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@#[摘 要] 目的：探讨COUP-TFⅡ在胃癌中对淋巴转移相关因子VEGFR3-NRP2轴的调控分子机制。方法：收集2015年3月至2015年8月在滨州医学院附属医院手术切除的60例胃癌组织和相应的癌旁组织及正常胃黏膜活检组织，用免疫组化和qPCR法检测COUP-TFⅡ、VEGFR3和NRP2的表达；培养胃癌细胞SGC7901和BGC823，构建过表达和siRNA-COUP-TFⅡ质粒后转染SGC7901细胞，用WB和qPCR检测转染后SGC7901细胞中COUP-TFⅡ、VEGFR3、NRP2的表达，用免疫共沉淀（CHIP）和双荧光素酶报告基因实验验证COUP-TFⅡ与VEGFR3-NRP2轴的靶向关系。结果:免疫组化检测显示，VEGFR3、NRP2、COUP-TFⅡ在胃癌组织中呈高表达（P<0.01）；qPCR结果显示，与癌旁组织和正常组织相比，胃癌组织VEGFR3、NRP2和COUP-TFⅡ的mRNA呈高表达（P<0.05或P<0.01）；WB和qPCR法结果显示，与对照组相比，过表达COUP-TFⅡ组SGC7901细胞中COUP-TFⅡ mRNA和蛋白水平表达均显著升高（均P<0.01）；敲减组SGC7901细胞中COUP-TFⅡ mRNA和蛋白水平均显著下降（P<0.05或P<0.01），且VEGFR3和NRP2 mRNA水平也均显著下降（均P<0.01）；CHIP结果显示，SGC7901和BGC823细胞中COUP-TFⅡ抗体的免疫共沉淀物中含有VEGFR3和NRP2启动子DNA序列；双荧光素酶报告基因实验结果显示，COUP TFⅡ表达水平与VEGFR3和NRP2表达水平呈正相关。结论：COUP-TFⅡ在胃癌组织中高表达且对VEGFR3-NRP2轴存在正性调控作用，且在胃癌中高表达，COUP-TFⅡ可能为胃癌治疗的新靶点。
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Lymphangiomatosis, a rare disease, occurs in individuals of any age, regardless of gender, but is predominantly seen in younger individuals. It often presents with pulmonary involvement, although, the bones, spleen and liver can also be affected. Histologically, pulmonary involvement includes proliferation, complex anastomoses and secondary dilatation of the lymphatic vessels. Clinically presentation is often variable but pulmonary involvement is affected more common than other involvements. Diagnosis is made histologically but radiologically can suggest diseases. Treatment is only supportive and symptomatic.
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Objective To prepare targeted microbubbles which load anti-VEGFR-3 and observe the targeting effect in vitro.Methods Targeted microbubbles loaded VEGFR-3 antibody were prepared and the targeting effect in vitro were tested by direct and indirect methods.Direct method took off-line analysis by image analysis software IPP and statistical analysed with SPSS software package.Indirect method took direct observation the target binding effect after incubation and centrifugal sedimentation.Results Image software IPP results showed that the quantity of microbubbles in targeted-group was obviously more than ordinary group.The average microbubbles per field in targeted-group was (151 ±20).The average microbubbles per field in ordinary group was ( 12 ±4 ) microbubbles, the numbers of combined microbubbles between targeted-group and ordinary-group had significant differences ( t=19.19,P<0.01).The indirect method results showed that, compared with ordinary group, the microbubbles in targeted-group could combined with cell gradually.Conclusion Targeted microbubbles load anti-VEGFR-3 have a better contrast effect than common microbubbles.
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Objective To investigate effect of anthracyclines of VEGF-C, VEGFR-3, ER and E-cadherin in serum of patients with breast cancer.Methods 43 patients with breast cancer were selected and randomly divided into experimental group and control group, 40 cases in each group.According to the experimental program based on the treatment, serum VEGF-C, VEGFR-3 levels and breast tissue ER and E-cadherin were detected after the end of the treatment.Results Compared with the control group, VEGF-C, VEGFR-3 levels of the experiment group were lower( P<0.05),ER level was lower(P<0.05) and E-cadherin level was higher(P<0.05).Conclusion the ER, VEGFR-3 and VEGF-C levels in breast cancer patients can significantly decrease the E-cadherin level, and it has a guiding significance for clinical.
