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Objective To explore the mutation types and disciplines of STR commonly used in forensic in gynecologic and breast cancerand investigate the application of microdissection in forensic practice involving tumor tissue. Methods DNA of tumor tissues, adjacent normal tissues and peripheral blood from 62 patients with breast cancer, 62 patients with gynecologic cancer and 10 patients with benign gynecologic tumor were amplified by PowerPlex 21 System kit and Argus X-12 kit. Capillary electrophoresis of PCR products was carried out on an ABI 3130 Genetic Analyzer to obtain genotypes. Some tumor tissues with STR variation were microdissected. Results The genotype of peripheral blood in cancer patient was consistent with that of corresponding normal tissue. 4 types of STR variations were found in 46.77% gynecologic cancer tissues, compared with that in benign tumor tissues and breast cancer, the difference of STR variation was significant(P<0.01,P=0.009). The genotype of stromal cells separated by microdissection was consistent with that of corresponding adjacent normal tissue. Conclusion The STR loci detected in the study with poor stability are not suitable for forensic cases involving gynecologic cancer tissues. The genotype of stromal cells separated accurately from tumor tissues by microdissection could represent the normal DNA genotype of the individual with cancer. Microdissection is an effective solution in forensic cases with tumor tissues.
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Objective The purpose of this study was to detect the degradation degree of long-term formalin fixed tissue and to compare the detection rate of STR with SNP. Methods DNA was extracted from 24 formalin-fixed tissues stored at -20 ℃ for five years, and the concentration and degradation index of DNA was quantified with Quantifiler? Trio DNA Kit. A 55-SNP multiplex SNaPshot assay and PowerPlex? 21 system were used to amplify SNP and STR loci, respectively. Results The results showed that the degradation indexes of 24 specimens were ranged from 1~8. The SNP genotypes of the 24 specimens were completely consistent with the non-degraded DNA from the same individuals and the successful genotyping rate was 100%. However, 33 allele dropouts were observed with STR genotyping in 8 samples, of which the degradation index was higher than 2.6, and the fragment size of the 75.8% allele was longer than 300bp. The likelihood ratio based on 16 typable STR loci in the sample was close to that onthe basis of 54 SNPs. However, likelihood ratio based on more than 17 STR loci was over that accord to 54 SNPs. There was a negative correlation between the fragment size of STR and the allele detection rate, and a negative correlation also observed between the degradation index of samples and the allele detection rate except for two samples with mild degradation. Conclusion This study validated that the long-term formalin-fixed tissues were susceptible to degradation, and the SNP was more suitable for detecting these tissues than STR typing system. However more SNP loci are needed to test in order to increase the discrimination power.
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AIM: To investigate in vitro effects of cholecystokinin octapeptide(CCK-8) on the expressions of B7.1 and B7.2 and the costimulatory activity of T lymphocytes in unstimulated macrophages.METHODS: Mouse peritoneal macrophages were isolated and incubated with CCK-8(10-12-10-6 mol/L) for indicated time.The B7.1 and B7.2 expressions of murine peritoneal macrophages were analyzed by flow cytometry.CD4+T cells were isolated from mouse spleen using immunomagnetic beads,and cultured with 1/4 numbers of macrophages which were pretreated with CCK-8 and/or anti-B7.1 antibody,anti-B7.2 antibody,CCK1R antagonist CR1409,CCK2R antagonist CR2945 for 24 h.ConA was added into the culture medium to stimulate CD4+T cell proliferation.The proliferation was determined by measuring -TdR incorporation in a ?-scintillation counter.RESULTS: B7.1 and B7.2 expressions and costimulatory activity of peritoneal macrophages were enhanced by CCK-8 in a dose-dependent manner,and the maximal effects occurred at the concentrations of 10-9 mol/L to 10-7 mol/L.Anti-B7.2 antibody,but not anti-B7.1 antibody,reduced the modulatory role of CCK-8 on costimulatory activity.Both CR1409 and CR2945 reversed the effect of CCK-8 on costimulation,and the role of CR1409 was more significant.CONCLUSION: CCK-8 enhances macrophage costimulatory activity by upregulating B7.2 expression,which is mediated by CCK1R and CCK2R.CCK1R might be the major receptor responsible for the modulation of CCK-8 on costimulation.
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AIM: To investigate the inhibitory effect s of a synthetic CRE-transcription factor decoy oligodeoxynucleotide (CRE-decoy ODN) on the upregulation of the expression of cholecystokinin (CCK) and fosB mRN A induced by chronic morphine administration in SK-N-SH cells. METHODS: The CRE cis-element, TGACGTCA, was palindromic, a sy nthetic single-stranded phosphorothioate oligodeoxynucleotide composed of the CR E sequence self-hybridizes to form a duplex/hairpin. The CRE-palindromic decoy a nd control oligodeoxynucleotides were added to the medium (1 h before exposure t o morphine) at 150 nmol/L in the presence of cationic lipid DOTAP. After the cel ls were treated with 100 ?mol/L morphine for 48 h, 10 ?mol/L naloxone was use d for 15 min. The effects of CRE-decoy ODN on the DNA-binding activity of CREB, the expression of CCK and fosB mRNA were detected by electrophoresis mobi lity shift assay (EMSA) and RT-PCR, respectively. The stability of cell-incorpo rated [ 32P]-labeled CRE-decoy ODN was extracted with phenol:chloroform a nd then subjected to 20% nondenaturing polyacrylamide gel electrophoresis and au toradiography. RESULTS: Chronic morphine administration and acute naloxone-prec ipitated withdrawal significantly activated the DNA-binding activity of CREB and the expression of CCK and fosB mRNA in SK-N-SH cells. The CRE-decoy ODN pen etrated into the cells, specifically downregulated these indexes. CONCLUSIONS: CRE-decoy ODN can significantly downregulates the e xpre ssion of CCK and fosB mRNA through specifically suppressing the DNA-binding activity of CREB activated by chronic morphine administration in SK-N-SH cells.