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淫羊藿素对人鼻咽癌细胞CNE-2抑制增殖、诱导凋亡及放射增敏作用的实验研究

发布时间:2018-09-17 11:40  文章来源:笔耕文化传播
【摘要】:研究背景:鼻咽癌(Nasopharyngeal carcinoma, NPC)是生长于鼻咽部位的最为常见的头颈部恶性肿瘤之一。NPC的发病是一个多因素共同作用的结果,其病因可能与EB病毒感染、吸烟、居住环境受污染、进食腌制食品有关。由于鼻咽周围的解剖结构相当复杂,因此不适合行手术治疗。治疗鼻咽癌的主要方法是放射治疗,但肿瘤的辐射抗性和大剂量照射对正常组织的损伤是鼻咽癌放射治疗中难以突破的瓶颈。因此,寻找新型抗肿瘤药物及高效低毒的肿瘤放射增敏剂是目前NPC治疗的主要研究目标。 淫羊藿素(Icaritin, ICT)作为中药淫羊藿提取物淫羊藿苷的衍生物,不仅具有抗氧化、防治骨质疏松、改善心血管功能、保护神经变性损伤等作用,近几年的研究表明,其对血液系统恶性肿瘤、乳腺癌、前列腺癌、子宫内膜癌及肝癌还具有一定的抗肿瘤作用。本研究用淫羊藿素作用于人鼻咽癌细胞CNE-2,观察其对CNE-2细胞是否具有抑制增殖、诱导凋亡及放射增敏的作用,并对淫羊藿素放射增敏作用的可能机制进行初步的研究与揭示,为进一步的临床研究提供实验证据。 第一部分淫羊藿素(Icaritin, ICT)对人鼻咽癌细胞CNE-2抑制增殖和诱导凋亡效应 目的:观察淫羊藿素对人鼻咽癌细胞CNE-2抑制增殖和诱导凋亡的效应。 方法:以不同浓度淫羊藿素处理CNE-2细胞,倒置显微镜下观察细胞贴壁情况和形态变化,四甲基偶氮唑蓝(MTT)比色法检测细胞生长抑制情况,Annexin V-FITC/PI双染流式细胞术检测细胞凋亡情况。 结果:1.倒置显微镜观察发现,随着药物浓度及作用时间的增加,经淫羊藿素处理后的CNE-2细胞增殖减慢,形态逐渐变小、变圆、浮起,折光度降低,细胞间连接松散。 2.MTT比色法检测不同浓度淫羊藿素作用于CNE-2细胞,发现随着药物浓度的增加、.作用时间的延长,其对CNE-2细胞的抑制率逐渐升高。CNE-2细胞经淫羊藿素作用24h、48h、72h时的IC50值(半抑制浓度)分别为(93.63±1.60)μmol/L、(52.90±0.32)μmol/L、(32.62±1.17) μmol/L。 3.流式细胞仪分析:不同浓度淫羊藿素0μmol/L、2.5μmol/L5μmol/L、10μmol/L、20μmol/L、40μmol/L)作用CNE-2细胞24、48h后,其细胞凋亡率分别由(4.59±1.49)%增至(30.73±4.06)%,(6.15±0.97)%增至(52.65±2.07)%,经统计学分析,其与对照组的差异具有统计学意义(P0.05)。 结论:体外实验证明,一定浓度的淫羊藿素对人鼻咽癌细胞CNE-2有抑制增殖和诱导凋亡作用,并呈时间浓度依赖性。 第二部分淫羊藿素(Icaritin, ICT)对人鼻咽癌细胞CNE-2的放射增敏作用及机制研究 目的:探讨淫羊藿素对人鼻咽癌细胞CNE-2的放射增敏作用及可能机制。 方法:在不同的照射剂量下,以低细胞毒性浓度的淫羊藿素处理CNE-2细胞,MTT比色法初步判断淫羊藿素对CNE-2细胞的放射敏感性,克隆形成实验观察其对CNE-2细胞的放射增敏作用。流式细胞术分析低浓度淫羊藿素作用前后CNE-2细胞的周期变化。 结果:1.MTT比色法发现在同一照射剂量下,经低细胞毒性浓度淫羊藿素(5.0μmol/L)作用24h后的CNE-2细胞,其细胞生长抑制率高于对照组(0.0μmol/L),差异具有统计学意义(P0.05)。 2.克隆形成实验显示:5.0μmol/L淫羊藿素组对CNE-2细胞具有放射增敏作用,其D0值、Dq值、N值及SF2值均较单纯照射组降低,其增敏比SER值为(1.71+0.05) 3.流式细胞仪分析:低细胞毒性浓度的淫羊藿素作用CNE-2细胞24h后,G2/M期细胞明显多于对照组,G0/G1期则减少,其差异均具有统计学意义(P0.05),而S期无明显变化(P0.05) 结论:在一定的低细胞毒性浓度下,淫羊藿素对人鼻咽癌细胞CNE-2具有放射增敏作用,其机制可能与抑制CNE-2细胞亚致死性损伤修复和促进细胞周期再分布有关。
[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.
【学位授予单位】:中南大学
【学位级别】:博士
【学位授予年份】:2012
【分类号】:R739.63

【参考文献】

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1 杨光伟;毛志达;;乏氧细胞放射增敏剂[J];癌症;1997年01期

2 郭颖;鼻咽癌遗传学的研究进展[J];癌症;1998年04期

3 王雯s,

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