人mGluR1基因mRNA实时荧光定量PCR方法的建立

目的建立代谢型谷氨酸受体1（mGluR1）基因mRNA表达水平的TaqMan real-time PCR检测方法。方法以争actin为内参基因，根据GenBank中人mGluR1及β-actin基因序列，分别设计了两套特异性引物和TaqMan探针，接着对反应的退火温度、引物浓度、探针浓度、Mg^2＋浓度进行优化，然后以优化的条件建立相对定量标准曲线，并对该方法的稳定性进行分析。结果mGluR1及争actin基因的real-time PCR扩增效率分别为99．7％和100．0％；相对定量标准曲线的CT值线性范围分别为8．1～30．9和11．9～32．1，相关系数分别为0．999及1．000；批内及批闻变异系数〈6．4％。结论本研究所建立的针对mGluR1 mRNA表达水平的Taqman real—time PCR检测方法具有扩增效率高、稳定性好等特点，为进一步探索mGluR1的功能及其mRNA表达水平的变化和各种疾病发生、发展的相关性提供了方法学基础。     

人mGluR1基因mRNA实时荧光定量PCR方法的建立

目的建立代谢型谷氨酸受体1(mGluR1)基因mRNA表达水平的Taq Manreal-time PCR检测方法。方法以β-actin为内参基因,根据GenBank中人mGluR1及β-actin基因序列,分别设计了两套特异性引物和TaqMan探针,接着对反应的退火温度、引物浓度、探针浓度、Mg2 浓度进行优化,然后以优化的条件建立相对定量标准曲线,并对该方法的稳定性进行分析。结果mGluR1及β-actin基因的real-time PCR扩增效率分别为99.7%和100.0%;相对定量标准曲线的CT值线性范围分别为8.1～30.9和11.9～32.1,相关系数分别为0.999及1.000;批内及批间变异系数<6.4%。结论本研究所建立的针对mGluR1 mRNA表达水平的Taqman real-time PCR检测方法具有扩增效率高、稳定性好等特点,为进一步探索mGluR1的功能及其mRNA表达水平的变化和各种疾病发生、发展的相关性提供了方法学基础。     

SYBR荧光实时定量PCR检测不同病理类型颅咽管瘤ICAM-1 mRNA表达

目的定量研究细胞间粘附分子基因(ICAM—1 mRNA)在不同病理类型颅咽管瘤的表达差异及意义。方法收集30例经手术治疗的颅咽管瘤标本,采用SYBR荧光实时定量PCR法检测 ICAM-1 mRNA在肿瘤组织的表达,并对表达结果行统计学分析。结果造釉细胞型颅咽管瘤 ICAM-1mRNA表达量为(62．18±6．43)×103 copies／μg,鳞状乳头型颅咽管瘤ICAM-1 mRNA表达量为 (1．13±0．17)×103 copies／μg,造釉细胞型颅咽管瘤ICAM-1 mRNA表达量显著性高于鳞状乳头型颅咽管瘤(P<0．01)。结论两种病理类型颅咽管瘤ICAM—1 mRNA表达存在显著性差异,此差异性可能与两种病理类型颅咽管瘤不同的肿瘤炎症有关。     

参照多重PCR片段分析法进行实时荧光PCR法对ABCB1三等位基因测定结果读取方法的确定

目的 为解决实时荧光PCR法对三等位基因测定结果无法正常读取的问题,通过参照多重PCR片段分析法,确定实时荧光PCR法读取方法,实现临床对ABCB1三等位基因准确、简便且价格低廉的检测。方法 收集西安市精神卫生中心2020年3月-2021年3月DNA样本2 794例,抽取5%作为实验样本,分别进行实时荧光PCR法和多重PCR片段分析法测定。根据PCR曲线Ct值的比较以及多重PCR片段分析法碱基峰位强度的比较,对比分析两种方法的判读结果,对其中报告结果不相同的样本进行数据核查并确定PCR读取方法。结果 共抽取139例样本,其中存在120例等位基因及19例三等位基因,实时荧光PCR法和多重PCR片段分析法对等位基因的检测结果完全一致。根据多重PCR片段分析结果,对19例三等位基因制定了实时荧光PCR法的读取方法：扩增曲线图中,当∣∣Ct._G-Ct._T∣-∣Ct._G-Ct._A∣∣<3,分别读取两组碱基Ct值小的碱基,将其组合形成判读结果;当∣∣Ct._G-Ct._T     

