褪黑素通过下调自噬增强索拉菲尼的抗肝癌作用

发布时间：2018-06-16 20:49

本文选题：肝细胞癌 + 索拉菲尼　；参考：《安徽医科大学》2017年硕士论文

【摘要】：研究背景原发性肝癌是消化道常见肿瘤之一,全球第六位,每年新发约75万人次,在肿瘤所致的死因中排在第二位,治疗效果欠佳。近年来分子靶向药物的发展非常迅速。索拉非尼是一种多靶点抗肿瘤药,已被FDA批准用于晚期肝癌的治疗,是目前治疗肝癌相对有效的一种酪氨酸及被抑制剂。但是,患者对索拉菲尼的有效率仅有大约30%,并且通常在使6个月内发生耐药。因此,寻找提高索拉非尼治疗效果的方法是目前研究的热点。褪黑素是由松果体分泌的一种吲哚类生物活性物质,有昼夜节律的改变,生物学功能包括生理调节、抗氧化、抗炎、免疫调节、抑制肿瘤、增强传统化疗药物疗效等作用。而且,有临床研究表明褪黑素可以增强化疗效果并减少副作用,并提高肿瘤患者的生存时间和生活质量。已有索拉非尼对肺、前列腺、恶性黑色素瘤及乳腺癌的研究,国外文献也有索拉非尼联合化疗对肾、结肠肿瘤治疗的报道,这些都说明褪黑素可以作为潜在的联合使用的抗肿瘤药物。我们在前期研究中发现,褪黑素可增强阿霉素的抗肿瘤作用。但是褪黑素对靶向药物的增效作用报道较少,褪黑素联合索拉非尼对肝癌细胞的影响暂未见报道。自噬是回收不必要或功能障碍的细胞组分(比如蛋白质或者是细胞器)并加以降解可用以维持细胞代谢的稳定。但是自噬具有双刃剑的作用,过度的自噬可能使细胞产生自噬性死亡而不是保护作用。索拉菲尼可以调节自噬活性,可能对细胞起保护作用,这就可能解释了临床上索拉菲尼治疗效果欠佳。自噬抑制剂氯喹(CQ)可以增强药物的抗肿瘤作用或逆转耐药。因此索拉菲尼联合褪黑素对自噬的影响值得研究。目的本实验通过观察索拉菲尼、褪黑素单独用药、联合用药对人肝癌细胞增殖的影响,来证实二者联合用药在抗肿瘤治疗方面是否更具有优势。为进一步临床实验及治疗提供基础理论依据。方法(1)5个浓度的索拉菲尼作用于肝癌细胞24,48,72小时后,用CCK-8法检测抑制率,并计算索拉菲尼的IC50.(2)肝癌细胞与两种药物或分别与一种药物共培养48小时后,同样用CCK-8法检测细胞抑制率。并用药物相互作用指数(CDI)评价联合抗肿瘤作用。并用流式细胞术检测两种药物单独或联合作用于肝癌细胞株后的凋亡比率。并选取两药协同效应明显且能在人体达到的浓度进行后续实验。(3)采用筛选的两药物浓度,两种药物单独或联合作用于肝癌细胞后使用Western-Blot检测Bcl-2、Bax、LC3、P62蛋白的表达情况。(4)使用自噬抑制剂CQ(选取能明显升高自噬水平并且不影响细胞生长的合适浓度)加入索拉菲尼单药组以及联合用药组,观察加入CQ后细胞凋亡情况以自噬蛋白表达的情况。(5)加入自噬抑制剂CQ后用CCK-8法检测索拉菲尼或联合用药对肿瘤细胞的影响,并用流式细胞术检测凋亡。结果(1)结果显示索拉菲尼在HepG2和Bel-7402细胞株上表现为时间和剂量依赖的抗肿瘤效应。并且在两个细胞株上的IC50分别是13.21μmol/L和11.83μmol/L。(2)联合用药对HepG2和Bel-7402细胞的具有协同抑制作用,索拉菲尼(10μmol/L)与褪黑素(10-5mol/L)联合用药作用于HepG2和Bel7402细胞时,联合作用指数分别是0.827±0.09和0.91±0.05。(3)Annexin V/FITC-PI染色后,流式细胞术检测联合组的HepG2细胞的凋亡率明显升高,褪黑素单药组为6.79%±2.34%,索拉菲尼单药组为36.8%±1.51%,联合组为52.5%±12.56%,同样的结果在Bel-7402细胞株上也可观察到。(4)联合组与两种药单药组相比,Bcl-2蛋白水平明显下降而Bax蛋白的水平升高,并且联合组的Bcl-2/Bax比值与对照组以及索拉菲尼或褪黑素单药明显降低。(5)Western-Blot方法检测自噬蛋白LC3和P62表达情况,索拉菲尼单药组的自噬水平明显升高,但是在联合用药组,索拉菲尼引起升高的自噬水平被褪黑素降低,并伴有P62的相应变化。(5)加入自噬抑制剂氯喹(CQ)抑制后,索拉菲尼或联合组的自噬水平下降,并且细胞抑制率升高。用流式细胞术同样发现,加入氯喹后的索拉菲尼和联合组两组的细胞凋亡率也升高。结论(1)褪黑素联合索拉菲尼具有协同抗肿瘤作用。(2)褪黑素联合索拉菲尼是通过抑制自噬发挥协同抗肿瘤作用。(3)索拉菲尼单药组或联合组在加入自噬抑制剂CQ后细胞受到进一步抑制,并且细胞凋亡率比不加CQ前有明显升高。
[Abstract]:Background primary hepatocellular carcinoma (HCC) is one of the common digestive tract tumors, sixth in the world, about 750 thousand new times a year and second in the cause of death. The treatment effect is not good. In recent years, the development of molecular targeting drugs is very rapid. Sorafeni is a multi target antitumor drug, which has been approved by FDA for the treatment of advanced liver cancer. It is a relatively effective tyrosine and inhibitor for the treatment of liver cancer. However, the patient's effective rate to Sola Feeney is only about 30% and usually occurs within 6 months. Therefore, finding a way to improve the effect of Sola Fini is a hot spot. Melatonin is a kind of indole biological activity secreted by the pineal body. Sexual substances, with changes in circadian rhythms, biological functions including physiological regulation, antioxidant, anti-inflammatory, immunoregulation, tumor suppression, and enhancement of the efficacy of traditional chemotherapeutic drugs. Moreover, clinical studies have shown that melatonin can enhance chemotherapeutic effects and reduce side effects and improve the survival and quality of life of cancer patients. The study of lung, prostate, malignant melanoma and breast cancer, and foreign literature also reports on the treatment of kidney and colon tumors by sorafeni combined with chemotherapy. These indicate that melatonin can be used as a potential combined antitumor drug. In our previous study, we found that melatonin could enhance the antitumor effect of adriamycin. The synergistic effect of melanin on targeted drugs is less reported. The effects of melatonin combined with Sola Fini on hepatoma cells have not been reported. Autophagy is a cell component (such as protein or organelle) that reclaims unnecessary or dysfunction, and can be degraded to maintain the stability of cell metabolism. However, autophagy has a double-edged sword. Autophagic autophagy may cause autophagic death instead of protective action. Sola Feeney can regulate autophagy activity