| Objective1. To detect the expression of the inhibitor of apoptosis gene, survivin, and its correlation factors, caspase -3 and cyclin D2 in human osteosarcoma, and investigate the relationship of theses factors on malignant biological behaviour of osteosarcoma.2. To investigate antisense oligodeoxyribonucleotide ( ASODN ) blocking the expression of survivin gene, inducing apoptotic effect, and its synergistic effect on chemotherapeutics, DDP, in human osteosarcoma cell line in vitro.3. To explore the use of antisense gene technique combined with traditional chemotherapy to treat the nude mice bearing human osteosarcoma xenograft.Methods1.38 cases of osteosarcoma, 15 cases of osteochondroma and 5 cases of normal osseous tissue were involved. In situ hybridization, Immunohistochemis-try and Tdt - mediated dUTP nick end labeling ( TUNEL method) were used to detect the expression of survivin mRNA and the protein expression of caspase -3 and cyclin D1 and apoptosis; and then compared survivin mRNA with the major clinical pathological parameters of osteosarcoma and the relationship and significant of other factors expression.2. Targeted ASODN of survivin was designed and synthesized, liposome as carrying agent, and then transfected to osteosarcoma OS - 732 cells at different concentration and time; and at the same time normal control and positive control(sense oligonucleotides, SODN group) were set for comparison. Cell growth were observed under the microscope; reverse tanscriptionase - Polymerase chain reaction ( RT - PCR) and immunocytochemical technique were used to detect the expression of survivin mRNA and protein in each group; acridine orange / ethid-ium bromide ( AO/EB ) staining and flow cytometer ( FCM ) for the detection of the level and apoptotic morphology in each group; MTT assay for the detection of cell growth suppression; kinase activity assay for the detection of the degree of caspase - 3 activation.On the base of transfection ASODN alone to osteosarcoma OS - 732 cells to block the expression of survivin gene, DDP treatment group and DDP combination with ASODN group were set up. Compared cell growth of the combination group with that of each single - agent group. Reverse tanscriptionase - Polymerase chain reaction ( RT - PCR) and immunocytochemical technique were used to detect the expression of survivin mRNA and protein in each group; acridine orange / ethidium bromide ( AO/EB) staining and flow cytometer ( FCM ) were used to detect the level and apoptotic morphology in each group; MTT assay was used to detect cell growth suppression.3. Human OS -732 cells were implanted in the nude mice, osteosarcoma implanted tumor model was established. According to control group ( physiological saline by abdominal cavity injection) , ASODN group ( ASODN by intratu-moral injection) , DDP group ( DDP by abdominal cavity administration) and ASODN + DDP group ( combined administration) , the tumor bearing mice were treated respectively for 7 days. Comparison with each single - agent therapy and control group was performed in aspects such as tumor growth condition, pathological changes of tumor tissues; survivin protein expression in tumor tissues by immunohistochemistry and tumor apoptosis by Tdt - mediated dUTP nick end labeling (TUNEL).Results1. The positive rate of survivin mRNA in osteosarcoma was 65. 8% (25/ 38) , but rarely expressed in ostechondroma (6.1%) and normal osseous tissue(0%). Apoptotic cells were detected in 24 osteosarcoma cases (63. 2%), mean value of AI was 1.1% (0% ~ 17% ) , AI of negative group( 11. 3% ) of survivin was significantly higher than positive group(4. 7% , x =3. 87,P <0. 01); caspase -3 protein expressed in 27 cases(71.1% ) and its expression was not correlated with AI and survivin expression (r = 0. 267, P = 0. 105 ) . Cyclin Dj protein were detected in 27 osteosarcoma cases (71.1% ) , and its expression was positive correlated with survivin (r = 0. 370, P = 0. 022 ).2. Cell morphous was basically normal in the normal control and SODN group at different transfection phase, but in ASODN transfection group, cell growth condition gradually decay as transfection time prolonged, especially in 400nM and 600nM ASODN groups; cell exfoliation and death was more obvious when treated with ASODN in combination with DDP. The expression of survivin mRNA and protein in OS - 732 cells of each ASODN transfection groups were weaker than those of normal control and SODN group with different degree, which showed gradient suppression as transfection concentration and time increased. 12 hours after transfection, survivin mRNA and protein in ASODN groups were significantly lower than those of the normal control and SODN group; but there was no obvious difference between the normal control and SODN groups; otherwise, the expression of survivin mRNA and protein did not decreased obviously in DDP group and SODN + DDP group.Flow cytometry detection showed, apoptotic peak was seen in ASODN groups 6h after transfection, which get obviously as transfection time prolonged, apoptosis index (AI) of each ASODN groups was significantly higher than thai of the normal control and SODN groupsafter 12h transfection; AO/EB fluorescent staining showed the characteristic of typical apoptotic changes, including cell atrophy, condensed chromatin, condensed and heavily stained nuclei. At the same time, caspase - 3 activity increased correspondingly, and showed concentration and time depended. 6h after transfection, MTT light absorption ( A492 vale) of each ASODN group decreased, but inhibition ratio (IR) increased; 12h after transfection, the index above-mentioned exhibited more obviously in ASODN + DDP group, which AI and IR were significantly higher than those of each single agent group and SODN + DDP group. The major apoptotic cells were... |
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