【摘要】：研究背景：鼻咽癌(Nasopharyngeal carcinoma, NPC)是生長于鼻咽部位的最為常見的頭頸部惡性腫瘤之一。NPC的發(fā)病是一個多因素共同作用的結(jié)果,其病因可能與EB病毒感染、吸煙、居住環(huán)境受污染、進(jìn)食腌制食品有關(guān)。由于鼻咽周圍的解剖結(jié)構(gòu)相當(dāng)復(fù)雜,因此不適合行手術(shù)治療。治療鼻咽癌的主要方法是放射治療,但腫瘤的輻射抗性和大劑量照射對正常組織的損傷是鼻咽癌放射治療中難以突破的瓶頸。因此,尋找新型抗腫瘤藥物及高效低毒的腫瘤放射增敏劑是目前NPC治療的主要研究目標(biāo)。 淫羊藿素(Icaritin, ICT)作為中藥淫羊藿提取物淫羊藿苷的衍生物,不僅具有抗氧化、防治骨質(zhì)疏松、改善心血管功能、保護(hù)神經(jīng)變性損傷等作用,近幾年的研究表明,其對血液系統(tǒng)惡性腫瘤、乳腺癌、前列腺癌、子宮內(nèi)膜癌及肝癌還具有一定的抗腫瘤作用。本研究用淫羊藿素作用于人鼻咽癌細(xì)胞CNE-2,觀察其對CNE-2細(xì)胞是否具有抑制增殖、誘導(dǎo)凋亡及放射增敏的作用,并對淫羊藿素放射增敏作用的可能機(jī)制進(jìn)行初步的研究與揭示,為進(jìn)一步的臨床研究提供實驗證據(jù)。 第一部分淫羊藿素(Icaritin, ICT)對人鼻咽癌細(xì)胞CNE-2抑制增殖和誘導(dǎo)凋亡效應(yīng) 目的：觀察淫羊藿素對人鼻咽癌細(xì)胞CNE-2抑制增殖和誘導(dǎo)凋亡的效應(yīng)。 方法：以不同濃度淫羊藿素處理CNE-2細(xì)胞,倒置顯微鏡下觀察細(xì)胞貼壁情況和形態(tài)變化,四甲基偶氮唑藍(lán)(MTT)比色法檢測細(xì)胞生長抑制情況,Annexin V-FITC/PI雙染流式細(xì)胞術(shù)檢測細(xì)胞凋亡情況。 結(jié)果：1.倒置顯微鏡觀察發(fā)現(xiàn),隨著藥物濃度及作用時間的增加,經(jīng)淫羊藿素處理后的CNE-2細(xì)胞增殖減慢,形態(tài)逐漸變小、變圓、浮起,折光度降低,細(xì)胞間連接松散。 2.MTT比色法檢測不同濃度淫羊藿素作用于CNE-2細(xì)胞,發(fā)現(xiàn)隨著藥物濃度的增加、.作用時間的延長,其對CNE-2細(xì)胞的抑制率逐漸升高。CNE-2細(xì)胞經(jīng)淫羊藿素作用24h、48h、72h時的IC50值(半抑制濃度)分別為(93.63±1.60)μmol/L、(52.90±0.32)μmol/L、(32.62±1.17) μmol/L。 3.流式細(xì)胞儀分析：不同濃度淫羊藿素0μmol/L、2.5μmol/L5μmol/L、10μmol/L、20μmol/L、40μmol/L)作用CNE-2細(xì)胞24、48h后,其細(xì)胞凋亡率分別由(4.59±1.49)%增至(30.73±4.06)%,(6.15±0.97)%增至(52.65±2.07)%,經(jīng)統(tǒng)計學(xué)分析,其與對照組的差異具有統(tǒng)計學(xué)意義(P0.05)。 結(jié)論：體外實驗證明,一定濃度的淫羊藿素對人鼻咽癌細(xì)胞CNE-2有抑制增殖和誘導(dǎo)凋亡作用,并呈時間濃度依賴性。 第二部分淫羊藿素(Icaritin, ICT)對人鼻咽癌細(xì)胞CNE-2的放射增敏作用及機(jī)制研究 目的：探討淫羊藿素對人鼻咽癌細(xì)胞CNE-2的放射增敏作用及可能機(jī)制。 方法：在不同的照射劑量下,以低細(xì)胞毒性濃度的淫羊藿素處理CNE-2細(xì)胞,MTT比色法初步判斷淫羊藿素對CNE-2細(xì)胞的放射敏感性,克隆形成實驗觀察其對CNE-2細(xì)胞的放射增敏作用。流式細(xì)胞術(shù)分析低濃度淫羊藿素作用前后CNE-2細(xì)胞的周期變化。 結(jié)果：1.MTT比色法發(fā)現(xiàn)在同一照射劑量下,經(jīng)低細(xì)胞毒性濃度淫羊藿素(5.0μmol/L)作用24h后的CNE-2細(xì)胞,其細(xì)胞生長抑制率高于對照組(0.0μmol/L),差異具有統(tǒng)計學(xué)意義(P0.05)。 2.克隆形成實驗顯示：5.0μmol/L淫羊藿素組對CNE-2細(xì)胞具有放射增敏作用,其D0值、Dq值、N值及SF2值均較單純照射組降低,其增敏比SER值為(1.71+0.05) 3.流式細(xì)胞儀分析：低細(xì)胞毒性濃度的淫羊藿素作用CNE-2細(xì)胞24h后,G2/M期細(xì)胞明顯多于對照組,G0/G1期則減少,其差異均具有統(tǒng)計學(xué)意義(P0.05),而S期無明顯變化(P0.05) 結(jié)論：在一定的低細(xì)胞毒性濃度下,淫羊藿素對人鼻咽癌細(xì)胞CNE-2具有放射增敏作用,其機(jī)制可能與抑制CNE-2細(xì)胞亞致死性損傷修復(fù)和促進(jìn)細(xì)胞周期再分布有關(guān)。
[Abstract]:BACKGROUND: Nasopharyngeal carcinoma (NPC) is one of the most common head and neck malignancies growing in the nasopharynx. The pathogenesis of NPC is the result of a combination of multiple factors. The etiology of NPC may be related to Epstein-Barr virus infection, smoking, contaminated living environment, and eating salted food. Radiotherapy is the main method to treat nasopharyngeal carcinoma, but the radiation resistance of tumor and the damage of normal tissues caused by high dose radiation are the bottlenecks in the radiotherapy of nasopharyngeal carcinoma. Main research objectives.