伴糖尿病的脑梗死患者血清组织因子与组织因子途径抑制物水平的研究

目的 探讨伴糖尿病的脑梗死患者血清组织因子(Tissue factor,TF)、组织因子途径抑制物(Tissuefactor pathway inhibitor,TFPI)水平的变化及其临床意义.方法 采用酶联免疫吸附法(ELISA)测定30例门诊体检者(正常对照组)和60例急性脑梗死患者(分为伴糖尿病脑梗死和不伴糖尿病脑梗死两组)血清TF及TFPI水平,并于发病后72h进行头部CT检查,计算梗死灶大小,同时进行神经功能缺损评分.结果 (1)脑梗死组较正常对照组血清TF及TFPI水平明显升高(P＜0.05).(2)伴糖尿病脑梗死与不伴糖尿病脑梗死相比,前者血清TF及TFPI的水平升高更明显(P＜0.05).(3)脑梗死体积、临床神经功能缺损评分均与血清TF、TFPI水平无明显相关性.结论 血清TF及TFPI水平与脑梗死的严重程度无明显相关性,但伴糖尿病脑梗死确实存在更为严重的凝血功能障碍.     

The relationship between plasma and placental tissue factor, and tissue factor pathway inhibitors in severe pre-eclampsia patients

Objective

To investigate the mechanism underlying the hypercoagulable state in severe pre-eclampsia.

Methods

Plasma tissue factor (TF) and tissue factor pathway inhibitor (TFPI) expression from pre-eclampsia patients and healthy pregnant controls were determined by ELISA. Placental TF and TFPI gene and protein expression were detected by quantitative RT-PCR, immunohistochemistry, and Western analysis.

Results

The plasma TF level in the pre-eclampsia group was significantly higher than the control group (p < 0.01), and surprisingly, the plasma TFPI-1 and TFPI-2 in the pre-eclampsia group were significantly lower (p < 0.01). Placental TF gene and protein expression levels in the pre-eclampsia group was significantly higher than the control group, while TFPI-1 and TFPI-2 levels were significantly lower (p < 0.05). Lastly, a significant correlation was found between plasma and placental TF protein levels in the pre-eclampsia group (p < 0.01).

Conclusion

Higher expression and/or release of TF from the placenta may contribute towards a pathological hypercoagulable state in pre-eclampsia patients.     

A new sensitive chromogenic substrate assay of tissue factor pathway inhibitor type 1

The present assay is a modification of our previously published two-stage chromogenic substrate assay of tissue factor pathway inhibitor type-1 (TFPI) activity [1]. In the first stage, the reaction mixture was made with factor VIIa (FVIIa) molecules in excess of tissue factor (TF) binding sites and contained diluted plasma, recombinant FVIIa (10 nM), recombinant TF (1/400 vol/vol), bovine factor Xa (1,1 nM), I-2882(R) (100 microg/ml), and CaCl(2) (10 mM). The fibrin polymerisation inhibitor I-2882(R) was added to the reaction mixture to prevent formation of cross-linked fibrin. In the second stage, residual TF/FVIIa catalytic activity was measured by the addition of a substrate mixture that contained bovine factor X and a chromogenic substrate (S-2222(R)). Standard curves were constructed using serial dilutions (0-1%) of pooled normal plasma. The dose-response relationship for serial dilutions of plasma was linear. The intra-assay coefficient of variations (CVs) for pre- and postheparin plasma samples (i.e., normal and high TFPI levels) were 1.7% and 9.9%, respectively; the inter-assay CVs were 10.0% and 19. 7%, respectively. The effect of variation in antithrombin activity on the assay was approximately 5%. The present assay correlated fairly well with our previously published assay (r=0.82, n=100) and with a commercial TFPI activity assay (Actichrome(R) TFPI Activity Assay, American Diagnostica, Greenwich, CT, USA; r=0.90, n=100), as well as with an antigen assay for TFPI total antigen (Imubind(R), American Diagnostica; r=0,96, n=100). Altman and Bland plots revealed that our previous assay underestimated TFPI activity at high TFPI levels (i.e., postheparin TFPI samples) compared with the other methods.     

