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ObjectiveTo investigate the value of 99Tcm labeled survivin mRNA antisense peptide nucleic acid (PNA) as an imaging agent in the specific diagnosis for carcinoma.MethodsSurvivin mRNA antisense PNA was labeled directly with 99Tcm by the ligand-exchange method.Twenty nude mice with lung carcinoma A549 xenografts were randomly divided into 4 groups.Three groups were used for biodistribution study and one group was used for imaging study.Other twenty mice infected by staphylococcus aureus underwent the same procedure.The biodistribution and imaging of 99Tcm-survivin mRNA antisense PNA was studied at 1,2 and 4 h respectively after the intravenous injection in nude mice bearing lung carcinoma A549 xenografts or inflammation models.SPSS 13.0 was used in the study and all data were analyzed by t test.ResultsBiodistribution results showed that the highest radioactivity was found in the liver,and then in the kidney.Four hours after the administration of the imaging agent,the radioactivity ratios of target-tonon target (T/NT,tumor or inflamumatory lesions to the contralateral regions) in tumor model group were significantly higher than those in inflammation model group ( 3.69 ± 1.13 vs 2.03 ± 0.47,t =3.01,P =0.02 ).Tumors were clearly visible in the tumor model groups at 0.5 h and still clearly seen at 4 h after the injection of antisense PNA.On the contrary,inflammatory lesions could not be seen clearly.Conclusion 99Tcm labeled survivin mRNA antisense PNA can be used to distinguish tumor from inflammation and it may provide a new feasible method for specific tumor diagnosis.
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Objective To prepare the 99Tcm-survivin mRNA antisense peptide nucleic acid (PNA)and investigate its value as a gene imaging agent in tumor bearing mice and early diagnosis in tumor.Methods Survivin mRNA antisense PNA and mismatch PNA were synthesized.Four amino acids (Gly- (D)Ala-Gly-Gly) and Aba (4-aminobutyric acid) were linked to the 5' end of PNA.Gly- (D)Ala-Gly-Gly served as a chelating moiety for strong chelation of 99Tcm and Aba acted as a spacer to minimize the steric hindrance.PNAs were labeled with 99Tcm by the ligand-exchange method.The labeling efficiency and radiochemical purity were measured by HPLC and ITLC methods.There were five BALB/c nude mice bearing human lung carcinoma ( A549 ) in each of antisense PNA and mismatch PNA groups.Gene imaging of 99Tcm-survivin mRNA antisense and mismatch PNAs were performed at 1,2 and 4 h post the injection,respectively,and the T/NT ratio was measured by the method of ROI.The statistical comparisons of average values were performed with the two-group t-test for independent sample by SPSS 13.0.Results The product kept stable in vitro.The labeling efficiency of 99Tcm-survivin mRNA antisense PNA was (95.48 ±1.92)％ and more than 85％ after the incubation for24 h in serum.The radiochemical purity was ＞ 95％.The labeling efficiency of mismatch PNA was similar to the antisense PNA.99Tcm-survivin mRNA antisense PNA was especially uptaken by tumor lesion,and its accumulation reached the top at 4 h post the injection.T/NT ratios at 1,2,and 4 h were 2.70 ± 0.28,3.44 ± 0.35,4.21 ± 0.63,respectively.In the comparison,the T/NT ratio of 99Tcm-survivin mRNA mismatch PNA at 4 h (3.12 ±0.50) was significantly lower (t =2.918,P =0.019).Conclusions 99Tcm-survivin mRNA antisense PNA has high labeling efficiency,good stability and no need of purification.Its characteristic of especial uptake by tumor lesion provides the potential value in early diagnosis of tumor.
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Objective To establish a specific,sensitive,simple human respiratory adenovirus detection method to put a good experimental foundation for developing universal and specific diagnostic reagents of human respiratory adenovirus.Methods The nucleotide sequences of 10 serotype of human respiratory adenovirus were obtained from GenBank.Highly consewed five pairs of universal and specific adenovirus primers were designed on the evaluation of multiple sequence alignment of the 10 full genomic sequences with the software DNAMAN 5.2.2,Gene Runner 3.05,BLAST,and to ensure that the polymerase chain reaction(PCR)products were type-specific.NP-40 sample lytic method was employed to prepare the template.The effectiveness,specificity and sensitivity of primers were evaluated.And PCR was carried out to test 64 samples of throat swabs of acute respiratory infection children.The positive PCR products were sequenced directly to identify the adenovirus serotypes.The positive specimens was inoculated on HeLa cells to observe cytopathie effect(CPE)under light microscope,and virus morphology were observed under electron microscope.Results BLAST results indicated that the five pairs of primers were specific adenovirus primers with low homology to the others.The primers were identified as PCR positive fragments obtained by using five pairs of primers to amplify the human adenovirus type 3 DNA,which did not react to the respiratory syncytial virus and Coxsackie virus.the effectiveness and specificity of the primer were thus indicated.PCR sensitive results showed 10-5 dilutin of adenovirus culture and DNA sample could be deteeted which meant the method was sensitive and stable.Two PCR positive specimens were detected in 64 clinical samples.the positive rate was 3.13%(2/64).Using the two PCR positive specimens to inoculate the HeLa cells,the typical adenovirus CPEs of rounded and aggregated,detatched cells were observed under light microscope.And a large number of adenovirus typical particles with characteristic lattice arrangement were observed in the infected cells under electron microscope.PCR product sequencing results showed that these two isolated adenoviruses were typed in human adenovims group B.Conclusions Universal and specific adenovirus PCR primers with the serotype specificity of the PCR products were designed successfully.The PCR primers was sensitive and specific and could be routinely applied in clinical adenovirus diagnosis for respiratory specimens.