Icaritin (ICT), as a derivative of icariin extracted from icariin, not only has antioxidant effect, prevention and treatment of osteoporosis, improvement of cardiovascular function, and protection of neurodegenerative damage, but also has certain effects on hematological malignancies, breast cancer, prostate cancer, endometrial cancer and liver cancer. In this study, Icariin was used to treat human nasopharyngeal carcinoma cell line CNE-2 to observe whether it can inhibit proliferation, induce apoptosis and radiosensitize CNE-2 cells. The possible mechanism of radiosensitizing effect of Icariin was preliminarily studied and revealed, providing experimental evidence for further clinical research.
Part one: Icaritin (ICT) inhibits proliferation and induces apoptosis in human nasopharyngeal carcinoma cell line CNE-2.
Objective: To observe the effect of Icariin on inhibiting proliferation and inducing apoptosis of human nasopharyngeal carcinoma cell line CNE-2.
Methods: CNE-2 cells were treated with different concentrations of icariin. The adherence and morphological changes of CNE-2 cells were observed under inverted microscope. The inhibition of cell growth was detected by MTT colorimetry. The apoptosis was detected by Annexin V-FITC/PI double staining flow cytometry.
Results: 1. Inverted microscope observation showed that the proliferation of CNE-2 cells slowed down with the increase of drug concentration and acting time. The morphology of CNE-2 cells gradually became smaller, rounded, floated, refractive index decreased, and intercellular junction loosened.
2. MTT colorimetric assay showed that the inhibitory rate of Icariin on CNE-2 cells increased with the increase of the concentration of icariin. The IC50 values (semi-inhibitory concentration) of Icariin on CNE-2 cells at 24, 48 and 72 hours were (93.63 + 1.60) micromol/L, (52.90 + 0.32) micromol/L, (32.62 + 1.1) micromol/L, respectively. 7) mu mol/L.
3. Flow cytometry analysis: The apoptotic rate of CNE-2 cells treated with different concentrations of icariin (0.5, 2.5, 10, 20, 40, and 40) increased from (4.59 (+ 1.49)%) to (30.73 (+ 4.06)%, (6.15 (+ 0.97)%) to (52.65 (+ 2.07)) after 24 and 48 hours, respectively. The difference was statistically significant (P 0.0). 5).
CONCLUSION: In vitro, icariin at a certain concentration can inhibit proliferation and induce apoptosis of human nasopharyngeal carcinoma cell line CNE-2 in a time-concentration dependent manner.
Part II Radiosensitization of Icaritin (ICT) on Human Nasopharyngeal Carcinoma Cell Line CNE-2 and Its Mechanism
Objective: To investigate the radiosensitizing effect of Icariin on human nasopharyngeal carcinoma cell line CNE-2 and its possible mechanism.
METHODS: CNE-2 cells were treated with low cytotoxic concentration of icariin at different irradiation doses. MTT colorimetry was used to determine the radiosensitivity of icariin to CNE-2 cells. The radiosensitization effect of Icariin on CNE-2 cells was observed by clone formation assay. Change in time.
Results: 1. MTT colorimetric assay showed that the growth inhibition rate of CNE-2 cells treated with low cytotoxic concentration of icariin (5.0 micromol/L) for 24 hours was higher than that of the control group (0.0 micromol/L), and the difference was statistically significant (P 0.05).
2. Clone formation assay showed that 5.0 micromol/L icariin could enhance the radiosensitivity of CNE-2 cells. The D0 value, Dq value, N value and SF2 value were lower in the 5.0 micromol/L icariin group than those in the simple irradiation group, and the SER value was (1.71+0.05).
3. Flow cytometry analysis: After 24 hours of low cytotoxic concentration of Icariin on CNE-2 cells, G2/M phase cells significantly more than the control group, G0/G1 phase decreased, the difference was statistically significant (P 0.05), but S phase did not change significantly (P 0.05).
CONCLUSION: Icariin has radiosensitization effect on human nasopharyngeal carcinoma cell line CNE-2 at a certain low cytotoxic concentration. The mechanism may be related to inhibiting the repair of sublethal damage and promoting cell cycle redistribution of CNE-2 cells.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R739.63

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本文編號：2245784