Hypoxic Imaging And The Experimental Study Of Hypoxic Factor HIF-1α

Objective: To elucidate the applicability of (99m)^Tc-HL91 and (18)^F-FDG in hypoxic diagnosis by animal experiment.Method and Materials: Tumor cells experiment: VX₂ cell line separated by ice-digested method was inoculated into 12 multiwell plates at 5×10⁴ cells per well in 1 ml of medium and was cultured at 37°C, 95% 0₂ , 5% C0₂ and at 37°C, 0.5% 0₂ , 5% C0₂, respectively. The cells were incubated with 0. 64MBq of (99m)^Tc-HL91 and 2μCi (18)^F-FDG in 0. 1 ml of medium. The reaction was stopped at 1, 2, 3, 4h and the radioactivity was counted. Animal experiment: (99)^Tc(m)^-HL91 and (18)^F-FDG was injected intravenously, images were acquired 3h and 1h, respectively, then the rabbits were killed. The specimen of muscle, brain, heart, liver, spleen, kidney, lung, necrotictumor and non-necrotic tumor were. weighted and radioactivity was counted by y-counting well. The character of tumor was observed by HE stained.Results: The uptakes of (99)^Tc(m)^-HL91 and (18)^F-FDG in hypoxic cells were higher than nomorxia cells. The uptakes increased with time prolonged. The tumors were found clearly in (99)^Tc(m)^-HL91 SPECT and (18)^F-FDG PET/CT images. But the radioactivity of venter was higher than that of tumor. The radioactivity of non-necrotic tumor was 4 times or 3. 5 times of that of necrotic tumor using (99)^Tc(m)^-HL 91 or (18)^F-FDG respectively. HE stained indicated that there was a lot of necrotic tissue in tumor.Conclusion: The uptake of (99)^Tc(m)^-HL 91 and (18)^F-FDG were higher in hypoxic VX₂ cells than normoxic one; (99)^Tc(m)^-HL91 SPECT and (18)^F-FDG PET/CT could identify regional tumor in rabbit bearing VX₂cell. The radioactivity was higher in non-necrotic tumor than necrotic tumor; they could not identify hypoxic regional clearly.AbstractObjective: About 80% of all lung cancer cases are NSCLC and there is a difficult in increasing the effect. Hypoxia is one of the reasons, so during this process hypoxia-induced factor-1 (HIF-1) plays a key role. The objective of this study is to investigate the probability of RNA-interference targeting HIF-la on the lung adenocarcinoma cells (SPCA -1) in radiosensitivity.Method and materials: 1) According to HIF-la in GenBank data base,we designed the sense and antisense strands of RNAi. 2) We insert the siRNA into the vector psuper at the restriction nuclease sites Bglll and Hindlll orientately, and the recombinant plasmid was evaluated with enzyme digestion and gene sequencing. 3)We transfected SPCA-1 cell with pSUPER-HIF-la by lipofectamine 2000. 4) The expression of mRNA is examined by realtime RT-PCR under hypoxia and normoxia condition . 5) The expression of protein was examined by Western blot. 6) The radiosensitivity of hypoxic cell of RNA-interference targeting HIF-la was evaluated by constructing the cell survival curve.7) The weight, volume and the survival time of the rats treated by RNA-interference targeting HIF-la was calculated.Results: 1) we have inserted the HIF-la DNA to the vector Psuper at the restriction nuclease site Bglll and Hindlll orientately and the recombinant plasmid was evaluated with enzyme digestion and gene sequencing. 2) HIF-la mRNA was decreased as the expression of realtime RT-PCR under the condition of hypoxia and normoxia. 3) HIF-la protein level was decreased as the expression of Western blotting. 4) The radiosensitivity of SPCA-1 cell was increased by RNA-interference targeting HIF-la, the sensitivity enhance ratio was 1. 6. 5) The weight and volume of the tumors decresed significantly in the rat treated with transfection combined with radioation than other groups, the survival time prolonged also.Conclusion: RNA-interference targeting HIF-la may increase the radiosensitivity of hypoxic cell.Part threeEffect of RNAi for HIF-la on SPCA-1 cell in the uptake of 18F^FDGAbstractObjective: To investigate the probability of RNA-interference targeting HIF-la on the lung adenocarcinoma cells (SPCA -1) in the uptake of 18F-FDG.Method and materials: 1) According to HIF-la in GenBank data base, we designed the sense and anti-sense strands of RNAi. 2) We insert the siRNA into the vector psuper at the restriction nuclease sites Bglll and Hindlll orientately, and the recombinant plasmid was evaluated with enzyme digestion and gene sequencing. 3)We transfected SPCA-1 cell with pSUPER-HIF-la by lipofectamine 2000. 4) The expression of mRNA is examined by realtime RT-PCR under hypoxia and normoxia condition. 5) The expression of protein was examined by Western blotting. 6) The expression of GLUT-1 was examined by immunohistochemistry. 7) The uptake of 18F-FDG of cell under different time of hypoxia was measured by y-counting well.Results : 1) we have inserted the HIF-la DNA to the vector psuper at the restriction nuclease site Bglll and Hindlll orientately and the recombinant plasmid was evaluated with enzyme digestion and gene sequencing. 2) HIF-la mRNA was decreased as the expression of realtime RT-PCR under the condition of hypoxia and normoxia. 3) HIF-la protein level was decreased as the expression of western blotting. 4) The GLUT-1 level was decreased as the expression of immunohistochemistry. 5) The uptake of18F^FDG of cell was decreased as measured by y-counting well.Conclusion: RNA-interference targeting HIF-la may increase the...