RESUMEN
<p><b>OBJECTIVE</b>To construct a recombinant adenovirus vector for SH2-DED fusion gene and assess its inhibitory effect on the proliferation of K562 cells.</p><p><b>METHODS</b>SH2-DED fusion gene and its mutant SH2mt-DED were amplified by splicing PCR and cloned into pAdTrack-CMV plasmid separately to construct the shuttle plasmids pAdT-SD-EGFP and pAdT-SmD-EGFP, respectively. After Pme I digestion, the shuttle plasmids were transformed into ultra-competent pAd5F35-BJ5183 cells to generate defective adenovirus vectors pAd5F35-SD-EGFP and pAd5F35- SmD-EGFP by homologous recombination. The vectors, linearized by Pac I digestion, were further transfected into AD293 cells for packaging and amplified by infecting AD293 cells repeatedly. K562 cells were then infected by the recombinant adenoviruses and the expression of SD was detected by Western blotting. MTT assay and flow cytometry were used to investigate the effect of Ad5F35-SD-EGFP and Ad5F35-SmD-EGFP on the proliferation of K562 cells.</p><p><b>RESULTS</b>The recombinant adenovirus vectors pAd5F35-SD-EGFP and pAd5F35-SmD-EGFP were constructed correctly, with a titer reaching 1.5×10(12) pfu/ml after amplification. Western blotting demonstrated that the target proteins were effectively expressed in transfected K562 cells. MTT assay and flow cytometry showed that transfection with pAd5F35-SD-EGFP resulted in growth inhibition rate of 55.21% in K562 cells, significantly higher than the inhibition rate of 17.95% following transfection with pAd5F35- SmD-EGFP and 7.33% following PBS treatment (P<0.05).</p><p><b>CONCLUSION</b>The recombinant adenovirus vector Ad5F35-SD-EGFP we constructed can significantly inhibit the proliferation of K562 cells in vitro.</p>
Asunto(s)
Humanos , Adenoviridae , Genética , Metabolismo , Proteínas Reguladoras de la Apoptosis , Genética , Proliferación Celular , Clonación Molecular , Vectores Genéticos , Proteínas Fluorescentes Verdes , Genética , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva , Patología , Proteínas Mutantes , Genética , Proteínas Recombinantes de Fusión , Genética , Proteínas Represoras , Genética , Proteínas Adaptadoras de la Señalización Shc , Genética , TransfecciónRESUMEN
<p><b>OBJECTIVE</b>To observe the effect of recombinant adenovirus-mediated wild-type p53 gene on the number and proteins of centrosome in K562 cells. To explore the possibility of application of wild-type p53 gene therapy in the treatment of chronic myeloid leukemia.</p><p><b>METHODS</b>The recombinant adenoviruses carrying wild-type p53 gene (Ad5 wtp53), mutant p53 gene (Ad5 mtp53) or the green fluorescent protein (GFP) gene was repeatedly amplified and co-infected into K562 cells with cation polybrene. The optimal infection titer and infection time of the recombinant adenoviruses were determined by MTT assay, p53 mRNA and protein expression were determined by RT-PCR and Western blot respectively. The centrosomal structural protein gamma-tubulin and the spindle protein alpha-tubulin were marked simultaneously by indirect immunofluorescence staining, and the expression of the centrosomal gamma-tubulin protein, the mitosis and the number of centrosome were observed under the laser confocal microscopy.</p><p><b>RESULTS</b>Infection efficiency with recombinant adenoviruses was facilitated by polybrene in K562 cells, and 4 microg/ml polybrene was chosen. The optimal adenovirus infection titer was 20,000 MOI and the optimal infection time was 72 hours. p53 mRNA and P53 protein can be expressed in K562 cells by Ad5wtp53 and Ad5mtp53. Both the expression of the centrosomal gamma-tubulin protein and the number of centrosomes were decreased after Ad5wtp53 infection.</p><p><b>CONCLUSION</b>There is sustained expression of P53 protein in K562 cells after its infection by Ad5wtp53. Wild-type P53 protein can lead to the down-regulation of the number of centrosomes and the expression of centrosomal gamma-tubulin protein in K562 cells.</p>
Asunto(s)
Humanos , Adenoviridae , Genética , Centrosoma , Metabolismo , Genes p53 , Genética , Vectores Genéticos , Células K562 , Transfección , Tubulina (Proteína) , Metabolismo , Proteína p53 Supresora de Tumor , MetabolismoRESUMEN
Objective To establish a quantitative RT-PCR method with self-quenched fluorogenic probe for detection of bcr/abl mRNA in patients with chronic myeloid leukemia for providing a useful tool for diagnosis of CML,evaluation of therapeutic effect and monitoring of minimal residual disease(MRD). Methods bcr/abl gene from cultured K562 cells was amplified by conventional RT-PCR.The standard quantitative plasmid was constructed by A-T clone method.The self-quenched fluorogenic quantitative RT- PCR method(FQ-RT-PCR)for determination of bcr/abl mRNA was established successfully using the ABI PRISM 7000 PCR Detector.The linear range,sensitivity,stability,and repetitiveness of the method were determined.The marrow samples from 25 CML patients and 3 ALL patients were assessed.Results The sensitivity of the FQ-RT-PCR was 10 copies/?l recombined plasmid,and bcr/abl mRNA can be detected from 1 K562 cell in 10~5 normal cells.The linear range was 10~2-10~9 copies/?l recombined plasmid.The coefficient variation(CV)value was 2.1% in intra-assay and 6.1% in inter-assay.The median ber/abl mRNA expression level was 4.50?10~4 copies/?g RNA [(0.45-89.00)?10~4],5.45?10~4 copies/?g RNA [(2.95-19.30)?10~4 ],13.00?10~4 copies/?g RNA [(4.10-89.00)?10~4] and 2.35?10~4 copies/?g RNA [(0.45-5.12)?10~4] in 25 CML patients,11 patients in the incipient chronic phase,6 patients in blastic crisis,8 patients in chronic period after treatment,respectively.The bcr/abl mRNA level in blastic crisis was significantly higher than that in chronic phase(q= 3.41,P