Utilization of a two-standard system in real-time PCR for quantification of gene expression in the brain

In this study, we applied for real-time PCR the two-standard system that we had worked out previously for PCR with gel-detection of products. Genomic DNA of a known concentration was used as external standard and mRNA of the DNA-dependent RNA-polymerase II was used as internal standard. It was shown that PCR with gel-detection of products and real-time PCR provide similar results and demonstrate almost identical accuracy and repeatability when the two-standard system is used. With the help of the both methods and using the two-standard system we have confirmed the link between the genetically determined freezing reaction in mice and reduced 5-HT_1A receptor mRNA level in the midbrain. We have also found that the genetically determined freezing reaction in mice is not connected with changes in Tph2 gene expression.     

Changes in tissue factor and the effects of tissue factor pathway inhibitor on transient focal cerebral ischemia in rats

INTRODUCTION: To determine the contribution of tissue factor (TF) to focal cerebral ischemia/reperfusion injury, we investigated the changes in TF in rat brains with transient focal cerebral ischemia and also assessed the effect of TF pathway inhibitor (TFPI). MATERIALS AND METHODS: Spontaneous hypertensive rats were subjected to 90-min of middle cerebral artery occlusion (MCAO) and then were reperfused for up to 24 h. Immediately after MCAO, recombinant human TFPI (rhTFPI) (50 or 20 microg/kg/min) was administered by means of a continuous intravenous injection for 4.5 h. RESULTS AND CONCLUSIONS: TF immunoreactivity decreased or scattered in the ischemic area after reperfusion, however, an increased TF expression was observed in the microvasculature with the surrounding brain parenchyma and it peaked at 3 to 6 h, which coincided with the start of fibrin formation. On the other hand, total TF protein in ischemic area continued to exist and did not remarkably change until 24 h after reperfusion. At 24 h after reperfusion, the total infarct volume in the group treated with 50 microg/kg/min rhTFPI was significantly smaller than that in the controls (saline). Western blotting and immunohistochemical studies showed that rhTFPI treatment resulted in a decrease of fibrin in the ischemic brains and microvasculature. TF-mediated microvascular thrombosis is thus considered to contribute to focal cerebral ischemia/reperfusion injury. The continuous infusion of rhTFPI until a peak of TF-mediated microvascular thrombosis therefore attenuates the infarct volume by reducing fibrin deposition in the cerebral microcirculation.     

阿尔茨海默病患者外周血APP mRNA水平的变化

目的建立荧光定量PCR方法检测Alzheimer病(AD)患者外周血中淀粉样蛋白前体 (APP)的 mRNA水平,并探讨该基因在AD患者外周血中的表达及意义。方法根据APP的基因序列,设计并合成引物和荧光标记探针。将PCR扩增目的片段用AT克隆的方法克隆入T载体,重组质粒经筛选、鉴定后,作为阳性模板,用于标准曲线的制定和样品检测。用该方法检测30例AD患者和 23例正常老年人对照组外周血中APP基因的mRNA水平。结果应用重组质粒制作的定量曲线循环阈值与模板浓度具有良好的线性关系。AD组APP基因平均表达水平为(2．54×105±1．53×105) copies/μgRNA,高于对照组(6．03×104±7．58×105)copies／μgRNA(P<0．001)。结论荧光定量PCR检测AD患者APP mRNA水平的方法较常规PCR技术更为简便、快速、准确。用该法测得APP在AD患者外周血中的mRNA水平有所增加。     

Atorvastatin reduces thrombin generation after percutaneous coronary intervention independent of soluble tissue factor

