MRI Study On Vascular Endothelia Growth Factor Antisense Gene Regulation For Meningeal Metastasis

Purpose1. To establish an animal model of hematogenous meningeal metastasis.2. To discuss MRI study of the model and to analyze the influencing factors of meningeal metastasis.Method and materials30 New Zealand rabbits were divided into 3 groups and inoculated with VX2 carcinoma with or without opening up blood brain barrier (BBB) respectively. The last group was inoculated with physiological saline only. MRI examinations of SE sequence T1WI (TR/TE=340/10ms) and T2WI (TR/TE=3000/80ms) with the knee coil were performed at different times after inoculation on a SIGNA? 1.5 Tesla scanner . DCE-MRI were acquired using a 2D spoiled gradient echo pulse sequence(TR/TE/Flip angle=9.7ms/1.7ms/20°) . MSI and PEI of time-signal curve were analyzed. Pathological study was made under microscope by HE and immunohistochemical stains. Results1. The rate of growth was 72 % for meningeal metastasis when BBB was opened.2. MRI showed the tumor appeared isointense on T1WI, hypo or isointense on T2WI with marked enhancement after intravenous injection of Gd-DTPA. Foci lager than 2 mm can be seen accurately on enhanced MRI. DCE-MRI can show early changes on meningeal enhancement and the values of PEI had a correlation with VEGF expression of all the meningeal foci.Conclusion1. The animal model is able to provide a good method for medical imaging study.2. The BBB may play an important role for meningeal metastasis.3. T1 WI with Gd-DTPA is highly sensitive for detecting the metastasis and DCE-MRI may correlate with VEGF expression.Purpose1. To construct eukaryotic expression vector of rabbit antisense VEGF cDNA by molecular biology technique.2. To verify in vivo the antiangiogenesis effect on rabbit VX2 tumor model in the hind leg.Method and materials1. Construction of eukaryotic expression vector of rabbit antisense VEGF cDNA: Total RNA was isolated from rabbit kidney tissue with the trizol method. VEGF cDNA was amplifyed by RT-PCR from total RNA and was sequenced. PCR product was digest and was inserted into polylinker site of plasmid pcDNA3.1. By using restricion enzyme Nco I and Hind III digestion and DNA sequence, we successfully constructed rabbit antisence VEGF165 cDNA eukaryotic expression vector pcDNA3.1-ASVEGF.2. Verifying in vivo the modulation effect of antisense VEGF cDNA on rabbit VX2 tumor model in the hind leg:30 New Zealand white rabbits were divided into 5 groups, with 6 in group A for 150ug pcDNA3.1-ASVEGF and VX2 cells in the same time; 6 in group B for 75(xg DNA:Liposome injected directly into the tumor for 3 times; 6 in group C for 150(xg DNA:Liposome injected directly into the tumor for 3 times ; 6 in group D for only VX2 cells; 6 in group E for Liposome and VX2 cells. DCE-MRI examinations were performed 0, 3, 7 days after the last intervention. DCE-MR images were acquired using a 3D spoiled gradient echo pulse sequence (TR/TE/Flip angle=10.8ms/4.3ms/45°). Time-intensity cures were obtained and the Steepest Slops of enhancement in the first minute were calculated. Use Western Blot and immunohistochemistry stain to assay VEGF expression of tumor tissue.Results1. We successfully constructed eukaryotic expression vector of rabbit antisense VEGF cDNA(pcDNA3.1-ASVEGF).2. On (K 3 -. 7 days after intervention, ANOVA analysis showed that the tumors size were significantly different between group A and control groups (P <0.05) .On day 7 after intervention, ANOVA analysis showed that the tumors size were significantly different between groupB,C and control groups (P <0.05) .3. In group B and C, the DCE-MRI parameter (SS) were significantly decreased when compared with group C and D on day 3 after intervention. In group A, this phenomina was seen on day 0 and 3.4. Western Blot and IHS assay showed that the VEGF expression were decreased in group A ,B and C when compared with group D and E.5. The gross pathology showed that the vascular net around tumor were obviously decreased in theraputic groups with a distinct border between tumor and around tissue under microscopy study.Conclusion1. We successfully constructed eukaryotic expression vector of rabbit antisense VEGF cDNA(pcDNA3.1-ASVEGF).2. Antisense VEGF cDNA can inhibit the VEGF expression by VX2 tumors effectively in an early way and inhibit tumor growth.3. The semi-quantitative parameters of DCE-MRI can be used effectively on surveillance of the early pathophysiology changes of the antiangiogenesis therapy.PARTIII MRI study on antisense VEGF cDNA regulation formeningeal metastasisPurposeTo monitor antisense VEGF cDNA regulation for meningeal metastasis by MRI and discuss the possibility in prevention and cure of meningeal metastasis by antiangiogenesis therapy.