activities than those with wild homozygous in the same TNM stage.Conclusion G20210A and C399T gene mutations are the risk factors in the invasion and metastasis of esophageal carcinoma.Significant interactions between G20210A and C399T mutations increase the risk of invasion and metastasis of esophageal carcinoma,which may be closely related to their increased thrombin activities in plasma.

Puerarin attenuates the expressions ofHUVECs tissue factor and its inhibitor induced by ox-LDL / 基础医学与临床

Objective To investigate the effects of puerarin on the expression of human umbilical vein endothelial cells (HUVECs) tissue factor (TF) and tissue factor pathway inhibitor (TFPI) induced by oxidized low-density lipoprotein (ox-LDL).Methods After HUVECs were incubated with different concentrations of puerarin and 50 mg/L ox-LDL,the expression of TF and TFPI mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blot respectively.Results Compared with control,treatment with ox-LDL caused the augment of TF mRNA and protein expression (P<0.01),and the decrease of TFPI mRNA and protein expression.However,50,100,and 200 μmol/L puerarin blunted the augment of TF mRNA and protein expression and weakened the inhibition of TFPI mRNA and protein expression induced by ox-LDL(P<0.01).Conclusions Puerarin reduces HUVECs TF and TFPI mRNA and protein induced by ox-LDL.

rTFPI reduces ischemic necrosis in random pattern skin flap avulsion injuries rat model / 中华显微外科杂志

Objective To determine whether rTFPI could inhibit vascular thrombosis and salvage random pattern skin flaps following AIRC in rat models.Methods From April,2013 to June,2015,30 adult male Sprague-Dawley rats were randomized into 3 groups;a control group,an avulsion injury with roll compaction (AIRC) group,and an AIRC plus rTFPI therapy group.An 8.0 cm× 2.5 cm random flap was raised on the dorsum of each rat.The AIRC and AIRC plus rTFPI flaps were then altered with a device designed to simulate avulsion injury with roll compaction.After flap closure primarily,treatment was initiated immediately and continued for 3 days.Phosphate buffered saline was used in the control group and the AIRC group,while the AIRC plus rTFPI group received the recombinant Tissue Factor Pathway Inhibitor.Laser Doppler flowmetry and infra-red thermalgraphy were used on postoperative day three to assess nicrocirculatory blood flow and viability of the avulsed flaps.At postoperative day seven,final flap survival was determined.Using SPSS19.0 statistical analysis.Results The flap survival in AIRC group was only (32.7 ± 5.2)％ versus (62.5 ± 6.5)％ in control group,but the flap survival significantly increased (51.6 ± 8.2)％ after topical injecting rTFPI in experimental group.Statistically significant differences exist (P ＜ 0.05) between every two groups.The detection results of Laser-Doppler flowmetry and infra-red thermography showed that perfusion arrived the centre of the flaps in experimental group,while perfusion only arrived the proximal part of the flaps in the AIRC control group.Conclusion rTFPI therapy is effective in reducing ischemic necrosis of random pattern flaps following avulsion injury in the rat model.It suggests that rTFPI therapy may play an important role in clinical salvage of the failing avulsion injuries with roll compaction.

Pig bone marrow stromal cells transfected with human tissue factor pathway inhibitor combat species incompatibility and regulate the human TF pathway in vitro / 中华器官移植杂志

Objective To observe the reception of using pig bone marrow stromal cells (BMSCs) that were transfected with human tissue factor pathway inhibitor (TFPI) to resolve the dysregulation of coagulation after liver xenotransplantation.Methods Pig BMSCs were immortalized by transfection with lentivirus containing SV40T and then transfection with human TFPI.At last the cells were tested for their ability to inhibit clotting in a model by co-incubation of human plasma,human monocytes and pig aortic endothelial cells.Results After transfection with SV40T,pig BMSCs were immortalized and similar to primary cells.The immortalized pig BMSCs showed a stable TFPI expression after transfection with human TFPI by lentivirus.Moreover,they showed the potential of regulating coagulation dysregulation in vitro.Conclusion Pig BMSCs transfected with human TFPI could solve the regulation dysregulation caused by TF activation effectively,and have the potential of resolving coagulation dysregulation in liver xenotransplantation.

