The Correlation Study Of TF(+) Circulating Tumor Cells To Promote The Formation Of Portal Vein Thrombosis

[Objective and background]HCC (hepatocellular carcinoma, HCC) is one of the common malignant tumors in China, most patients diagnosed at advanced stage, the prognosis is very poor, 5-year survival rate is still below 5%. HCC tend to invade the portal vein to form the portal vein tumor thrombus (Portal vein tumor thrombus, PVTT), the incidence is about 30%-60%, more researchers found that the incidence of endoscopic of PVTT up to 90%. HCC once violated portal and formed PVTT, bad prognosis, with a median survival rate of only 2.7 months. PVTT can not only promote liver blood spread, and the formation pressure extrahepatic metastases,but PVTT can be further increasing, leading to upper gastrointestinal bleeding,and ascites formation, accelerate clinical stage of disease progression. However, the current state of PVTT formation mechanism is still unclear, and lack of early prevention and treatment methods.Trousseau found that there is a relationship between tumor and tumor thrombus formation in patient, called "Trousseau’s syndrome". Tissue factor (tissue factor, TF) is the initiator of extrinsic coagulation pathway, studies have shown that patients with TF and tumor thrombus formation. Tissue factor, also known as clotting factor III or tissue thromboplastin clotting factor is only present in the cell membrane type I single chain transmembrane glycoprotein. Studies have shown that, TF expression in a variety of solid tumors, breast, lung and colorectal cancer, et al.And TF can inhibit tumor cell differentiation, promote thrombus formation and tumor metastasis, and the prognosis of patients. More study have show that, TF is an important factor in promoting the formation of CTCs, can be used as a surface marker of CTCs, TF expression in tumor tissue and its number of CTCs in the blood showed a positive correlation. Therefore, our study was designed to study that whether there is a relationship of TF (+)CTCs and PVTT formation, as well as the possibility of TF can be as a prognostic factors in HCC patients with PVTT and Preliminary study by in vitro experiments to research the machanism of TF promote PVTT formation. Hope for targeted cancer therapy strategies of TF/CTCs can provide new research directions.[Methods]Part 11、Expression of TF in HCC by immunohistochemistryDetect the expression of TF in 128 patients of hepatocellular carcinoma, PVTT and 30 patients with Hemangioma by immunohistochemistry.2、Expression of TF phenotypes of CTCs by multicolor-immunofluorescence(1) 128 patient 5ml peripheral venous middle blood was extracted with EDTA tubes anticoagulant. Ficoll centrifugation+ CD45 immunomagnetic negative sorting method is used to get CTCs.(2) Produce the CTCs smears and incubate with TF、CK、CD45 antibody for multicolor-immunofluorescence. CTCs number and TF phenotypic was counted in the fluorescence microscope.(3) Using X-tile software to group all those patient into categories.(4) Using SPSS software to Analyze the correlation of TFexpression between HCC tissue and CTCs.(5) Chi-square test and Fisher’s exact test and the statistical analysis of the relationship between TF group and sex, age, hepatitis virus B surface antigen, AFP expression, capsule, tumor size, Edmondson-steiner grade, PVTT.3-. Expression of TF in PVTT by immunohistochemistry(1) Detect the expression of TF in those patients with PVTT.(2) Using SPSS software to Analyze the correlation of TFexpression between TF group and PVTT.4、Analysis of prognostic factors for liver cancerUsing SPSS software to analyze relationship between TF (+) CTCs and prognosis of HCC.Part 21、Epression of kinds of hepatoma cell linesDetecting the expression of TF in Hep3B、HepG2、MHCC97L, MHCC97h、 PLC/PRF/5、SMMC77216 kinds of hepatoma cell lines by flow cytometry.2、Hepatoma cells in vitro procoagulant activity experiments and TF expression in CTM(1) Choose TF expression highest and lowest cell line from All above of the six kinds of tumor cells were respectively counted. Different concentrations of the cells were putted into the platelet-free plasma and took "pick silk" method to detect the difference procoagulant activity in vitro as soon as added Ca2+. Analysis whether the procoagulant ability is Corresponding to the TF differential expression of tumor cell’s.(2) Show TF phenotypes of CTM by multicolor- immunofluorescence.[Results]1、Expression of TF in HCC by immunohistochemistryTF epression is 69.53% in 128 patients with HCC and 6.67% in 30 patiets with Hemangioma by immunohistochemistry. There are differences between the two groups, the difference is statistically significant (P<0.05).2、Expression of TF phenotypes of CTCs by multicolor- immunofluorescence(1) CK was stained red,located in the cell membrane;TF was stained green,located in the cell membrane; DAPI was stained blue, located in the nucleus;CD45 was stained orange; located in the cell membrane.(2) We find that there were 5-112 CHCC form 5mL peripheral blood in those HCC patients,and the average number was 39.95±19.61.The number of TF-positive CHCC number was 0-103 with the average 20.52±16.79. positive rate 0-91.96% and the average rate48.59±24.41%.(3) Obtained to the number of CTCs 12 is bounded by two groups prognosis biggest difference by using X-Tail software.(4) TF group have significant correlation with tumor size (P= 0.014), the Edmondson-steiner grade grade (P= 0.000), and PVTT correlation (P= 0.000);TF group have non-significant correlation with sex, Age, HBSAg, AFP, tumor capsule.(5) there are correlations of TFexpression between HCC tissue and CTCs by SPSS software Analyzing. 3、Expression of TF in PVTT by immunohistochemistryTF epression is 91.18% in patients with PVTT. 4、Analysis of prognostic factors for liver cancerUnivariate analysis using SPSS 18.0, prognostic factors in patients with liver cancer is that:TF immunohistochemical staining, TF (+) CTCs, CTCs and PVTT prognostic factor in patients with liver cancer; multivariate analysis of variance shows: TF (+) CTCs and CTCs is an independent prognostic factor in patients with liver cancer. 5、Hepatoma cells in vitro procoagulant activity experiments and TF expression in CTM(1) The result of flow cytometry show that TF is high exspressed in Hep3B and MHCC7721 cells with the respectively rate are 89.16±6.21% and 83.73±8.15%, while low expressed in PLC/PRF/5、MHCC97H、MHCC97L and HepG2 cells as the respectively rate are 46.96±18.14%、67.83±18.58%、59.63±10.16%、41.76±6.21%.(2) By comparison with the undoped hepatoma cells of normal human plasma, Hep3B and HepG2 cells procoagulant time, find that Hep3B cells with high expression of TF clotting times shorter than the HepG2 cells, the difference between the two groups was statistically significance. Clotting time between normal HepG2 have not statistically significant.(3) Only two cases of the CTM isolated from 128 patients.[Conclusion](1) TF (+) CTCs have relationship with PVTT formation of HCC patiet.(2) TF (+) CTCs is an independent prognostic factor of patients with liver cancer.(3) TF promote the PVTT formation of liver cancer by promoting the circulation. of local hypercoagulable.