本文選題：肝細胞癌 + 索拉菲尼��； 參考：《安徽醫(yī)科大學》2017年碩士論文

【摘要】：研究背景原發(fā)性肝癌是消化道常見腫瘤之一,全球第六位,每年新發(fā)約75萬人次,在腫瘤所致的死因中排在第二位,治療效果欠佳。近年來分子靶向藥物的發(fā)展非常迅速。索拉非尼是一種多靶點抗腫瘤藥,已被FDA批準用于晚期肝癌的治療,是目前治療肝癌相對有效的一種酪氨酸及被抑制劑。但是,患者對索拉菲尼的有效率僅有大約30%,并且通常在使6個月內(nèi)發(fā)生耐藥。因此,尋找提高索拉非尼治療效果的方法是目前研究的熱點。褪黑素是由松果體分泌的一種吲哚類生物活性物質(zhì),有晝夜節(jié)律的改變,生物學功能包括生理調(diào)節(jié)、抗氧化、抗炎、免疫調(diào)節(jié)、抑制腫瘤、增強傳統(tǒng)化療藥物療效等作用。而且,有臨床研究表明褪黑素可以增強化療效果并減少副作用,并提高腫瘤患者的生存時間和生活質(zhì)量。已有索拉非尼對肺、前列腺、惡性黑色素瘤及乳腺癌的研究,國外文獻也有索拉非尼聯(lián)合化療對腎、結(jié)腸腫瘤治療的報道,這些都說明褪黑素可以作為潛在的聯(lián)合使用的抗腫瘤藥物。我們在前期研究中發(fā)現(xiàn),褪黑素可增強阿霉素的抗腫瘤作用。但是褪黑素對靶向藥物的增效作用報道較少,褪黑素聯(lián)合索拉非尼對肝癌細胞的影響暫未見報道。自噬是回收不必要或功能障礙的細胞組分(比如蛋白質(zhì)或者是細胞器)并加以降解可用以維持細胞代謝的穩(wěn)定。但是自噬具有雙刃劍的作用,過度的自噬可能使細胞產(chǎn)生自噬性死亡而不是保護作用。索拉菲尼可以調(diào)節(jié)自噬活性,可能對細胞起保護作用,這就可能解釋了臨床上索拉菲尼治療效果欠佳。自噬抑制劑氯喹(CQ)可以增強藥物的抗腫瘤作用或逆轉(zhuǎn)耐藥。因此索拉菲尼聯(lián)合褪黑素對自噬的影響值得研究。目的本實驗通過觀察索拉菲尼、褪黑素單獨用藥、聯(lián)合用藥對人肝癌細胞增殖的影響,來證實二者聯(lián)合用藥在抗腫瘤治療方面是否更具有優(yōu)勢。為進一步臨床實驗及治療提供基礎理論依據(jù)。方法(1)5個濃度的索拉菲尼作用于肝癌細胞24,48,72小時后,用CCK-8法檢測抑制率,并計算索拉菲尼的IC50.(2)肝癌細胞與兩種藥物或分別與一種藥物共培養(yǎng)48小時后,同樣用CCK-8法檢測細胞抑制率。并用藥物相互作用指數(shù)(CDI)評價聯(lián)合抗腫瘤作用。并用流式細胞術(shù)檢測兩種藥物單獨或聯(lián)合作用于肝癌細胞株后的凋亡比率。并選取兩藥協(xié)同效應明顯且能在人體達到的濃度進行后續(xù)實驗。(3)采用篩選的兩藥物濃度,兩種藥物單獨或聯(lián)合作用于肝癌細胞后使用Western-Blot檢測Bcl-2、Bax、LC3、P62蛋白的表達情況。(4)使用自噬抑制劑CQ(選取能明顯升高自噬水平并且不影響細胞生長的合適濃度)加入索拉菲尼單藥組以及聯(lián)合用藥組,觀察加入CQ后細胞凋亡情況以自噬蛋白表達的情況。(5)加入自噬抑制劑CQ后用CCK-8法檢測索拉菲尼或聯(lián)合用藥對腫瘤細胞的影響,并用流式細胞術(shù)檢測凋亡。結(jié)果(1)結(jié)果顯示索拉菲尼在HepG2和Bel-7402細胞株上表現(xiàn)為時間和劑量依賴的抗腫瘤效應。并且在兩個細胞株上的IC50分別是13.21μmol/L和11.83μmol/L。(2)聯(lián)合用藥對HepG2和Bel-7402細胞的具有協(xié)同抑制作用,索拉菲尼(10μmol/L)與褪黑素(10-5mol/L)聯(lián)合用藥作用于HepG2和Bel7402細胞時,聯(lián)合作用指數(shù)分別是0.827±0.09和0.91±0.05。(3)Annexin V/FITC-PI染色后,流式細胞術(shù)檢測聯(lián)合組的HepG2細胞的凋亡率明顯升高,褪黑素單藥組為6.79%±2.34%,索拉菲尼單藥組為36.8%±1.51%,聯(lián)合組為52.5%±12.56%,同樣的結(jié)果在Bel-7402細胞株上也可觀察到。(4)聯(lián)合組與兩種藥單藥組相比,Bcl-2蛋白水平明顯下降而Bax蛋白的水平升高,并且聯(lián)合組的Bcl-2/Bax比值與對照組以及索拉菲尼或褪黑素單藥明顯降低。(5)Western-Blot方法檢測自噬蛋白LC3和P62表達情況,索拉菲尼單藥組的自噬水平明顯升高,但是在聯(lián)合用藥組,索拉菲尼引起升高的自噬水平被褪黑素降低,并伴有P62的相應變化。(5)加入自噬抑制劑氯喹(CQ)抑制后,索拉菲尼或聯(lián)合組的自噬水平下降,并且細胞抑制率升高。用流式細胞術(shù)同樣發(fā)現(xiàn),加入氯喹后的索拉菲尼和聯(lián)合組兩組的細胞凋亡率也升高。結(jié)論(1)褪黑素聯(lián)合索拉菲尼具有協(xié)同抗腫瘤作用。(2)褪黑素聯(lián)合索拉菲尼是通過抑制自噬發(fā)揮協(xié)同抗腫瘤作用。(3)索拉菲尼單藥組或聯(lián)合組在加入自噬抑制劑CQ后細胞受到進一步抑制,并且細胞凋亡率比不加CQ前有明顯升高。
[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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.7

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