| Objective: To investigate the possibility of gene imaging technology and provide theoretical evidence that it can be applied to early lung carcinoma diagnosis, through preparing the 99Tcm-survivin mRNA antisense peptide nucleic acid (PNA), and experimenting of distributions and antisense imaging on BALB/c nude mice bearing human lung carcinoma A549 xenografts.Methods: As the No. 240-251 of survivin mRNA for the target sequence, survivin mRNA antisense PNA (5'-CCCAGCCTTCCA-3') was synthesized while four amino acids (Gly-(D)-Ala-Gly-Gly-) andγ-Aba(4-aminobutyric acid) were linked to the 5′terminal of PNA. Of these, Gly-(D)Ala-Gly-Gly- served as a chelating moiety for strong chelation of 99Tcm andγ-Aba as a spacer to minimize steric hindrance. Then labeled it with 99Tcm by ligands exchange method. The quality assurance of radiolabeling PNA was measured by high performance liquid chromatography (HPLC). Labeling rate, radiochemical purity and stability of antisense probe were assessed by instant thin-layer chromatography (ITLC). The experiments of uptaken rates and retention rates with A549 cells were studied for investigated initially the pharmacokinetics of antisense PNA. Then the distribution study was in progress. 30 nude mice were divided into two groups randomly, antisense PNA and mismatch PNA groups. 99Tcm-survivin mRNA antisense PNA (740KBq) was tail intravenously injected to animal models. The mice were killed at 1,2,4h postinjection respectively and tissue biodistributions were measured. %ID/g and tumor to muscle ratios (T/M) were calculated. The antisense gene imaging experiment of 99Tcm-survivin mRNA antisense PNA in vivo was studied. 10 nude mice were divided into two groups randomly, antisense PNA and mismatch PNA group.99Tcm-survivin mRNA antisense PNA (37MBq) was injected to nude mice bearing human lung carcinoma A549 xenografts via the tail wein. The value of target to nontarget ratios (T/NT) at 1,2,4h was measured by the method of region of interest method (ROI). The antisense gene imaging and tissue distributions of 99Tcm-survivin mRNA mismatch PNA in BALB/c nude mice bearing A549 xenografts were progressed similar to the groups of 99Tcm-survivin mRNA antisense PNA. The statistical comparisons of average value were performed with the two-group t test for independent samples by SPSS13.0. P<0.05 was considered significant.Results: The results of HPLC confirmed that 99Tcm was joined to the PNA, with a high radiochemical purity (>95%). The labeling rate of 99Tcm-survivin mRNA PNA was 95.48%±1.92% (n=5) by instant thin-layer chromatography (ITLC), and that radiolabeling rate of antisense PNA was also above 85% with human fresh serum after 24 hours at 37℃. Similar results were obtained for 99Tcm-survivin mRNA mismatch PNA. The results of Rt-PCR confirmed that both human lung carcinoma A549 cells and tumor lesions overexpressed the oncogene of survivin. 99Tcm-survivin mRNA antisense PNA was specifically uptaken by tumor lesions, and reached the top at 4h postinjection. %ID/g of 99Tcm-survivin mRNA antisense PNA at 1,2,4h were 2.51±1.20, 1.52±0.16, 1.35±0.27. The T/M (tumor to muscle) ratio of 99Tcm-survivin mRNA mismatch PNA at 4h were significantly lower(t=2.506, p=0.041)。In addition, The kidney and liver uptaken more radionuclide than other tissues.The T/NT (target to nontarget) ratios of 1,2,4h were 2.70±0.28, 3.44±0.35, 4.21±0.63 by method of ROI. The T/NT ratio of 99Tcm-survivin mRNA mismatch PNA at 4h were significantly lower than the antisense group (t=2.918, p=0.019). which was at equal with the result of distribution studies.Conclusions: 99Tcm-survivin mRNA antisense PNA labeling rate was high, stable, and antisense probes were specifically uptaken by the tumor lesions which overexpressed the oncogene of survivin. Gene imaging technology will be perspective, and it provided theoretical evidence that antisense imaging can be applied to early lung carcinoma diagnosis.
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