INTRODUCTION: Statins were previously shown to suppress cellular tissue factor (TF) in vitro. Here, we investigated the effect of atorvastatin on the TF-pathway and thrombin generation after coronary angioplasty and stenting in vivo. MATERIALS AND METHODS: A cohort of 30 patients with coronary artery disease (CAD) was randomised to treatment with either none (n=10), 10 mg (n=10) or 80 mg (n=10) atorvastatin per day for the postinterventional period of 6 months starting the day before percutaneous coronary intervention (PCI). Fasting blood samples were collected on admission and after 6 weeks and 6 months of statin therapy to determine sTF, free tissue factor pathway inhibitor (TFPI) and prothrombin fragment F1.2 by immunoassay. RESULTS: Soluble TF (sTF) significantly correlated with thrombin generation as measured by prothrombin fragment F1.2 at baseline. This correlation was lost 6 weeks and 6 months after initiation of statin therapy. In vivo, F1.2 was significantly lowered after 6 months of statin therapy by both, low dose (0 vs. 10 mg: 1.3+/-0.3 vs. 0.7+/-0.2 ng/ml; P<0.05) and high dose (0 vs. 80 mg: 1.2+/-0.3 vs. 0.6+/-0.2 ng/ml; P=0.01) atorvastatin compared to control. However, sTF and free TFPI did not change significantly with atorvastatin therapy when compared to baseline or control. CONCLUSIONS: Our results demonstrate reduced in vivo generation of thrombin six months after percutaneous coronary intervention and statin therapy independent of sTF and free TFPI.     

Tumour expresion of tissue factor and tissue factor pathway inhibitor in ovarian cancer- relationship with venous thrombosis risk

Introduction

Ovarian cancer is known to display a particular association with venous thromboembolism (VTE) with reports up to 42% of patients developing thromboembolic complications. Tissue Factor (TF) and its inhibitor Tissue Factor Pathway Inhibitor (TFPI) have been implicated in VTE risk in cancer. The aim of this study was to measure tumour derived TF and TFPI and to investigate their potential role in VTE in ovarian cancer patients.

Methods

TF and TFPI mRNA expression was measured using TaqMan real time PCR in 99 ovarian tumour samples. Nineteen cases complicated by VTE were matched to 19 cases without VTE. TF and TFPI protein levels were measured using ELISA and immunohistochemistry was used to localize TF expression. The role of TF expression on overall survival was also determined.

Results

TF mRNA and protein expression was increased in tumours from patients with clear cell carcinoma (p < 0.001). TF protein expression was also increased in endometroid carcinoma (P < 0.01) compared with benign tumours. TFPI mRNA expression was increased in clear cell carcinoma (P < 0.01). TF mRNA and antigen level was increased in malignant tumours of patients who developed VTE compared with matched malignant õtumours of patients who remained thrombosis free (P < 0.01). There was no difference in TFPI expression between the two groups.

Conclusion

TF expression in ovarian cancer is significantly higher in patients who develop VTE. TF expression was increased in clear cell ovarian cancer and endometroid cancer and this may explain the higher risk of VTE in these subgroups. TF derived from these tumours may be the trigger for VTE in ovarian cancer.     

The extrinsic coagulation cascade and tissue factor pathway inhibitor in macrophages: A potential therapeutic opportunity for atherosclerotic thrombosis

Objectives

The coagulation protease cascade plays the central requisite role in initiation of arterial atherothrombosis. However, the relative participation of the extrinsic as compared to the intrinsic pathway is incompletely resolved. We have investigated in vivo the relative importance of the extrinsic and intrinsic pathways to define which is more essential to atherothrombosis and therefore the preferable prophylactic therapeutic target. We further addressed which type of plaque associated macrophage population is associated with the thrombotic propensity of atherosclerotic plaques.

Methods

Both photochemical injury and ferric chloride vascular injury models demonstrated arterial thrombosis formation in ApoE deficient mice. We found that direct interference with the extrinsic pathway, but not the intrinsic pathway, markedly diminished the rate of thrombus formation and occlusion of atherosclerotic carotid arteries following experimental challenge. To explore which plaque macrophage subtype may participate in plaque thrombosis in regard to expression tissue factor pathway inhibitor (TFPI), bone marrow derived macrophages of both M and GM phenotypes expressed tissue factor (TF), but the level of TFPI was much greater in M- type macrophages, which exhibited diminished thrombogenic activity, compared to type GM-macrophages.

Results and conclusions

Our works support the hypothesis that the TF-initiated and direct extrinsic pathway provides the more significant contribution to arterial plaque thrombogenesis. Activation of the TF driven extrinsic pathway can be influenced by differing colony-stimulating factor influenced macrophage TFPI-1 expression. These results advance our understanding of atherothrombosis and identify potential therapeutic targets associated with the extrinsic pathway and with macrophages populating arterial atherosclerotic plaques.     