Relationship between TF,TFPI and severity in patients with diabetic cerebral in-farction / 中国免疫学杂志

Objective:To explore the relationship between tissue factor (TF),tissue factor pathway inhibitor (TFPI) and the severity in patients with diabetic cerebral infarction.Methods: 226 patients with diabetic cerebral infarction were included into the study,National Institute of Health Stroke Scale ( NIHSS) was used to evaluate the severity of CIS.The single factor analysis and multiple regression analysis were used to explore the relationship between TF , TFPI and the severity.Results: The concentrations of TF,TFPI,TF/TFPI,cholesterol and triglyceride in the NIHSS≤12 group were significantly lower than that in the NIHSS>12 group ( P<0.05);the NIHSS was significantly positive correlate with TF (r=0.354,P=0.012),TFPI (r=0.302,P=0.027),TF/TFPI (r=0.410,P=0.000),cholesterol (r=0.364,P=0.006) and triglycerides (r=0.334,P=0.018);Multiple linear regression analysis showed that the TF , TFPI, TF/TFPI, cholesterol were independent risk factors of the severity in patients with diabetic cerebral infarction.Conclusion:The level of TF and TFPI could reflect the severity in patients with diabetic cerebral infarction according to the NIHSS.

Effect of α-melanocyte stimulating hormone and its novel analogue on the production of tissue factor pathway inhibitor in mice with endotoxemia / 中华危重病急救医学

Objective To evaluate the effect of α-melanocyte stimulating hormone (α-MSH) and its novel analogue STY39 on the production of tissue factor pathway inhibitor (TFPI) in mice with endotoxemia.Methods Female BALB/c mice were randomly divided into eight groups with 9 mice in each group.Endotoxemia was reproduced by intraperitoneal injection of lipopolysaccharide (LPS,25 μg/kg) and D-galactosamine (D-Gal,100 mg/kg).The animals of the control group were given phosphate buffered solution (PBS) instead.In the experimental groups,the mice were injected intraperitoneally with 2.5 mg/kg α-MSH or STY39 at 1,2 or 3 hours following LPS injection.The orbital blood was collected at different time points,and tissues of lung,liver,and kidney were collected 8 hours after the administration of LPS.The plasma TFPI levels were determined by enzyme linked immunosorbent assay (ELISA),and the expression of TFPI mRNA in different tissues was determined with reverse transcription-polymerase chain reaction (RT-PCR).Results The plasma TFPI levels (μg/L) began to increase (11.84 ± 1.55) in the endotoxemia mice 4 hours after LPS challenge and reached the peak (23.49 ± 1.12) at 8 hours.α-MSH or STY39 treatment at 1,2 or 3 hours after LPS challenge could significantly increase the TFPI content,with the best drug effect at 1 hour after LPS challenge (the blood was collected 8 hours after LPS challenge,α-MSH group:58.79 ± 2.67 vs.28.49 ± 1.69,STY39 group:71.08 ± 2.13 vs.28.49 ± 1.69,both P＜0.01),and the effect of STY39 was better than that of α-MSH (P＜0.01).A small amount of TFPI mRNA expression was observed in each tissue of the healthy mice.After LPS challenge,TFPI mRNA expression was increased in all the tissues,especially in the lung,liver and kidney.α-MSH or STY39treatment at 1 hour after LPS challenge could significantly up-regulate the expression of TFPI mRNA in the lung and liver (A value,α-MSH in lung:51.10 ±2.89 vs.32.43 ±2.51,STY39 in lung:72.11 ±3.48 vs.32.43 ±2.51;α-MSH in liver:43.21 ± 2.12 vs.29.29 ± 2.06,STY39 in liver:66.82 ± 1.76 vs.29.29 ± 2.06,both P＜0.01).The treatment with STY39 at 1 hour after LPS challenge could significantly up-regulate the expression of TFPI mRNA in the kidney (A value:45.21 ± 1.80 vs.30.44 ± 2.23,P＜0.01),but the treatment with α-MSH had no obvious effect (A value:24.61 ± 1.98 vs.30.44 ± 2.23,P＞0.05).The enhancing effect of early administration of STY39 on TTPI mRNA expression in the lung,liver and kidney tissues of endotoxemia mice was more powerful than that of α-MSH (all P＜0.01).Conclusion The early administration of α-MSH or STY39 may up-regulate TFPI production in the mice with endotoxemia,and the effect of STY39 is superior to α-MSH.

