Effect Of VEGF Anti-sense CDNA On Tumor Radiotherapy And Its Correlative Pathological Study And MR Imaging Detection

Part I MRI characteristics and Establishment of rabbit VX2 maxillofacial tumor modelsPurposeTo introduce two methods of building animal model of maxillofacial tumors and to analyse their rate of growth and MRI features. Materials and Methods28 New Zealand rabbits were randomly divided into two groups.Group A: 12 rabbits were inoculated with 0. 5ml (1× 10~8/ml) VX2 tumor cells in the maxillofacial region subcutaneously.Group B:16 rabbits were inoculated with 0. 5ml (1 × 10~8/ml) VX2 tumor cells in the masseter.One week later, MRI examinations including spin-echo sequence TlWI(TR/TE=350ms/15ms), fast spin-echo sequence T2WI (TR/TE=3000ms/90ms) and T1WI in all three imaging planes after intraveneous injection of Gd-DTPA at a dose of 0. 2ml/Kg at different times. Rate of growth of the two groups were compared. MRI characteristics and the correlative pathological observations were summarized. Results 1. MR features1) Both group had a growth rate of 100%. Group A:Relative growth of tumors were slowly between the 10th to 19th day, and faster after the 19th day. Group B:Growth of tumors were faster after the 15th day.2) Group A: Tumors had relatively clear margin and compressed the adjacent tissues. Two tumors invaded the near muscles and had indefinite demarcation. 3/12 tumors(25%) infiltrated the adjacent skin and occurred ulcer. All tumors appeared hypointensity or isointensity on T1W images, and isointensity or hyperintensity on T2W images.Tumors manifested a rim-like enhancement and nonenhanced necrosis in the central area at early stage. One rabbit (8.3%) occured neck lymph node enlargement on the inoculated side.3) Group B: Tumors had a relatively indefinite border. The tumors appeared isointensity or slight hyperintensity on T1W images, hyperintensity on T2W images and appeared as prominent enhancement after intraveneous injection of Gd-DTPA with smaller central necrosis. One tumor invaded the isolateral mandible. 8 tumors showed isolateral lymph node metastasis on the neck. The minimum tumor to be found lymph metastasis is about 1. 5cm in diameter.2. Pathology1) Group A: Tumors were encased by aboundant superficial tumor vascular net. Tumor cells were pleomorphism with many infiltrated lymph cells at the periphery by microscope. Karyopyknosis, karyorrhexis and fragmentation of the tumor cells were often found near tumor centers, and many tumor structures disappeared in the central area.2) Group B: Tumors infiltrated and destroyed the adjacent muscles with plenty of blood vessels. Derangement, atypia and pleomorphism as well as fragmental necrosis were found under the microscope.3. There is significant difference between the two groups in lymphatic metastasis rate(P<0.05).Conclusion1. Masseter inoculated tumors are not liable to necrosis and inclined to have lymph metastasis, which is more analogical to the biological features of human maxillofacial tumors. The masseter inoculated tumors can provide a better tumor animal model for clinical and medical imaging study.2. MRI could detect tumor location, diameter and lymph metastasis accurately.Part II Dynamic contrast enchanced MRI of maxillofacial tumor and its relationship between tumor hypoxia and VEGF expressionPurposeTo analyse the correlation of DCE-MRI with tumor hypoxia and VEGFexpression. To study the value of DCE-MRI in tumor biology and the relationship between tumor hypoxia staining and VEGF expression. Materials and Methods8 New Zealand rabbits were inoculated with 0. 5ml (IX 108/ml) VX2 tumor cells in the maxillofacial masseter. DCE-MRI(TR10ms/TE2ms/Flip angle20°) was performed every 2 days since the 10th day on. MER and SLE were calculated.After DCE-MRI examination of the rabbit, 60mg/Kg Pimonidazole were injected through the ear margin vein. 1 hour later, the rabbit was sacrificed and the corresponding tumor specimen was harvested. HE staining, hypoxia and VEGF immunohistochemical staining were carried out.Tumor hypoxia degree was judged according to the ratio of positive hypoxia staining area in the tumor and VEGF expression level was evaluated by means of IHS hemi-quantification. Results1. The hypoxia positive staining of the tumor was distributed in tumor cells far away from vessels and staining degree gradually increased along with the increase of distance from vessels. Tumor hypoxia degree had a negative correlation with MER (P<0. 01) by Spearman' s rank correlation statistical method.2. VEGF positive staining was localized in tumor cytoplasms and cell membranes, and some epithelial cells also had a positive expression. VEGF immunohistochemical score bore a positive correlation with SLE(P<0. 05) by rank correlation statistical method.3. We compared the continuous sections of hypoxia and VEGF staining of the tumor, and found that where the more hypoxia staining positive, the stronger VEGF expression . But VEGF staining was not always coincident with the staining of hypoxia. Many tumor cells near vessels also had a very strong VEGF expression. Tumor hypoxia degree bore no correlation with VEGF immunohistochemical score (P>0. 05) by rank correlation statistical method.Conclusions1. DCE-MRI had a good correlation with tumor hypoxia degree and VEGF expression.2. Pimonidazole hypoxia staining can accurately reflect tumor hypoxia degree.3. Tumor hypoxia had a close relationship with the expression of VEGF, and VEGFcould promote tumor angiogenesis.Part III Evaluation of VEGF antisense cDNA for radiosensitizing effect on rabbit VX2 maxillofacial tumorPurposeTo monitor the effect of VEGF antisense cDNA on rabbit VX2 maxillofacial tumor growth and its radiosensitizing effect under the detection of DCE-MRI. Materials and Methods24 rabbits were inoculated with 0.5ml (lX108ml) VX2 tumors cells in the unilateral maxillofacial masseter, and DCE-MRI was performed on the 12th day.Bearing tumor rabbits were randomly divided into 4 groups (6 for each group). Group A: made with 16Gy irradiation. Group B: injected with 150ug VEGF antisense in tumor. Group C: made with 16Gy irradiation just before the injection of 150ug VEGF antisense cDNA in tumor. Group D: without any treatment. DCE-MRI was performed on the third day, the fourteenth day after the treatment and MER and SLE value were calculated. Specimen were harvested and HE staining and VEGF immunohistochemical staining were carried out after the last MRI examination. Results1. Volume of tumors before and 3 days after treatment for each group had no statistical difference(P>0.05)by ANOVA analysis. Tumor volumes of 14 days after treatment for each group were compared by t-test. Group A> B^ C had statistical difference with group D (P<0.05) . Group C had statistical difference with group A and B (P<0. 05) . Group A and B also had statistical difference (P<0.05) .2. On the 3rd day after treatment, tumors in group A and C had stronger enhancement with nonenhanced central necrotic area. The enhancement degree decreased on the 14th day. No significant signal changes were observed before...