Elevated levels of leukocyte tissue factor mRNA in patients with venous thromboembolism

Tissue factor (TF) mRNA levels in leukocyte and TF antigen in plasma were examined in patients with deep vein thrombosis (DVT). Although TF mRNA levels in leukocytes were higher in patients with DVT than in healthy volunteers, they were lower in patients with DVT than in those with solid cancer and those with disseminated intravascular coagulation (DIC). On the other hand, the plasma levels of TF antigens were markedly high in patients with DVT/pulmonary embolism (PE). Analysis of the role of underlying disease of DVT showed no significant difference in TF mRNA levels and TF antigens among patients with solid cancer, post-surgical, other diseases and those free of underlying diseases. In patients with VTE, plasma levels of D-dimer, soluble fibrin, GE-XDP and plasminogen activator inhibitor-1 did not correlate with TF mRNA or TF antigen. In analysis of 18 patients with PE with and without DVT, TF mRNA levels in leukocytes correlated with the plasma levels of D-dimer. These findings suggest that TF in leukocytes is more likely to be involved in the development of thrombosis in PE than DVT.     

Murine strain differences in hemostasis and thrombosis and tissue factor pathway inhibitor

Introduction

Differences among murine strains often lead to differential responses in models of human disease. The aim of the current study was to investigate whether differences exist among strains in models of hemostasis and thrombosis and whether these differences are reflected in differences in the tissue factor (TF) pathway.

Methods

We examined baseline hemostatic parameters and the response to FeCl3-induced arterial thrombosis and a tail vein bleeding model in C57BL/6J (C57), 129S1/SvImJ (129S), and Balb/cJ (BalbC) mice. Finally, we examined TF and tissue factor pathway inhibitor (TFPI) activities in blood and expression in vascular tissue to determine whether these factors covary with a thrombotic phenotype.

Results

No differences were observed in PT or aPTT among strains. 129S mice had lower platelet counts (p < 0.001). BalbC had an increased rate of occlusion (mean occlusion time of 330 ± 45 sec) in a FeCl₃-induced model of thrombosis when compared to C57 (1182 ± 349 sec) or 129 S (1442 ± 281 sec) (p < 0.05). Similarly, BalbC demonstrated reduced blood loss in tail bleeding experiments when compared to C57 and 129S. Vascular expression of TF and TFPI content did not correlate with the thrombotic phenotype of BalbC. However, circulating TFPI activities were lower in BalbC compared to both C57 and 129S mice. When normalized to circulating TF activities, BalbC had lower circulating TFPI activity than C57 and 129S, and there was a significant correlation between tail bleeding and normalized TFPI activity (r = 0.67).

Conclusions

These data suggest that there are significant differences among strains in thrombosis and hemostasis and that circulating TFPI activity correlates with these differences.     

New allele-specific real-time PCR system for warfarin dose genotyping equipped with an automatic interpretative function that allows rapid, accurate, and user-friendly reporting in clinical laboratories

Introduction

The optimal dose of the oral anticoagulant warfarin varies with polymorphisms of the vitamin K epoxide reductase complex subunit 1 (VKORC1) and cytochrome P450 2C9 (CYP2C9) genes. A fast and reliable method of warfarin dose adjustment is required to prevent serious hemorrhagic or thrombotic complications. The aim of this study is to develop and validate a new warfarin dose genotyping system with an automatic interpretation function.

Materials and methods

Four VKORC1 and two CYP2C9 SNPs were genotyped by real-time PCR using allele-specific primers and probes. Multiple reactions that included internal positive controls were performed in each well, and an automatic interpretative algorithm was developed. This system was validated using 82 clinical specimens previously genotyped by PCR-direct sequencing. The analytical time of the method was calculated.

Results

No interference was observed when multiple samples were included in each reaction, with all internal positive control reactions being successful. In the genotyping algorithm, Ct differences < 2 and ≥ 2 identified heterozygotes and homozygotes, respectively. All results obtained were concordant with those of the reference method. The overall analytical time for assay of 12 specimens was around 3 hours.

Conclusion

This rapid, accurate, and user-friendly genotyping system improves the efficacy and safety of anticoagulation therapy in clinical practice.