Expressions of TFPI-2 and MMP-9 in esophageal squamous cell carcinoma tissue and their relationship with vasculogenic mimicry / 吉林大学学报(医学版)

Objective To investigate the expressions of tissue factor pathway inhibitor-2 (TFPI-2 ) and matrix metalloproteinase-9 (MMP-9)in esophageal squamous cell carcinoma(ESCC)tissue and their relationship with vasculogenic mimicry (VM).Methods 162 cases of esophageal squamous cell carcinoma tissues were collected. CD34/periodic acid-schiff double staining was performed to observe the distribution of VM in ESCC tissue,and immunohistochemical staining was used to detect the expressions of TFPI-2 and MMP-9 in ESCC tissue. The relationship between VM and the clinicopathologic parameters of ESCC, the expressions of TFPI-2 and MMP-9 were analyzed.Results The positive rate of VM in ESCC tissue was 20.37%.The positive rate of VM in poorly differentiated group(40.38%)was significantly higher than those in moderately differentiated group(11.76%)and well differentiated group (7.14%)(χ2 = 20.915, P < 0.01 ). The positive rate of VM in TNM Ⅰ-Ⅱ(11.59%)group was significantly lower than that in TNM Ⅲ group(26.88%)(χ2=5.707,P=0.017).The positive rate of TFPI-2 in VM(+)group(33.34%)was significantly higher than that in VM(-)group(6.45%) (χ2=4.582,P=0.032)in poorly differentiated ESCC.The positive rate of MMP-9 in VM(+)group(78.79%) was significantly higher than that in VM(-)group(44.96%)(χ2=12.05,P=0.001).The expression level of TFPI-2 in poorly differentiated group was positively correlated with VM (r= 0.166,P= 0.032 ), and the expression level of MMP-9 was positively correlated with the VM(r=0.183,P=0.018).The five-year survival rate in VM (-) group was significant higher than that in VM (+) group (χ2 = 22.84, P < 0.001 ). Conclusion VM exists in ESCC tissue,especially in poorly differentiated and advanced ESCC tissue.VM is related to poor prognostis of ESCC.TFPI-2 and MMP-9 might involve in the formation of VM in ESCC.

Inhibitory effect of alpha-melanocyte stimulating hormone and its novel analogue on the production of tissue factor in mouse brain microvascular endothelial cells / 中国脑血管病杂志

Objective To study the effect of alpha-melanocyte stimulating hormone (α-MSH) and its novel analogue ( STY39 ) on the production of tissue factor ( TF ) and tissue factor pathway inhibitor (TFPI) stimulated by lipopolysaccharide (LPS) in primary mouse brain microvascular endothelial cells (MBMECs). Methods Female BALB/c mice were selected,purified and primarily cultured for 5 to 7 days. Immunofluorescence assay was use to detect the Ⅷ factor related antigen and identify the MBMEC model. The MBMECs were divided into eight groups:PBS control group, LPS stimulation group, after LPS stimulation 1,2,and 3 h adding 10 -7 mol/Lα-MSH groups or STY39 group (LPS+α-MSH,LPS+STY39) ( n=4 wholes in each group) . The cell culture supernatant and cells were collected at 6 and 8 h after LPS stimulation. An enzyme-linked immunosorbent assay was used to detect the concentrations of TF and TFPI in cell supernatant. RT-PCR was used to detect the expression levels of TF mRNA. Results (1) LPS could induce MBMEC to produce TF and TFPI proteins. The level of TF in the cell culture supernatant reached the peak at 6 h,and the level of TFPI reached the peak at 8 h. (2) At 1,2,and 3 h after LPS stimulating MBMEC,10 -7mol/L α-MSH or STY39 were given. They could significantly decrease the TF protein content in the cell supernatant (P<0. 01),especially the effects of giving α-MSH or STY39 were most significant at 1 h after LPS stimulation (P<0. 05). The effect of STY39 for decreasing TF content was more significant than that of α-MSH (P<0. 05);however,α-MSH and STY39 did not have significant up-regulating effects for LPS inducing MBMEC to produce TFPI. (3) After LPS stimulation,10 -7 mol/Lα-MSH or STY39 were given at different time points. They significantly down-regulated the expression level of MBMEC TF mRNA (P<0. 01). The effect was most significant at 1 h time point (P<0. 05),but there was no significant difference in the effects betweenα-MSH and STY39. Conclusion Bothα-MSH and STY39 can suppress LPS-induced primary MBMEC to produce TF protein and express TF mRNA,and the effect of administration is better after 1 h LPS stimulation. The suppressive effect of STY39 on the production of TF protein is superior toα-MSH.

