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A Research of the Mechanisms of Radiosensitizing Effect of Taxotere on Human Nasopharyngeal Carcinoma Cell Line CNE-1

Author: GuFei
Tutor: LiGuang
School: China Medical University
Course: Oncology
Keywords: Taxotere Radiosensitization Human nasopharyngeal carcinoma cell lines Cell cycle Apoptosis
CLC: R739.63
Type: Master's thesis
Year: 2003
Downloads: 61
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Abstract

Objective To analyze the Taxotere human nasopharyngeal carcinoma cell line CNE-1 radiosensitizing effect of whether to change the cell cycle distribution, and observe whether the ability to induce apoptosis of CNE-1 cells. Materials and methods. Cell lines and culture methods: human nasopharyngeal carcinoma cell line CNE-1 (purchased from the Institute of Virology of the Chinese Academy of Preventive Medicine). 5% CO, ₂, 100% humidity, 37 ° C thermostat box, containing 10% fetal calf serum PRMI 1640 culture medium (GIBCO BRL, USA), CNE-1 cells were seeded in the culture flask monolayer subculture. Take the exponential growth phase cell experiments. 2. Drugs and irradiation method: experimental preparation of Taxotere (AVENTIS) PRMI 1640 culture medium to the desired concentration of 5 × 10 ^{-8 mg / ml. PRIMUS linear accelerator using SIEMENS 6MV X-ray irradiation, SSD = 100cm, dose rate 200cGy/min, beam 40 × 40cm. 3. Flow cytometry analysis: take the exponential growth phase cells trypsinized to count the number of viable cells, seeded in flasks at a certain number of adherent culture 12h after removal of the culture medium. (1) control group: Add fresh medium (4ml) of the drug-free culture 12h; once again for fresh culture fluid (4 ml); (2) medication alone group: Join with Taxotere (5 × 10 ^{-8 mg / ml) of the culture fluid (4ml) for 12h to drug-containing culture solution was discarded, washed 2 times with PBS, and fresh medium (4 ml); (3) simple irradiation group: drug-free fresh culture medium (4ml) cultured 12h, and then immediately to 2 Gy single irradiation for-the fresh medium NInl people again; the N) Teso Emperor plus irradiation group: Canadian people with Taxotere O x 10 a m Kuang ml) of discarded together drug culture liquid culture fluid NML) for 12h, washed twice with PBS, add fresh medium NInlX followed immediately single irradiated with 2 Gy. The cells in each group mentioned above, after treatment, to continue cultured for 0, 12,18,24 J6/8h, were trypsinized, cells were harvested, -20 ° C 70% ethanol precooled 4t fixing at least 0 · SH. Discarded before testing of ethanol, washed with tion the X-100 of 0.1% IInl * the RNase 200 ... J0Pg/Inl PI dyebath 100gi processed by flow cytometry (FACScan) excitation light wavelength 488run. Each sample for at least detecting lx 10 'cells, the data acquisition using CELLQuest software, and cell cycle was analyzed using the the ModF't 2.0 software. 4. Immunohistochemical staining: the coverslip a 6cm diameter glass dish, place two coverslips per culture dish. Take the exponential growth phase cells trypsinized to count the number of living cells, dropping by a certain number of single-cell suspension on coverslips, covered petri dish superstructure. Adherent culture after about half an hour, to a petri dish plus the the people 4Inl culture medium, cultured for 12h after removal of the culture medium. O) control group: Canadian people free of drugs of fresh medium kInl), cultured 12h; plus fresh medium KInlh K) treatment group again: plus people with Taxotere OX 10 - 'mgilnl) culture medium NInl) for 12h to the drug-containing culture solution was discarded, washed 2 times with PBS, for a fresh culture fluid (4ml); hood) the cells in each group treated culture was continued 48h, remove the coverslip washed 3 times with PBS solution (x smin human fixed in 95% ethanol 10ndn, PBS solution was washed 3 times (x sndn). O) at 0 the coverslip. 20% Triton X-100 solution dip 20ndn washed 3 times with PBS liquid (x 5 dn later step by immunohistochemistry kit instructions. Using S-P method, an anti-rabbit anti-human use. CasPase the-Pn Polyclonal · 2 · antibody (MB-0303, OL NO: 20526303A), mouse anti-human h-2 monoclonal antibody (MAB-0014, nt.NO: 11224014C) and S-P detection kit (KIT-97 10, oL N ... 021) are the Fuzhou step development of new biotechnology companies. 5. data and image processing: Using statistical software SPSS10.0 radiography chart. photomicrograph of immunohistochemical results with the control group compare, 5 x 10 m sand ml Taxotere or two X-ray irradiation, all can make the CNE a cell G + M phase arrest, the H Joint Application of G + M phase arrest the synergy. with the removal of the processing factors extend G + M phase cell percentage gradually increased, and the removal of processing factors 18h after the effect of increasing the treatment of different factors 0, 12, 18 on 4,36 / the 8h, processing factors induced by the order of the intensity of ** E apoptosis: Taxotere 10 irradiation> Taxotere or irradiation control apoptosis rate gradually increased with increasing time and removal process is becoming more apparent factors 18h. compared with the control group, removal of 48 h after treatment factors, Taxotere group positive expression of pro-apoptotic proteins CasPase-Pan enhanced, while the anti-apoptotic protein h twelve positive expression weakened Conclusion Taxotere can human nasopharyngeal carcinoma cell line CNE beans cells Gi + M phase arrest and induced apoptosis of nasopharyngeal carcinoma cell line CNE cells, may be the important mechanism of CNE cell radiosensitizer.}}

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