To confirm the amplification of VEGF165 and VEGF165b, we performe

To confirm the amplification of VEGF165 and VEGF165b, we performed sequence analysis of these PCR products. Real time PCR was performed on a LightCycler (Roche, Basel, Switzerland) for the semi-quantitation of VEGF165 and VEGF165b mRNA levels. The primer sequences were the same as those of the primers used for RT-PCR. The calculated amounts of VEGF165 and VEGF165b mRNAs were normalized to the endogenous reference control gene, human glyceraldehyde-3-phosphate dehydrogenase (h-GAPDH). All data were presented as the ratio of the target gene/GAPDH expression. Statistical analysis The ��2 test and Mann-Whitney U test were used to examine the association between the expression status of VEGF and clinicopathological characteristics. To analyze the risk factors for recurrence, logistic regression analysis was conducted.

Survival curves were computed according to the Kaplan-Meier method. The log-rank test was used to compare the survival curves. P < 0.05 was considered statistically significant. RESULTS Expression of VEGF-A in tumor and stromal cells VEGF-A expression in tumor cells was positive in 53.9% (89/165) of the cases (Figure (Figure1A).1A). VEGF-A immunoreactivity was observed mainly in the cytoplasm of tumor cells. VEGF-A expression in stromal cells was observed in 42.4% (73/165) of the cases (Figure (Figure1B1B). Figure 1 Immunohistochemical of colorectal cancer tissues used the anti-vascular endothelial growth factor-A antibody. A: Vascular endothelial growth factor (VEGF)-A was expressed in tumor cells but not in stromal cells; B: VEGF-A was expressed in stromal cells .

.. Association between VEGF-A expression status and clinicopathological characteristics A summary of the correlation between VEGF-A expression and clinicopathological characteristics is shown in Table Table2.2. Tumor VEGF-A (t-VEGF-A) expression rates in tumors were 11.1% (1/9) in stage 0, 12.5% (2/16) in stage I, 58.2% (32/55) in stage II, 57.6% (38/66) in stage III, and 84.2% (16/19) in stage IV. t-VEGF-A expression was associated with the clinical stage (P < 0.0001). VEGF-A (s-VEGF-A) expression rates in stromal cells were 77.8% (7/9) in stage 0, 37.5% (6/16) in stage I, 60.0% (33/55) in stage II, 33.3% (22/66) in stage III, and 26.3% (5/19) in stage IV. The s-VEGF-A expression rate increased in the earlier clinical stage (P = 0.004).

The t-VEGF-A expression rate increased with the depth of invasion (P = 0.0002). Conversely, the s-VEGF-A expression rate decreased with the depth of invasion (P = 0.01). There was no significant association between VEGF-A expression and the histological type. t-VEGF-A expression became significantly higher with the grade of venous and lymphatic invasion, while s-VEGF-A expression became significantly lower GSK-3 with the grade of venous and lymphatic invasion.

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