Research progress of tissue factor pathway inhibitor-2 gene in non-small-cell lung cancer / 肿瘤研究与临床

Tissue factor pathway inhibitor-2 (TFPI-2) that inhibits plasmin,trypsin and matrix metalloproteinases is a broad-spectrum inhibitor of serine proteinase.TFPI-2 can accommodate the invasion and metastasis of human non-small-cell lung cancer by maintaining the integrity of extracellular matrix and regulating the angiogenesis and apoptosis of tumor cells.It has been a new target for gene therapy of cancers.

Effect of 5-Aza-dC on the methylation and expression of TFPI-2 gene In pancreatic cancer PANCI cell line / 中华胰腺病杂志

ObjectivesTo investigate the effects of 5-aza-2-deoxycytidine(5-Aza-dC),a methylation inhibitor,on the expression and methylation of tissue factor pathway Inhibitor (TFPI-2) gene in PANC1 cell line of pancreatic cancer.MethodsPANC1 cell lines were treated with different dosages of 5-Aza-dC ( 1× 10-7,5 × 10-7,1× 10 -6 mol/L).The status of TFPI-2 methylafion and expressions of TFPI-2 mRNA and protein were determined by MSP,RT-PCR,and Western blot.Results TFPI-2 gene CpG island was completely methylated,and there was no expression of TFPI-2 mRNA and protein without 5-Aza-dC treatment.After treatment with different dosages of 5-Aza-dC( 1 × 10-7,5 × 10 -7,1 × 10-6 mol/L),TFPI-2 gene CpG hypermethylation was reversed from incomplete methylated to complete non-methylated.The relative expressions of TFPI-2 mRNA were 0.211± 0.087,O.327 ± 0.068,0.609 ± 0.017; and the relative expressions of TFPI-2 protein were O.429 ± O.121,O.675 ± O.044,1.132 ± O.124 in a dose-dependent manner ( P ＜0.05 ).ConclusionsThe hypermethylatien of promoter region may be the primary reason for TFPI-2 gene expression down-rogtdation and inactivation.5-Aza-dC may reverse the hypermethylation of TFPI-2 gene,and induce the m-expression of TFPI-2 mRNA and protein.

Effect of tissue factor pathway inhibitor-1 on no-reflow phenomenon in rabbit / 中华急诊医学杂志

Objective To observe the effects of different doses of human recombinant tissue factor pathway inhibitor-1 (TFPI-1) on no-reflow (NR) phenomenon in rabbit.Methods Fifty-two New Zealand white rabbits were subjected to coronary artery occlusion for 120 min and followed by reperfusion for 60 min,and then were randomly (random number) assigned into four groups:control group,large,moderate and low doses TFPI-1 groups ( 1000 ng/kg,100 ng/kg,10 ng/kg bolus and thenl0 ng/kg,1 ng/kg and 0.1 ng/kg per minute infusion for maintenance,each group n =13).The no-reflow area (NA) and ischemic area (IA) was measured by thioflavin S and Evan's blue.The NR severity was expressed by NA/IA.The difference in NR severity was compared between groups.The thrombi and myocardial injury were observed under light microscope.The infarction and NR severity in different groups were compared by using one-way ANOVA followed by LSD procedure.Results There were no significant differences in IA and body weight among four groups (P＞0.05).NR severity in the large,moderate,low doses TFPI-1 groups and control group were (0.210 ±0.061 ),(0.389 +0.110),(0.478 ±0.077) and (0.536 ±0.061 ),respectively.NR severity in the large dose TFPI-1 group was slightest among the four groups (P ＜0.01 ).NR severity in the moderate dose TFPI-1 group was significantly decreased than that in control group ( P ＜ 0.01 ) and in low dose TFPI-1 group (P ＜0.05 ).There was no significant difference in NR severity between the low dose TFPI-1 group and control group ( P ＞ 0.05 ).There was less thrombus formation and lower grade myocardial injury found in the large dose TFPI-1 group. Conclusion Human rTFPI-1 might lessen NR severity in rabbit in dose-dependent,suggesting the option on human rTFPI-1 for treatment of NR phenomenon.