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Subject(s)
Blotting, Western , Carcinoma, Squamous Cell , Cell Line , Enzyme-Linked Immunosorbent Assay , Gene Expression , Immunoprecipitation , Lymph Nodes , Protein-Tyrosine Kinases , Proteins , Receptors, Vascular Endothelial Growth Factor , RNA, Messenger , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factor C , Vascular Endothelial Growth Factor D , Vascular Endothelial Growth Factor Receptor-3ABSTRACT
Objective To investigate vascular endothelial growth factor-c (VEGF-C) and vascular endothelial growth factor receptor-3(VEGFR-3) mRNA expression, microvessels density (MVD) and lymphatic microvessels density (LVD) in human hepatocellular carcinoma and normal liver tissue. Try to illuminate the relationship among VEGF-C,VEGFR-3,MVD,LVD and the clinical pathological features of hepatocellular carcinoma. Methods Liver tissue of 60 cases definitely diagnosed as hepatocellular carcinoma and 20 normal cases were collected. VEGF-C and VEGFR-3 mRNA expression were examined by RT-PCR, MVD and LVD were examined by immunohistochemistry staining. Relationship between these indexes and clinical pathological features of hepatocellular carcinoma was also analysed. Results VEGF-C and VEGFR-3 mRNA expression, MVD and LVD in hepatocellular carcinoma were higher than those in normal liver tissue (P<0.01); In hepatocellular carcinoma tissue, expression of VEGF-C mRNA positively related with VEGFR-3 mRNA, MVD and LVD(P<0.01). VEGF-C and VEGFR-3 expression positively related with portal vein tumor thrombus, intrahepatal metastasis and lymph node metastasis (P<0.01). MVD positively related with portal vein tumor thrombus and intrahepatal metastasis (P<0.01). LVD positively related with lymph node metastasis (P<0.01). Conclusion VEGF-C and VEGFR-3 expression increase in hepatocellular carcinoma tissue. They might play roles in tumor invasion and metastasis by inducing angiogenesis and lymphangiogenesis.
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Objective To detecte the expression of COX-2,VEGF-C and lymphatic vessel density (LVD)in pancreatic cancerous and paracancerous tissues,and investigate their correlation.Methods The expression of COX-2.VEGF-C and LVD in 40 cases of pancreatic cancer tissues and paracancerous tissues and 12 cases of normal pancreas was detected by tissue chip and immunohistochemical assays,and the relationship between them and the cljnicopathological parameters was analyzed. Results The expression of COX-2,VEGF-C in pancreatic cancer tissues were 70.0%(28/40)and 67.5%(27/40),respectively,which were significantly higher than that in paracancerous tissues(42.5%,17/40)and(35.0%,14/40),and that in normal pancreas(8.3%,1/12)and(25.0%,3/12).The LVD in pancreatic cancerous,paracancerous and normal pancreatic tissues were 4.75±2.77,15.2 ±4.70 and 1.67±1.15,respectively.The expression of COX-2 in cancerous tissues and LVD in paracancerous tissues was correlated with tumor differentiation and lymph metastasis;the expression of VEGF-C Was correlated with lymph metastasis.LVD in paracancerous tissues was correlated with the expression of COX-2 and VEGF-C.Conclusions Pancreatic cancer lymphangiogenesis mainly existed in paracancerous tissues,COX-2 and VEGF-C may play an important role in the lymphangiogenesis.
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Objective To explore the influence of ICAM-1 and LFA-1 on the breast cancer metastasis and provide scientific base for studying the molecular mechanism of breast cancer metastasis by observing the expression of ICAM-1(intercellular adhension molecule1) and LFA-1(Lymphocyte function related antigen 1) in different time different metastasis stage of breast cancer. Methods We observed the expression of ICAM-1 and LFA-1 in breast cancer tissue as well as the expression of ICAM-1、LFA-1 and VEGFR-3 in Lymphatic vessel endothelial cell of nearby the breast cancer tissues from the patients of the operation in the defferent stages with the methods of HE staining and immunohistochemistry staining. Results The positive rate of ICAM-1 and LFA-1 expression in cancer cell and cancer nest of breast cancer was increased gradually along with the increase of clinical stages and the occurrence of lymph node metastasis. And the positive rate of ICAM-1、LFA-1 and VEGFR-3 expression in lymphatic vessel endothelial cell of nearby the breast cancer tissues was increased gradually too along with the increase of clinical stages and the occurrence of lymph node metastasis,which has the positive correlation,and it has the positive correlation that the ICAM-1 and LFA-1 expressed in Lymphatic vessel endothelial cell of nearby the breast cancer tissues(P0.05).Conclusion The ICAM-1 and LFA-1 were related with breast cancer lymph metastasis,and the ICAM-1 and LFA-1 promoted breast cancer lymph metastasis through synergism each other.The increase or decrease of ICAM-1 and LFA-1 expression in the breast cancer tissue and lymphatic vessel endothelial cell of nearby the breast cancer tissues could as the data target to display certain instruction function for prevention and treatment of breast cancer lymph metastasis.The Lymphatic vessel quantity of nearby the breast cancer tissues was increased along with tumor progress, which may be related with breast cancer lymph metastasis.VEGFR-3 has the high specificity in the lymphatic vessel endothelial cell expression,and may act as the lymphatic vessel as the lymphatic vessel endothelial cell specific marker.