and may protect the cell, which may explain the poor clinical Sola Feeney treatment effect. The autophagic inhibitor CQ can enhance the antitumor effect of the drug or reverse the drug resistance. So Sola Feeney Lian The effect of melatonin on autophagy is worth studying. The purpose of this experiment is to verify whether the combination of Sola Feeney, melatonin, and combined use of drugs on the proliferation of human hepatoma cells, to confirm whether the combination of the two drugs is more advantageous in the antitumor treatment. It provides a basic theoretical basis for further clinical trials and treatment. Method (1) 5 concentration. The inhibition rate was detected by CCK-8 method after 24,48,72 hours of Sola Feeney in the liver cancer cells. The inhibitory rate of Sola Feeney's IC50. (2) hepatoma cells with two drugs or a drug was co cultured with a drug for 48 hours, and the inhibition rate of the cells was evaluated by the drug interaction index (CDI). Cytometry was used to detect the apoptosis ratio of two drugs alone or in combination with liver cancer cell lines. The synergistic effect of two drugs was selected and the concentration reached in the human body was observed. (3) the concentration of two drugs was selected and the two drugs were used separately or combined to detect Bcl-2, Bax, LC3, P62 eggs after hepatoma cells. (4) the use of autophagy inhibitor CQ (selecting the appropriate concentration of autophagy and not affecting cell growth) added to the Sola Feeney single drug group and the combination group, and observed the expression of autophagic protein in cell apoptosis after the addition of CQ. (5) after adding the autophagic inhibitor CQ, the CCK-8 method was used to detect the expression of autophagy. The effects of combined drugs on tumor cells and apoptosis were detected by flow cytometry. Results (1) Sola Feeney showed a time and dose dependent anti-tumor effect on HepG2 and Bel-7402 cells, and the IC50 on two cell lines was 13.21 mu mol/L and 11.83 mu mol/L. (2) for HepG2 and Bel-7402 cells. The combined action index of Sola Feeney (10 mu mol/L) and melatonin (10-5mol/L) in HepG2 and Bel7402 cells was 0.827 + 0.09 and 0.91 + 0.05. (3) Annexin V/FITC-PI, respectively. The apoptosis rate of HepG2 cells in the combined group was significantly higher than that of the combined group of melatonin, 6.79% + 2.3 in the melatonin single drug group. 4%, the Sola Feeney single drug group was 36.8% + 1.51% and the combined group was 52.5% + 12.56%. The same results were also observed on the Bel-7402 cell line. (4) the level of Bcl-2 protein was significantly decreased and the level of Bax protein in the combination group was significantly lower than that of the two single drug groups, and the Bcl-2/ Bax ratio in the combined group and the control group and the monotherapy of melatonin or melatonin. Significantly lower. (5) the Western-Blot method detected the expression of autophagic protein LC3 and P62, the autophagy level of the Sola Feeney single drug group increased significantly, but in the combined drug group, the elevated autophagy level was reduced by Sola Feeney and associated with the corresponding changes in P62. (5) after the inhibition of the autophagic inhibitor chloroquine (CQ), Sola Feeney or union The level of autophagy decreased and the rate of cell inhibition increased. The apoptotic rate of Sola Feeney and two groups after chloroquine was also increased by flow cytometry. Conclusion (1) melatonin combined with Sola Feeney has synergistic antitumor effect. (2) melatonin and Sola Feeney combined with inhibition of autophagy to play a synergistic antitumor effect. (3) in the Sola Feeney monotherapy group or the combined group, the cells were further inhibited after the addition of autophagy inhibitor CQ, and the rate of apoptosis was significantly higher than that before CQ.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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