| 1.The evaluation of the effects of cationic lipid-mediated uptake of C-erbB-2-specific antisense oligonucleotides and toxicity in mouse breast cancer cellsObjective To determine the optimal effect of cationic lipids on oligonucleotides uptake and their toxicities on mouse breast cancer TM40D cells. Methods Oligonucleotides were manually synthesized and completely phosphorothioate-modif ied. FAM was conjugated to them at 5 ' -end for flow cytometry and fluorescence microscopy. TM40D cells were incubated with oligonuclotides with or without liposome. The transfection ratios of antisense ODNs in different time were detected by flow cytometry with cultured cells as negative control. ODNs distribution in cells was observed through fluorescence microscopy. Lactate dehydrogenase in cell-culture mediums was examined. Results The transfection ratio of antisense ODNs without liposome in cells was 30.06% at 1h, gradually increased with time progress, and reached 63% at 6h. Thetransfection ratio of liposome-mediated delivery of ODNs was from 50.23% to 72.23%. Flow cytometric analysis revealed that the time of the optimal effect was 4h, and the ratio maintained at a relatively high level at 6h. Intracellular fluorescence intensity correlated with transfection ratio. Fluorescence microscopy permitted the analysis of intracellular distribution of FAM-labeled ODNs. In the absence of liposome, fluorescence staining in cytoplasm, not in nucleus, was observed. In presence of liposome, cells showed fluorescence staining in cytoplasm and nucleus. There was no statistical difference about lactate dehydrogenase concentrations among three groups. Conclusions Cationic lipid as a transfection carrier enhanced uptake of ODNs in TM40D cells and the peak of uptake appeared earlier. Cationic lipid and antisense ODNs had no apparent toxicity to cells during short contact.2. The study about inhibitory effect of C-erbB-2-specific antisense oligonucleotides on C-erbB-2 over-expressing mouse breast cancer TM40D cellsObjective To study the effect of C-erbB-2-specific antisense oligonucleotides on C-erbB-2 expression, cell proliferation and apoptosis of C-erbB-2 over-expressing breastcancer cells. Methods The C-erbB-2 status of mouse breast cancer TM40D cells was determined by immunohistochemistry. Cells were incubated with liposome-mediated ODNs for 4h and cultured for another 72h, then the effects of ODNs on C-erbB-2 expression, cell proliferation and activation of apoptosis were examined by western blot, MTT assay and flow cytometry. Results Immunohistochemistry staining showed that cytoplasm and cell membrane were stained yellow, which indicated that TM40D cells overexpressed C-erbB-2. Western Blotting showed treatment of TM40D cells with C-erbB-2-specif ic antisense ODNs resulted in inhibition of C-erbB-2 expression. The effects of antisense ODNs on C-erbB-2 protein levels correlated with their effects on cell proliferation. MTT Assay showed antisense ODNs inhibited cell growth by about 50%. Flow cytometry analysis revealed that antisense ODNs increased cell apoptosis by38. 5%, compared with cultured cells group 9.13% and liposome group 9.29%. Conclusions (1)mouse breast cancer TM40D cells overexpressed C-erbB-2 protein; (2) Antisense ODNs reduced C-erbB-2 expression, inhibited cell proliferation and induced cell apoptosis.3. Establishment of Breast Cancer Model in BALB/c Mice andDetection of C-erbB-2 by Immunohistochemistry Objective To establish a model of breast cancer TM40D cells in BALB/c Mice and study their C-erbB-2 expression by immunohistochemistry staining. Methods TM40D cells were implanted hypodermically between 5th and 6th ribs in 30 BALB/c Mice. TM40D cells in each mouse reached 1 × 10~6. Two of tumors were removed for pathological examination. Results The tumor masses were formed on 14th day after implantation, their diameters ranged from 3mm to 5mm. Tumor-formation rate was 93.33% (28/30). Tumor masses were breast cancer, and C-erbB-2 of tumor cells was positive by immunohistochemistry staining. Pathologic examination: the tumor... |
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