| Objectives: To observe the expression of survivin gene in human hepatocellular carcinoma and its relationship to the clinical pathological features. To inhibit the expression of survivin gene in human hepatocellular carcinoma line SMMC-7721 by antisense technology, to study its inhibitory effect on the growth of 7721 cell line and its mechanism. To explore the changes of expression and activity of p38MAPK and caspase-3 in 7721 cell line after transfection with survivin antisense oligonucleotide(ASODN), to learn the signal transduction in the inhibitory effects of survivin ASODN.Methods: The present study was divided into three parts. In the first part, immunohistochemical method has been used to assess the expression of survivin and proliferating cell nuclear antigen(PCNA) protein in a series of 48 surgically resected, formalin-fixed, and paraffin-embedded human primary hepatocellular carcinoma tissue samples. Apoptosis was assessed by means of the Tdt-mediated dUTP-biotine nick end labeling(TUNEL) technique in combination with morphological criteria. The results obtained were analysed compared with some clinical histopathological factors. In the second part, we designed and synthesized a 20mer ASODN target to the promoter region of survivin mRNA. The ASODN was transfected into 7721 cell line at variety concentration using DOTAP liposomal transfection reagent. The change ofexpression of survivin protein after transfection was assessed by Western blot and immunohistochemical methods. In situ hybridization method was used to detect the change of expression of survivin mRNA. The changes of cell adherent rate, cell growth activity, and the inhibitory rate of cell growth were also studied in this part. Furthermore, in order to explore the mechanisms of the inhibitory effect on the cell growth of survivin ASODN, we observed the changes of ultrastructure, apoptosis, and cell skeleton microfilament system after transfection by electron microscope, flow cytometer, and confocal microscope, respectively. In the last part, cell cycle and the expression of cyclinBl mRNA were assessed by flow cytometer and reverse transcription polymerase chain reaction(RT-PCR), respectively. We detected the changes of expression and activity of p38MAPK and caspase-3 after transfection with survivin ASODN. SB202190, a specific inhibitor of p38MAPK, was used to study the relationship between the activity of p38MAPK and caspase-3, to explore the signal pathway after survivin ASODN transfection. cl 011DResults: 1.Survivin protein expressed positively in 31 of 48 cases of primary hepatocellular carcinoma patients, the positive rate was 64.6%. The expression of survivin protein in hepatocellular carcinoma tissues has no relationship to the clinical histopathological features but the differentiation of the tumor. The expression of survivin protein in patients of Edmondson grade III-IV were significantly higher than that in patients of grade I ~ II .The ratio of cell proliferation to apoptosis in the patients of survivin protein expression positively were significantly higher than that in patients of survivin expression negatively. The survive rate of three years in the patients of survivin protein expression positively was significantly lower than that in patients of survivin expression negatively.2.The expression of survivin protein and mRNA were decreased markedly after survivin ASODN transfection by DOTAP liposomal transfection reagent. Meanwhile, the cell adherent rate also decreased markedly.The inhibitory effect on the cell growth could maintain about a week after transfection, and the highest inhibitory rate could be 71.82% three days after transfection. 3.The ultrastructure presented apoptotic changes after transfection such as chromatin condensed and apoptotic body formation.4.Great changes of cell skeleton microfilament system had taken place after survivin ASODN transfection. The well-arranged structure of microfilament was broken or destroyed, and the average intensity of fluorescent of microfilament system decre...
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