淫羊藿素联合顺铂抗非小细胞肺癌作用机制研究

发布时间：2018-06-29 02:39

本文选题：淫羊藿素 + 顺铂　；参考：《昆明医科大学》2016年硕士论文

【摘要】：背景：淫羊藿素(icaritin,ICT)作为一种中药成分,研究发现淫羊藿素对多种实体和非实体肿瘤具有一定抗肿瘤作用,同时也具有保护心血管系统、促进骨骼生长发育、提高免疫力。而在非小细胞肺癌化疗中,以铂类为基础的化疗,其毒副作用大,且存在部分患者对铂类化疗不敏感。目的：本研究通过从细胞水平,研究淫羊藿素是否能增强顺铂抗非小细胞肺癌抑制作用,并研究其可能的作用机制,同时研究淫羊藿素和顺铂对正常支气管上皮细胞的毒性作用。方法：用不同浓度的淫羊藿素、顺铂及淫羊藿素联合顺铂处理A549细胞不同时间后观察A549细胞形态学变化；MTT法测定不同浓度的淫羊藿素、顺铂及淫羊藿素联合顺铂处理A549细胞、支气管上皮细胞BEAS-2B细胞株不同时间后细胞增殖抑制影响,计算抑制率和IC50值；细胞克隆实验检测不同浓度的淫羊藿素、顺铂及淫羊藿素联合顺铂处理A549细胞处理A549细胞24h后对细胞克隆形成的影响；流式细胞仪检测不同浓度的淫羊藿素、顺铂及淫羊藿素联合顺铂处理A549细胞处理A549细胞24h后对细胞细胞周期及凋亡的影响；Western blotting实验检测不同浓度的淫羊藿素、顺铂及淫羊藿素联合顺铂处理A549细胞24h后对细胞中AKT、p-AKT、Bcl-2、Bim、Caspase-9蛋白质表达水平的影响。结果：(1)淫羊藿素、顺铂及淫羊藿素联合顺铂能使A549细胞的细胞形态发生明显改变,且随作用时间延长细胞形态变化更加明显；联合组的细胞形态变化得更加明显,。(2)淫羊藿素可抑制A549细胞增殖,其抑制作用呈时间-剂量依赖性。(3)顺铂可抑制A549细胞增殖,其抑制作用呈时间-剂量依赖性。(4)淫羊藿素联合顺铂对A549细胞增殖抑制作用明显高于单药组。各联合指数CI均小于0.9,淫羊藿素可协同增强顺铂抑制A549细胞增殖作用。(5)淫羊藿素组对BEAS-2B细胞的活性抑制较低,细胞毒性较少；顺铂组BEAS-2B细胞活性抑制相对较大,细胞毒性较大；淫羊藿素联合顺铂后并不会大幅度增加顺铂对BEAS-2B细胞的细胞毒性。(6)淫羊藿素、顺铂单药可抑制A549细胞克隆形成,联合组与单药组比较,联合组明显抑制A549细胞克隆形成。(7)淫羊藿素诱导A549细胞周期阻滞于G2/M；顺铂诱导A549细胞周期阻滞于G0/G1期和S期；淫羊藿素联合顺铂诱导A549细胞周期阻滞于S期。(8)淫羊藿素、顺铂及淫羊藿素联合顺铂可明显诱导A549细胞发生凋亡,且联合组与单药组比较,联合组凋亡率明显高于单药组。(9)Western blotting实验结果显示：联合组与单药组比较,联合组能够显著下调AKT、p-AKT的表达水平、抗凋亡蛋白Bcl-2的表达水平,并上调凋亡蛋白Bim, Caspase-9蛋白的表达水平。结论：淫羊藿素可通过细胞毒性作用、抑制非小细胞肺癌细胞增殖,诱导细胞凋亡,与顺铂联合后对非小细胞肺癌细胞增殖具有协同抑制作用,其作用机制可能与PI3K/AKT信号通路介导的细胞凋亡途径相关。淫羊藿素联合顺铂应用治疗非小细胞肺癌,并不会大幅度增加顺铂的支气管上皮细胞的细胞毒性,提示淫羊藿素联合顺铂治疗非小细胞肺癌具有一定可行性和临床应用价值。
[Abstract]:Background: icaritin (ICT) is a traditional Chinese medicine component. It has been found that icariin has a certain antitumor effect on various solid and non solid tumors, and it also protects the cardiovascular system, promotes the growth and development of bone and enhances immunity. In the chemotherapy of non-small cell lung cancer, platinum based chemotherapy, its toxic and side effects Objective: To investigate whether icariin can enhance the inhibitory effect of cisplatin against non-small cell lung cancer and to study the possible mechanism of action of cisplatin and study the toxic effect of icariin and cisplatin on normal tracheal epithelial cells. The concentration of epimedium, cisplatin and cisplatin combined with cisplatin treated A549 cells at different time to observe the morphological changes of A549 cells; MTT assay was used to determine the different concentrations of icariin, cisplatin and icariin combined with cisplatin to treat A549 cells, and the inhibitory effects of cell proliferation on the BEAS-2B cell line of the bronchial epithelial cells after different time, and the inhibition of the inhibition of cell proliferation. The effects of different concentrations of icariin, cisplatin and epimedium combined with cisplatin on the treatment of A549 cell 24h were detected by cisplatin and cisplatin in A549 cells. The flow cytometry was used to detect different concentrations of icariin, cisplatin and epimedium combined with cis platinum to treat A549 cell 24h after A549 cells were treated with A549 cells. Effect of cell cycle and apoptosis; Western blotting test was used to detect the effects of different