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Experimental study of angiogenesis mechanism of EGCG inhibits angiogenesis

Author: ZhaoZhiCheng
Tutor: ZhengShuSen
School: Zhejiang University
Course: Surgery
Keywords: Angiogenesis Hepatocellular carcinoma Invasion and metastasis Signal Transduction Angiopoietins/Tie2 VEGF / KDR
CLC: R735.7
Type: PhD thesis
Year: 2004
Downloads: 290
Quote: 2
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Abstract


OBJECTIVE: In the process of tumor growth, angiogenesis plays a critical role, it is rich in nutrients and nutrients; and to create conditions for tumor invasion and metastasis. Tumor cells to release pro-angiogenic factors acting on the host endothelial cells, promote the formation of new blood vessels. Vascular endothelial growth factor (VEGF) and tyrosine kinase receptor (KDR) plays an important role in angiogenesis. The maturity and stability of the blood vessels depends mainly on the tyrosine receptor another subfamily Tie2 and its ligand angiogenesis hormone (Angiopioetins) in maintaining. The two signal transduction pathway is currently considered the most directly involved in the pathway of the vascular endothelial cell-specific angiogenesis. Liver cancer has the characteristics of fast growth, early metastasis and high mortality; also a strong vascular-dependent tumors. Angiogenesis is a malignancy of the first step, and a large number of angiogenic factors and anti-angiogenic factors involved in the angiogenesis process. Angiogenesis signaling pathway as a target for anti-angiogenic therapy may be a promising anti-tumor invasion, metastasis and recurrence treatment. However, in the process of angiogenesis, signal transduction molecules mechanism is unclear, this study using reverse transcription-polymerase chain methods and Western blotting methods to evaluate the VEGF / KDR and Angiopoietins/Tie2 hepatocellular carcinoma, the purpose is to explore in HCC growth and metastasis of these pro-angiogenic cytokines and their receptors role in the molecular mechanism. : 1, object: 23 cases of liver cancer specimens resected in our hospital from May 2002 to September 2002, respectively, cut to take cancer tissue in 23 cases, the edge of the cancer (from the cancer tissue less than 1cm) 23 cases and away from cancer liver tissue (from the cancer tissue more than 5 cm) in 23 cases, 8 cases of cirrhosis of the liver specimens, four cases of normal liver tissue (both resection liver transplant donor liver). The cut specimens placed immediately frozen in liquid nitrogen and relocated at -80 ℃ spare. This group of patients age 28-68 (average 50.86 ± 12.78). All specimens were confirmed by HE staining and pathology are clinical hepatocellular carcinoma diagnosis, envelope incomplete or non-enveloped nine cases, microscopic vascular invasion seven cases, accompanied by satellite nodules seven cases. At Edmonson method of grading Ⅱ 18 cases, III in 5 cases. 2, reverse transcriptase polymerase chain reaction: using Trizol reagent isolated from resected specimens RNA. Specimens weighing, frozen the professional degree dissertation bowl with research Zhejiang University in Trizol reagent grinding spectrophotometer concentration of total RNA. 1 flash DNase to remove contaminating DNA molecules. Tuo total RNA was used for reverse transcription. Product cDNAZ Sichuan and specific primers Ang-1, Ang a 2, VEGF, KOR, TieZ and reference primer the G3PDH polymerase chain reaction. 1.5% agarose gel separation 13 Society 1 per product, and shore-based B spin dyeing. KodaDigital Scienee 1 5 2.0 DNA analysis software system semi-quantitative analysis of PCR product. 3, the western blot: the sample in lysis buffer homogenate. Add to SDS sample buffer, boiled for 5 minutes. The injection volume for each sample hole 50 Lin g SDS PAGE electrophoresis protein isolate (6% the plastic separation TieZ and KDR, 8% plastic separation of Angl, AngZ and VEGF). After the separated proteins were transferred to PVDF membrane (1 mA / cm 1 .5 hrs). Incubated for 1 hour of the Ponceau staining protein transferase, 5% skim milk (T BST preparation) closed PVDF membrane for 1.5 hours, an anti-4 ° C overnight; the TBST wash the membrane 3 and 15 minutes, added to the secondary antibody (l: 1000) washing the membrane by the above method. And developing agents ECL tablet lithography. 4 immunohistochemistry: formalin-fixed paraffin-embedded specimens were serially sectioned, a thickness of about 6 m, xylene dewaxing, gradient alcohol dehydration, antigen retrieval. 3% hydrogen peroxide, 5 minutes to eliminate endogenous peroxidase activity, 10% sheep serum blocking non-specific antigen-binding sites, the murine anti-CD34 monoclonal antibody (1: 25) 37.4 ° C, incubated for l hour, marked by HRP manual operation. Select 5 200 vision CD34-positive cell counts. 5 Statistical analysis: all measurement data are expressed as mean ± standard deviation (beginning) RT-PCR the absorbance analysis and vascular density analyzes were performed using analysis of variance. SPSSll.O analysis software for data analysis. p lt; 0.05 existence of differences. Results 1 expression and tumor vessel density relationship of two paths: the experiment confirmed that the VEGF / KDR and AngioPoietinS / TieZ two signaling pathways are activated in all liver cancer specimens. Angl were expressed in all groups, and no significant differences, but in the presence of liver cancer tissues and adjacent liver tissue expression tended to increase. Liver tissue away from the cancer, the positive control and negative control group compared ANGZ expression was significantly increased in the cancer tissue and cancerous tissues, p lt; 0.01; VEGF expression in cancer tissues, and the other group comparison p LT ; 0.05. In each group, the expression of KDR and TieZ there is no significant difference, however, in the cancer tissue and adjacent tissue there is still significantly higher absorbance bands. Zhejiang University professional degree dissertation in hepatocellular carcinoma angiogenesis factor and its receptor protein synthesis is significantly active, which is consistent with their level of gene transcription. In cancer tissue, VEGF and Angl protein content was significantly higher than the normal liver tissue and liver cirrhosis, and their corresponding receptors KI] the R and TieZ expression with an increasing; cancerous tissues, KDR and An Sand expression was significantly increased. Immunohistochemistry was used to detect microvascular density results show that the density of blood vessels in cancer tissues and adjacent liver tissue was significantly higher than the normal liver and the positive and negative control group, p lt; 0.01. Also found in this experiment, vascular endothelial growth factor (VEGF) and AngZ expression and microvessel density have a close relationship, at the same time also have different degree of expression of its receptor. Plays a key role in angiogenesis, VEGF service nR, and Angiopoietins / TieZ two of pathway activation. 2, liver cancer invasion and metastasis and angiogenesis in signal transduction pathway relationship: Angiogenesis is required for liver cancer invasion and metastasis. Relevant factors including the integrity of the tumor capsule invasion and metastasis of liver cancer, liver vascular tumor thrombus, satellite nodule formation and pathological grading. This study analyzes of VEGF / KDR and the Angi knock ofetins / TieZ two signal transduction pathways in tumor invasion and metastasis related factors relationship found in vascular invasion specimens,

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Liver tumors
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