| Since effective neoadjuvant chemotherapy has been applied, the 5-year disease free survival of nonmetastatic resectable primary Osteosarcoma has been greatly improved (from 20% up to 70%). However, approximately 40% of the cases are going to get local recurrence or lung metastasis, how to improve the chemotherapy effect of these patients is a pressing task. Numerous attempts to recruit the tumoricidal effect of chemotherapy, such as multidrug resistance reverse, have not fulfilled the expectation. A recently developed novel approach for potentiation chemotherapy cytotoxity was offered by applying caffeine, which is a kind of biochemical modulator. The experiment was designed to observe the enhancement effect of caffeine combined with cisplatin in OS-732 cell line: to observe the apoptosis of OS-732 cell; to explore the molecular10mechanisms of caffeine' s enhanced cytocidal and antitumor effect; and to provide with experimental proofs for further research of clinical use.Materials and Methods1. Cell cultureHuman osteosarcoma cell line (OS-732) was cultured with RPMI-1640 medium containing 10% fetal bovine serum at 37 ℃ in a CO: incubator in 95% air:5% CO: at 100% humidity. A solution of 0.5% trypsin was used to detach OS-732 cells for subculture.2. Cell treatmentAt 43 ℃, the OS-732 cell line was treated with caffeine, cisplatin, caffein+cisplatin for Ihr, respectively, then the cell line was cultured at 37℃ in a CQz incubator in 95% air:5% CO: at 100% humidity for 72hrs. At 371C, the OS-732 cell line was treated with caffeine, cisplatin, caffein+cisplatin for 72hrs, respectively.3. Temperature controlThe 43 condition was controlled by which the cells were suspended in grass tubes containing 2ml of fresh growth medium and incubated at 43 for Ihr in water baths equipped with a themoregulator with precision control, <.1(S.D). While the 37X condition was controlled by which the cells were cultured with RPMI-1640 medium containing 10% fetal bovine serum at 37℃ in a COz incubator in 95% air:5% COi at 100% humidity.4. MTT assayllAfter being harvested, the OS-732 cells (at 2xlOs/ml) were seeded in 96 wells microculture plates, was added to 3 replicated wells .The plates were incubated at 37X! in a COj incubator in 95% air:5% COz at 100% humidity for 72hrs and then 50jig/well HTT was added, continued incubated for 4 hrs. The formaza crystal was dissolved in 10% DMSO solution, and shook for 5min. Finally, the plates were read on Sigma960 Elisa reader at 590nm.5. Flow cytometry analysisAt 43℃, the OS-732 cell line was treated with caffeine, cisplatin, caffein+cisplatin for Ihr, respectively, the cell cycle phases were analysed. At 37℃, the OS-732 cell line was treated with caffeine, cisplatin, caffein+cisplatin for 72hrs, respectively, the cell apoptotic rate were analysed. At 37X2, the OS-732 cell line was treated with caffeine, cisplatin, caffein+cisplatin for 72hrs, respectively, by using FITC anti-Fas antibody, the expression rate of Fas on OS-732 cell line was analysed.6. Electron microscope observationAt 3TC, the OS-732 cell line was treated with caffeine, cisplatin, caffein+cisplatin for 72hrs, respectively, the cells were harvested, followed by fixation in 2% glutaraldehyde buffered in 0. Imol/L NaCaC, ph7.4. All procedures were carried out at room temperature. Treatment with 1% Os04 in 0. Imol/L NaCaC at 4TC for Ihr ensued. The samples were dehydrated using a gradient of ethanol washes ranging from 50% to 90%, followed by propylene oxide treatment. The samples were then embedded in 100% Spurr's resin heated overnight at 6512℃ and sectioned to a thickness of l(im. Sections were placed on copper grids and examined using Philips TECNAI Biosystem electron microscope at 80Kv.7. Reverse Transcription Polymerase Chain Reaction (RT-PCR)RT-PCR was used to investigation the alteration of Fas expression in the level of message RNA. Total cellular RNA was extracted from OS-732 cell using Trizol reagent(GIBCO). Reverse transcription reactions were carri...
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