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Objective:To construct a small interfering RNA(siRNA)expression vector(psiRNA-VEGFR-3)targeting vascular endothelial growth factor receptor 3(VEGFR-3)and to investigate the effects of VEGFR-3 siRNA on the adherence and invasion of human colon cancer cells.Methods:A siRNA expression vector(psiRNA-VEGFR-3)targeting VEGFR-3 were constructed and was used to transfect LoVo cells via lipofectamine 2000.The mRNA and protein expression of VEGFR-3 were examined after transfection by reverse transcriptase polymerase chain reaction(RT-PCR)and Western blotting,respectively.The tumor adhesion ability was detected by cell-matrix adhesion experiment and the invasion ability of tumor cells was evaluated by millicell chamber model.Results:The VEGFR-3 siRNA expression vector was successfully constructed.The expression of VEGFR-3 mRNA and protein was inhibited after psiRNA-VEGFR-3 transfection.Seventy-two hours after psiRNA-VEGFR-3 transfection,Western blotting assay showed that the expression of VEGFR-3 protein was decreased from(1.26?0.19)to(0.39 s0.12)(P
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Objective To investigate of the expression of vascular endothelial growth factor receptor 3 (VEGFR-3) and its relationship with the prognosis of esophageal carcinoma. Methods VEGFR-3 in 156 cases of esophageal carcinoma tissues was detected by immunohistochemistry. The follow-up data for 3 years in 47 cases were analyzed. Results The VEGFR-3 expression in esophageal carcinoma tissues with lymph node metastasis was higher than that in those without lymph node metastasis. VEGFR-3 expression was associated with the clinicopathological stages. Analysis of the follow-up data also showed that there was a statistically positive correlation between VEGFR-3 expression and lymph node metastasis in primary tumors (P
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Objective:To investigate the expression of vascular endothelial growth factor-C(VEGF-C) and vascular endothelial growth factor receptor 3(VEGFR-3)and the correlation with the lymph node metastasis in esophagus squamous cell carcinoma.Methods:72 esophagus squamous cell carcinoma cases with complete clinical,pathological and following-up data were included in this study.Immunohistological method SABC was used to detect the expression of VEGF-C and VEGFR-3.Results:(1)The VEGF-C positive rate in esophagus squamous cell carcinomas is 59.72%(43/72).The mean number of VEGFR-3 vessel in VEGF-C positive group is 5.02?2.24 per high power field(HPF),while is 2.82?0.16 per HPF in VEGF-C negative group.The former is significantly higher than the later ( P
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Objective To investigate the relationship between the expression of vascular endothelial growth factor receptor 3 (VEGFR 3) and lymphangiogenesis in esophageal carcinoma. Methods VEGFR 3 expression in the esophageal carcinoma tissues from 73 cases was detected by RT PCR and immunobistochemical method. and the relationship between VEGFR 3 and lymphangiogenesis was analyzed. Results There was a statistically positive correlation between VEGFR 3 mRNA expression and lymph node metastasis in primary tumors ( P
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Objective To analyse the expression patterns of the vascular endothelial growth factor receptor 3(VEGFR-3) at the protein and mRNA level,and to detect the biological function of VEGFR-3 in the lymphangiogenesis of the embryos. Methods A total of 61 specimens of skin were investigated by immunohistochemical staining with a polyclonal antibody against VEGFR-3 in embryo 15-day-old(E15) and in embryo 21-day-old(E21).The expression of VEGFR-3 mRNA was studied in situ hybridization. Results The positive expression of VEGFR-3 can be seen in lymphatic vessels of the embryonic skin.The occurrences of VEGFR-3 protein in lymphatic vessels in E15 and in E21 were 38.71%(12/31) and 73.33%(22/30) respectively,the expression level of VEGFR-3 protein in E21 was significantly higher than that in E15(?~2=7.408,P