Impact of the tissue factor pathway inhibitor gene on apoptosis in human vascular smooth muscle cells

Tissue factor pathway inhibitor (TFPI) plays a vitally important role in the blood coagulation pathway. Recent studies indicated that TFPI induces apoptosis in vascular smooth-muscle cells (VSMCs) in animals. The present study investigated whether the TFPI gene could also induce apoptosis in human vascular smooth-muscle cells (hVSMCs). Such cells were isolated from human umbilical arteries and subsequently transfected with pIRES-TFPI plasmid (2 μg/mL). MTT assaying and cell counting were applied to measure cell viability and proliferation, RT-PCR was utilized to analyze TFPI gene expression in the cells. Apoptosis was analyzed by fluorescence activated cell sorting (FACS). Several key proteins involved in apoptosis were examined through Western blotting. It was shown that TFPI gene transfer led to its increased cellular expression, with a subsequent reduction in hVSMC proliferation. Further investigation demonstrated that TFPI gene expression resulted in lesser amounts of procaspase-3, procaspase-8 and procascase-9, and an increased release of mitochondrial cytochrome c (cyt-c) into cytoplasm, thereby implying the involvement of both extrinsic and intrinsic pathways in TFPI gene-induced apoptosis in hVSMCs.

Expression of heparanase and its coagulation proteins on the surface of leukemic cells / 白血病·淋巴瘤

Objective To explore whether the expression level of heparanase (HPA) and its coagulation proteins on leukemic blast membrane could determine the hemostatic balance on the surface of leukemia cells.Methods Forty patients of leukemia were studied,and 20 patients with iron dificient anemia as the control group.Expression of tissue factor (TF),heparanase (HPA),tissue factor pathway inhibitor (TFPI),and urokinase plasminogen activator receptor (UPAR) on leukemic blast surfaces were analyzed by flowcytometry.Results The expression of TF,UPAR,and HPA in AML,ALL,CML,CLL and CRAL groups were significantly higher compared with the control group (t =.3.289,3.507,2.701,P ＜0.05; t =2.498,0.802,3.090,P ＜0.05; t =2.642,3.308,2.696,P ＜0.05; t =3.417,3.434,2.382,P ＜0.05; t =2.193,2.272,2.263,P ＜0.05).There were no significantly differences between the leukemic cell expression of TFPI and the control group (P ＞0.05).Expression of TF,UPAR,HPA in AML patients were significantly higher than ALL,CML and CLL groups (t =2.463,2.179,2.276,P ＜0.05; t =2.637,2.402,2.095,P ＜0.05; t =2.548,2.425,2.412,P ＜0.05).The levels of TF,UPAR and HPA in M3,M4 and M5 patients were higher than that of M1,M2 groups (P ＜0.05).There were no significantly differences among M3,M4 and M5 (P ＞0.05).Conclusions These results suggest that TF,UPAR and HPA are predominately expressed on leukemic blast surface,particularly in M3and M4,5 subtypes.The expression of coagulation proteins on blast membrane might determine the hemostatic balance on the surface of leukemia cells.

High-copy expression of tissue factor pathway inhibitor in pichia pastoris / 国际生物医学工程杂志

Objective To generate recombinant Pichia pastoris for high-copy expression of human tissue factor pathway inhibitor (TFPI). Methods The cDNA encoding human TFPI was inserted into the expression vector pPIC9K and the constructed expression vector rhTFPI-pPIC9K was confirmed by restriction endonuclease analysis and DNA sequencing. The recombinant plasmids were subsequently transformed into Pichia pastoris GS115 cells, and the transformants were confirmed by PCR amplification of the genomic DNA.The recombinant Pichia pastoris with high copies of TFPI cDNA was screened by G418 selection. Western blot and TFPI ELISA Kit were employed to analyzing. The temperature, time and concentration of methanol for the induction of recombinant protein were optimized. Results PCR analysis and DNA sequencing confirmed the successful construction of the expression vector rhTFPI-pPIC9K. Real time quantitative PCR and Western blot analysis demonstrated the positive correlation between TFPI expression level and the copy number of TFPI cDNA in Pichia pastoris cells. Optimization of the induction condition significantly elevated the expression level and activity of TFPI (9.95±0.78 mg/L and 3.91±1.37 U/mL). Conclusion The Pichia pastoris strain with high copy of TFPI expression was successfully constructed, which lays a solid foundation for the further investigation on the function of TFPI and its application in the prevention and therapy of diseases.

