RESUMO
<p><b>OBJECTIVE</b>To construct deltaNp63 specific small hairpin RNA (shRNA) expressing plasmid,to examine its inhibitory effect to the expression of deltaNp63 protein and mRNA in transitional cell carcinoma of the bladder (TCCB) , its effect on TCCB cells cycle and proliferation.</p><p><b>METHODS</b>DeltaNp63 specific oligonucleotides were designed and synthesized. These oligonucleotides were annealed to form double strand DNA fragments and this fragment was cloned into Pgenesil-1 plasmid. The recombinant deltaNp63-shRNA expression construct was confirmed by using Pst I + Sal I double digestion and by sequencing. Fluorescence staining was used to confirm the success of transfection in TCCB cells under the fluorescence microscope. The inhibitory effect of deltaNp63-shRNA construct was examined with semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical staining assay. The cell cycle of TCCB cells was assayed by flow cytometry (FCM). The cellular proliferation of TCCB cells was assayed by tetrazolium bromide (MTT) colorimetry.</p><p><b>RESULTS</b>The deltaNp63-shRNA expression plasmid was successfully constructed and transfected into TCCB cells. It can effectively reduce the expression of deltaNp63 protein and mRNA. The reduction rate of deltaNp63 mRNA was 63.0%, and the G0/G1 ratio was increased and S phase was decreased in transfected TCCB cells. The cellular proliferation was also lower in transfected 5637 cells in comparrison with that of non-transfected TCCB cells.</p><p><b>CONCLUSION</b>A deltaNp63-shRNA expression plasmid, constructed from Pgenesil-1 plasmid, can successfully be transfected into TCCB cells and can effectively inhibit the expression of deltaNp63 protein and mRNA. It also can take part in regulation of the cell cycling and inhibit the cellular proliferation of TCCB cells.</p>
Assuntos
Humanos , Carcinoma de Células de Transição , Genética , Metabolismo , Patologia , Ciclo Celular , Genética , Fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA , Genética , Fisiologia , Imuno-Histoquímica , Microscopia de Fluorescência , Plasmídeos , Genética , Interferência de RNA , RNA Mensageiro , Genética , RNA Interferente Pequeno , Genética , Proteínas Recombinantes , Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores , Genética , Fisiologia , Fatores de Transcrição , Transfecção , Proteínas Supressoras de Tumor , Genética , Fisiologia , Neoplasias da Bexiga Urinária , Genética , Metabolismo , PatologiaRESUMO
<p><b>OBJECTIVE</b>To study the effect and mechanism of saffor injection on renal ischemia/reperfusion (I/R) injury in rats.</p><p><b>METHOD</b>Seventy-five SD rats were randomly divided into five groups (n = 15, in each), normal control groups, I/R control groups, low-dose treatment groups, middle-dose treatment groups and high-dose treatment groups. After rat's I/R injury model was established, renal function was assessed by measuring serum creatinine, blood urea nitrogen, urine osmotic pressure and urine osmotic pressure/blood osmotic pressure, the apoptosis rate in I/R renal tissure was measured by TUNEL method and caspase-3 concentration was measured by immunohistochemistry.</p><p><b>RESULT</b>Reperfusion of the ischemic kidney induced marked renal dysfunction. Saffor injection significantly inhibited the reperfusion-associated increase in apoptosis rate and caspase-3 protein absorbance value. Moreover, the renal dysfunction at all treatment groups was markedly ameliorated by Saffor injection. (P < 0.01).</p><p><b>CONCLUSION</b>The results show that saffor injection significantly reduces the renal dysfunction and injury caused by I/R of the kidney, And the protective effect of Saffor injection may be related to the inhibition of cell apoptosis and caspase-3 gene expression following renal I/R.</p>