| Objective:Survivin was blocked with the antisense technology to explore the effect of survivin antisense oligonucleotide on the proliferation and apoptosis of human breast cancer cell lines MCF-7. Its therapeutic effect on the transplanted human breast cancer in nude mouse was also investigated. Methods: This experiment included three parts. The first part: antisense oligonucleotide (ASODN), nonsense oligonucleotide (NODN) was transfected in Human breast cancer MCF-7 cell line and RPMI 1640 culture medium (normol control). Realtime RT-PCR, Western blot were applied in this study to detect and measure Survivin protein and mRNA expression in MCF-7 cell, The cell adherent rate after transfection was measured to investigate the effect of antisense oligonucleotide on MCF-7 cell growth restriction. The second part: the cell apoptosis rate was observed by Hoechst 33258/P1; the cell ultrastructure change was examined by transmission electron microscope; the change of cell cycle and cell apoptosis was monitored under flow cytometer; the apoptosis of DNA was inspected in gel electrophoresis. The further research was made by the study of the effect of survivin antisense oligonucleotide on the growth restriction of the cell MCF-7. The third part: nude mice were transplanted with human breast cancer MCF-7 cell to construct tumor model, which were divided into three groups (5 mice per group) Lip, ASODN/Lip (lipofectin), NODN/Lip were administrated in the tumor or peri-tumor respectively. Totally, it was injected three times, once every 5 days. The observation was made by tumor restriction rate, volume and the change of the weight of the animal. The tumors were segregated for different measurements: cell apoptosis, ultrastructure and survivin protein. The three groups were monitored by TUNEL method, transmission electron microscope and Western blot. Results : 1. After induction of survivin antisense oligonucleotide to transfection human breast cancer MCF-7 cell, the expression of survivin protein and mRNA decreased obviously. Real time RT-PCR showed the original copy rate of survivin-mRNA decreased notably. Meanwhile, the cell adherent rate was reduced to 32. 96%, and the cell growth restriction rate could be as high as 73.60%. There were statistical differences among the ASOND/Lipgroup, NODN/Lip group and Lip group (P<0. 05). 2. After Hoechst 33258/PI staining, we observed the cell apoptosis rate after transfection which could be as high as 73.36%. The cell apoptosis index (AD increased. There was statistical difference among the ASOND/Lip group, NODN/Lip group and Lip group (P<0. 05). Under transmission electron microscope, the change of cell ultrastructure after transfection was observe: the change was mainly apoptosis. 3. The flow cytometer showed the cell apoptosis after transfection and the blocking phenomena appeared in G2/M period. 4. The subcutaneous tumor model in nude mouse was successfully built. The volume of tumor in the group of ASODN/Lip decreased during treatment while the volume of the other two groups increased. The rate of restriction reached 70.10%. There was no statistical difference among the groups in the weight of the mice. The apoptosis rate in ASOND/Lip group was 38. 6% detected by TUNEL and the rate was statistically different from the other two groups. The western blot showed that survivin protein in NODN/Lip group decreased significantly. Conclusions: 1. ASODN can reduce survivin expression in MCF-7 cell of breast cancer significantly at level of protein and mRNA and it can effectively suppress the proliferation of MCF-7 cells when liposome was used to transfect antisense oligonucleotide in survivin mRNA. Survivin targeting therapy can be used as an important method in breast cancer treatment. 2. The restriction by the reduction of survivin expression in breast cancer cells is mainly functioned through its induction of cell apoptosis and prevention of mitosis at G2/M period. 3. Survivin antisense o oligonucleotide can effectively restrict human breast cancer cells growth rate in subcutaneou...
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