The expressions of TF mRNA and TFPI mRNA of liver in rats with Vibrio vulnificus sepsis and the intervention effects of antibacterial agents / 中华急诊医学杂志

Objective To investigate the expressions of TF mRNA and TFPI mRNA of liver in rats with Vibrio vulnificus sepsis and to assess the interventional effects of cefoperazone sodium along with levofloxacin lac-tate. Method One hundred and ten male SD rats were divided (random number) into normal control group (NC group, n = 10), Vibrio vulnificus sepsis group (VV group, five subgroups n = 10 in each), drug intervention model (AA group, five subgroups n = 10 in each). The Vibrio vulnificus sepsis models and drug intervention models of rat were made. The reverse transcription polymerase chain reaction (RT-PCR) assay was employed for the measurement of TF mRNA and TFPI mRNA. ANOVA and t-test performed with SPSS version 12.0 software. Results Compared with NC, the expressions of TF mRNA in liver increased markedly 2 h,6 h, 12 h and 16 h af-termodeling in VV groups (P<0.05), and reached peak 6 hours after modeling. The expressions of TF mRNA in liver of rats in AA groups were much higher than those in NC group 9 h and 12 h after modeling (P<0.05). The expressions of TFPI mRNA in liver of rats in VV groups and AA groups were not significantly different to those in NC group (P>0.05). Compared with VV groups, the expressions of TF mRNA in liver of rats in AA groups were greatly lowered 9 hours after administration of bactericide (P<0.05), and the expressions of TFPI mRNA in liver of rats in AA groups were significantly higher 12h and 16 h after intervention (P<0.05). Conclusions There is a obvious imbalance between coagulation and anticoagulation functions of circulation system during Vibrio vulnificus sepsis, and the imbalance can be corrected gradually after treatment with antibacterial agents.

Expressions of tissue factor pathway inhibitor-2 and synuclein gamma in esophageal cancer and their correlation with local invasion, lymph node metastasis and apoptosis / 肿瘤

Objective:To investigate the expression of tissue factor pathway inhibitor-2 (TFPI-2) and synuclein gamma (SNCG) in esophageal cancer and their correlation with local invasion, lymph node metastasis and apoptosis. Methods:The expression of TFPI-2, SNCG, and matrix metalloproteinase-9 (MMP-9) was detected by immunohistochemical SP methods in 82 cases of esophageal cancer tissues, 20 cases of atypical hyperplasia tissues, and 54 cases of para-cancerous tissues. The apoptosis of esophageal cancer cells was detected by TUNEL staining and apoptosis index (AI) was calculated. Results:The positive rates were 30.4%, 60.0%, and 87.0% for TFPI-2 protein and 63.4%, 30.0%, and 3.7% for SNCG protein in the tumor tissues, atypical hyperplasia tissues,and tumor-adjacent normal tissues, respectively. There was a significant difference between the three groups(P0.05). The expression of TFPI-2 and MMP-9 was negatively correlated (r=-0.636, P=0.000). The expression of SNCG and MMP-9 was positively correlated(r=0.393,P=0.000). AI was related with TFPI-2 and SNCG expression (P<0.05). Conclusion:TFPI-2 not only inhibited the expression of MMP-9 but also induces apoptosis of esophageal cancer to prevent tumor invasion and metastasis, however, SNCG plays a contradictory role in cancer development. TFPI-2 and SNCG might serve as new tumor markers and the new targets for tumor gene therapy.

Effects of Recombinant Tissue Factor Pathway Inhibitor on No-reflow after Acute Myocardial Infarction and Reperfusion in Rabbits / 中国医科大学学报

Objective To investigate the effects and the mechanisms of tissue factor pathway inhibitor（TFPI）on no-reflow（NR）after acute myocardial infarction（AMI）and reperfusion in rabbits.Methods Rabbits were randomly divided into sham operation group,saline control group and TFPI group.The model of NR after AMI and reperfusion was induced by ligating coronary artery for 60 minutes and reperfusion for 90 minutes.The expressions of interleukin-6（IL-6）,tumor necrosis factor alpha（TNF-α）and tissue factor（TF）were determined by immunohistochemistry.No-reflow area（NRA）was evaluated by thioflavine S staining.The ligation area（LA）and necrosis area（NA） were evaluated by Evans blue and triphenyltetra zolium chloride（TTC）staining.Results After 90 minutes of reperfusion,the levels of IL6 and TF in saline control group were all singnificantly higher than those of TFPI and sham operation group（P 0.05）.There was no statistical difference in the expression of TNF-α（P 0.05）.There was no statistical difference of LA between saline control group and TFPI group（P 0.05）,while NRA and NA were markedly reduced in TFPI group compared with saline control group（P 0.05,P 0.01）.Conclusion No-reflow happened after ligating coronary artery for 60 minutes and reperfusion for 90 minutes in rabbits.TFPI could reduce NRA and NA,and the thrombosis and inflammation might be involved in the mechanisms.