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Objective:To determine vascular endothelial growth factor C (VEGF-C) and its receptor VEGFR-3 expression in pancreatic carcinomas and the relationship of the expression of VEGF-C and VEGFR-3 with lymph angiogenesis and lymphatic metastasis.Methods:Specimens of pancreatic carcinoma were immunohistochemically stained for VEGF-C and VEGFR-3. Lymphatic vessels were displayed by immunohistochemical double staining.And lymphatic vessel density (LVD) were counted.The correlations among VEGF-C expression,VEGFR-3 expression and LVD in tumors with or without lymph node metastasis were statistically analyzed.Results:VEGF-C expression was observed in the cytoplasm of carcinoma cell,and VEGFR-3 expression was observed in the cytoplasm of endothelial cell of lymphatic and micro vessel.The VEGF-C expression rate,VEGFR-3 expression rate and LVD of tumors with lymph node metastasis were higher than those of tumors without lymph node metastasis.LVD correlated with the expression of VEGF-C and VEGFR-3.Conclusion:The high expressions of VEGF-C and VEGFR3 in pancreatic carcinomas suggest their involvement in lymph angiogenesis.VEGF-C,VEGFR-3 and LVD are associated with lymphatic metastasis and may besignificant prognostic factors for lymphatic metastasis in pancreatic carcinomas.This implies that it is an effective option to control lymphatic metastasis of pancreatic carcinomas to inhibit the effect of VEGF-C or VEGFR-3.
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Objective To investigate the expression of vascular endothelial growth factorC(VEGF-C),its receptor3(VEGFR-3) and podoplanin in tissue of esophageal cancer,and the relationship between their expression and tumor lymphatic metastasis.Methods Expression of VEGF-C,VEGFR-3 and podoplanin in 76 cases of ESC was detected by immunohistochemical SP method and lymphatic vessel density(LVD) was calculated.Results Positive immunostaining of VEGF-C in esophageal cancer tissue was 63.1%;the expression of VEGF-C was significantly associated with the depth of lymph node metastasis and tumor invasion(P
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Objective To study the changes of biological characteristics of CD34~+/CD133~+/VEGFR-3~+ lymphatic endothelial progenitor cells(EPCs) isolated from umbilical cord blood after induction with VEGF-C and investigate the mechanisms of EPCs differentiation toward lymphatic endothelial cells. Methods Mononuclear cells were isolated from umbilical cord blood using density centrifugation with Percoll solution.CD34~+/CD133~+/VEGFR-3~+ cells were sorted with FACS.Differentiation of the cells was induced with VEGF-C.The ultrastructural changes of the cells were viewed under scanning and transmission electron microscopes.Changes in expression of markers of the cells were viewed under confocal laser scanning microscope. Results Lymphatic EPCs isolated from umbilical cord blood expressed CD34,CD133 and VEGFR-3.At 7 day after induction with VEGF-C,CD34~+/CD133~+/VEGFR-3~+ cells were shuttle-like,there were a lamellipodium,numerous filopodia and many short microvilli.Caveolae were observed.Mitochondrion and rough endoplasmic reticulum were rich in cytoplasm.At 14 day after induction,the cells demonstrated appearance of the endothelial cell and expressed lymphatic endothelial specific makers LYVE-1 and 5'-nucleotidase,the expression of CD133 disappeared.Weibel-Palade body was observed in the cytoplasm of the cell.Conclusion There are CD34~+/CD133~+/VEGFR-3~+ lymphatic EPCs in human umbilical cord blood.The cells may differentiate into lymphatic endothelial cells through VEGF-C/VEGFR-3 signaling pathway after induction with VEGF-C.
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Objective To investigate the role of vascular endothelial growth factor D(VEGF-D) and vascular endothelial growth factor receptor 3(VEGFR-3) in lymphangiogenesis and lymphatic metastasis of colon carcinoma by observing the expression of VEGF-D and VEGFR-3,and calculated lymphatic vessel density(LMVD) in colon carcinoma. Methods The expressions of VEGF-D and VEGFR-3 were observed by SP immunohistochemistry in 55 colon carcinoma samples of different stages and differentiation degrees.LMVD in colon carcinoma was calculated by using Podoplanin as the specific marker of lymphatic endothelium. Results The positive expression of VEGF-D in colon carcinoma(54.5%) was higher than that in peritumoral tissues(P