concentrations of icariin, cisplatin and icariin combined with cisplatin on the expression of AKT, p-AKT, Bcl-2, Bim, and Caspase-9 protein in A549 cells. Results: (1) icariin, cisplatin and cisplatin combined with cisplatin can make the A549 cells thin. Cell morphogenesis was obviously changed and the morphological changes of cells were more obvious with the time of action; the cell morphology of the combined group changed more obviously. (2) Epimedium could inhibit the proliferation of A549 cells, and its inhibition was time dose dependent. (3) cisplatin inhibited the proliferation of A549 cells, and its inhibitory effect was time dose dependent. (4) prostitution sheep The inhibitory effect of Epimedium combined with cisplatin on the proliferation of A549 cells was significantly higher than that of the single drug group. The combined index CI was less than 0.9. Icariin could synergistically enhance cisplatin to inhibit the proliferation of A549 cells. (5) the inhibition of the activity of Icariin on the activity of BEAS-2B cells was lower and the cytotoxicity of cisplatin was less. The inhibition of the activity of BEAS-2B cells in cisplatin group was relatively large, and the cytotoxicity of cisplatin was relatively large. The combination of icariin and cisplatin did not significantly increase the cytotoxicity of cisplatin to BEAS-2B cells. (6) icariin and cisplatin could inhibit the formation of A549 cell clones. Compared with the single drug group, the combined group significantly inhibited the formation of A549 cell clones. (7) Herba Epimedium induced the A549 cell cycle in G2/M; cisplatin induced A549. The cell cycle was blocked in G0/G1 phase and S phase; icariin combined with cisplatin induced A549 cell cycle arrest in S phase. (8) icariin, cisplatin and cisplatin combined cisplatin could induce apoptosis in A549 cells, and the combined group was significantly higher than the single drug group in the single drug group. (9) the results of Western blotting test showed: Compared with the single drug group, the combined group could significantly reduce the expression level of AKT, p-AKT, the expression of anti apoptotic protein Bcl-2, and up regulate the expression level of apoptotic protein Bim and Caspase-9 protein. Conclusion: Epimedium can inhibit the proliferation of non small cell lung cancer cells and induce apoptosis through the cytotoxic effect of icepin, and combined with cisplatin. Non small cell lung cancer cell proliferation has a synergistic inhibitory effect, and its mechanism may be related to the apoptosis pathway mediated by PI3K/AKT signaling pathway. Icariin combined with cisplatin in the treatment of non small cell lung cancer does not significantly increase the cytotoxicity of cisplatin's bronchial epithelial cells, suggesting that icariin combined with cisplatin for the treatment of non - small cell lung cancer. Small cell lung cancer has certain feasibility and clinical application value.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R734.2

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