| Objective:Like most tumors, parts of cells of laryngocarcinoma had no enough oxygen supply. The newborn blood vessels are necessary for the growth and metastasis of the tumor, so the biological treatment of tumor targeting the newborn blood vessel has become the new research hot spot in recent years. Four vascular relevant factors VEGF, VEGFR-3, Ang-2, ES were observed on this study.The four factors were detected both in the clinical specimen of the throat squamous cancer and in the laryngeal squamous cell cultured under the condition of normoxia or hypoxia (HIF-1α) imitating the interior state of tumor.By investigating the role of each protein playing in the tumor growth and metastasis process, the experiment foundation may be built for the neck tumor's treatment.Methods:1.All clinical specimens were fixed by 4% neutral formaldehyde solution, embedded by paraffin, and sliced into 5μm serial sections. The expression of HIF - 1α, VEGF, VEGFR-3, Ang-2, ES were detected by immunohistochemistry PV (Polymer Conjugated) method instant using two-step (non-biotinylated) method.2.Human laryngeal cancer cell Hep-2 was cultivated in the medium of RPMI-1640 containing 10% newborn calf serum, penicilin (l00U/ml), Stmycin (l00μg/ml) under normoxic condition of 37℃, 5%CO2, 20%O2 or under hypoxia condition of 37℃, 5%CO2, 2%O2, 94%N2. At the time the cells grew into the exponential phase, they were diviced into 2 groups: group A (normoxia 24h),group B (hypoxia 24h). Each group of cells was collected. The protein level of HIF-1α,VEGF,VEGFR-3,Ang-2 and ES was measured by flow cytometry (FCM), and detected by immunohistochemistry. The mRNA level of HIF-1α, VEGF, VEGFR-3, Ang-2 and ES was determined by RT- PCR.Results:1.Immunohistochemistry results: The positive particles of HIF-1αwas brown in laryngocarcinoma,which mainly located in the cytoblastema and/or in the nucleus of the cancer tumour cell, and had significant difference compared with the normal tissue around the cancer (t=14.3932,P<0.01). The expression of HIF-1αat different levels of the pathological differentiation has significant difference, and that between tumor with and without lymph node metastasis has significant difference(p<0.01). The positive particles of VEGF and VEGFR-3 mainly located in the cytoplasm and/or nuclei of tumor cells, and was significantly higher than that of normal tissue adjacent to cancer(VEGF t=4.736 p<0.01;VEGFR-3 t=6.561 P<0.01 ) .The expression of VEGF and VEGFR-3 was correlated with lymph node metastasis (p<0.05) but not tumor differentiation (p>0.05). The positive particles of Ang-2was brown in laryngocarcinoma, which mainly located in the cytoblastema of cells at the edge of the tumour, where a few of vascular endothelial cells also expressed weakly. The expression of Ang-2 between the nearby normal tissue and the cancer has significant difference (t=3.474,P<0.01). There was no significant correlation between Ang-2 expression and tumor differentiation or lymph node metastasis (P>0·05). The positive particles of ES mainly located in the cytoplasm and/or nuclei of tumor cells, which was significantly higher than that of normal tissue adjacent to cancer(t=7.263 p<0.01).The expression of ES was correlated with tumor differentiation (p<0.05) but not lymph node metastasis (p>0.05).2. Immunocytochemical results:The positive particles of HIF-1αwas brown in Hep-2, which mainly located in the cytoblastema and/or in the nucleus of the cancer tumour cell. The expression of HIF-1αwas higher in hypoxia(24h) group than in normoxia (24h) group, and there was significant difference(P<0. 05). The VEGF protein were not colored in cells of both groups. The positive particles of VEGFR-3 and Ang- 2 were brown in cytoplasm, especially around the nucleus, uneven thickness. The expression of VEGFR-3 and Ang-2 was higher in hypoxia (24h) group than in normoxia (24h) group, and there was significant difference (P<0. 05). The positive granules of ES were brown in cytoplasm, especially in perinuclear area, uneven thickness. The expression of ES was higher in normoxia (24h) group than in hypoxia (24h) group, and there was significant difference (P<0. 05).3. Flow cytometry result show that:the express of HIF-1α,VEGF,VEGFR-3 and Ang-2 in the group of normoxia (24h) (HIF-1α1.35±0.05;VEGF1.09±0.03;VEGFR-3 1.22±0.03;Ang-21.20±0.12)are lower than that of hypoxia (24h)(HIF-1α1.53±0.06;VEGF1.39±0.07;VEGFR-3 1.93±0.14;Ang-2 2.04±0.09).The expression of ES in the group of normoxia (24h) (1.69±0.01) is higher than that of hypoxia (24h) (1.07±0.19).The difference has statistical significance (P<0.05).4.RT- PCR result: the express of HIF-1α,VEGF,VEGFR-3 and Ang-2 mRNA in the group of normoxia(24h) (HIF-1α1.33±0.10;VEGF0.94±0.02;VEGFR-3 0.53±0.02;Ang-2 0.32±0.02)was lower than that of hypoxia(24h)(HIF-1α1.74±0.13;VEGF1.00±0.02;VEGFR-3 0.62±0.03;Ang-20.43±0.01).There was statistically significant difference between them (P<0.05). The express of ES mRNA in the group of normoxia (24h)(0.49±0.02)are higher than that of hypoxia (24h) (0.38±0.02). There was statistically significant difference (P<0.05).5.Pearson correlation analysis showed :There was a positive correlation between the expression of HIF-1αand VEGF (r=0.8679,P<0.01),and between HIF-1αand Ang-2 (r=0.058,P>0.05);the expression of HIF-1αprotein was negative correlated with ES(r=-0.268 P>0.05). There was a positive correlation between the expression of VEGF and VEGFR-3(r=0.7365,P<0.01); the expression of VEGF protein was negative correlated with ES(r=-0.202 P>0.05).There was positive correlation between the expression of Ang-2 and VEGF(r=0.8193,P<0.01),and ES(r=0.38 P<0.05).Conclutions:1.The protein level of HIF-1αwas increased greatly under hypoxia condition in Hep-2 cell, and it occured in the mRNA level.2.The increased expression of HIF-1αenhanced the expression of the VEGF,VEGFR-3,Ang-2, and clearly a positive correlation.3.The expression of HIF-1αrestrained the expression of ES, and negatively correlated.
|
PDF Full Text Request