纳米层状双氢氧化物共负载体系的制备及其抑制肝癌氟尿嘧啶耐药的协同效应机制研究

发布时间：2018-05-13 06:17

本文选题：层状双氢氧化物 + 五氟尿嘧啶　；参考：《第二军医大学》2016年硕士论文

【摘要】：原发性肝细胞癌(Hepatocellular Carcinoma,HCC)位于肿瘤发病率的第五位,其死亡率在恶性肿瘤中居第三。目前,手术治疗是中早期HCC患者的最佳治疗措施。然而,HCC起病较为隐匿,确诊时多属晚期,已丧失手术切除机会。同时,有临床数据显示,对于部分早期手术切除的患者,术后复发率也居高不下。因此,临床上化学药物治疗及介入治疗对原发性肝癌病人治疗具有重要意义。然而,HCC病人大多数存在不同程度肝硬化,其肝脏解毒功能较差,化疗药物如五氟尿嘧啶(5-Fluorouraci,Fu)等直接暴露于组织,引起较大的毒副作用,同时随着经典化疗药物的广泛开展,肿瘤多药耐药(Multidrug Resistance,MDR)已经成为肿瘤病人化学药物治疗的巨大障碍。文献调研发现,多种机制参与肿瘤的多耐药,如细胞膜的糖蛋白机制,肿瘤的抗凋亡基因上调以及自噬等。因此,如何解决肿瘤耐药已经成为肿瘤病人化疗成功与否的关键因素。常见的措施包括增大药物的剂量从而达到药物有效浓度,但却带来了更大的毒副作用风险。近年来,化疗药物的联合疗法为肿瘤耐药病人带来希望,可以通过发挥不同药物的功能协同杀伤肿瘤,但多种化疗药的同时作用存在诸多不确定因素,联合药物本身也可能带来更大的毒副作用。姜黄素(Curcumin,Cur)是食物来源的传统型药物,具有廉价、无毒等诸多优点。研究表明,姜黄素具有抗炎、抗氧化、抗肿瘤等多重作用,文献报道显示,化疗药物与姜黄素联合能够有效逆转肿瘤多耐药。然而,姜黄素具有水溶性差,体内代谢快等不足,如何充分发挥姜黄素应有的强大功能是实现其抑制耐药效果的关键。近年来纳米技术的发展为肿瘤药物剂型提供新方向,纳米技术具有以下优点:纳米载体的pH敏感性、温度敏感性可以实现药物的控释,从而提高药物利用度和有效降低药物因直接暴露带来的毒副作用;纳米载体的温敏功能及不同配体的修饰可以实现被动及主动靶向;纳米技术的鸡尾酒疗法不仅仅局限于两种药物混合使用,可以实现化疗药物的时序性、协同性作用。层状双氢氧化物(Layered hydroxide,LDH)是一类阴离子型黏土,它具有双层板状结构,LDH板层外带正电荷,且富含大量氢氧根,便于形成氢键而载药。板间带负电荷,可用于阴离子物质交换,可负载电离为阴离子的药物以及小分子的核酸。LDH具有无毒、高载药量和pH敏感性等优点而引起广泛关注,是不同肿瘤药物的良好载体。本课题中,我们利用LDH层间负载Fu以及Cur构建纳米混合剂型,通过Bel 7402/Fu肝癌耐药细胞为模型,研究纳米LDH剂型对于氟尿嘧啶耐药的肝癌肿瘤作用。细胞水平首先考察了 LDH-Fu-Cur对于正常肝脏细胞LO_2及Bel 7402/Fu的增殖抑制作用。采用流式凋亡手段检测LDH-Fu-Cur的促凋亡作用,并采用Western blot技术检测凋亡相关机制。通过共聚焦检测耐药细胞对于纳米药物的吞噬作用以及纳米药物引起的自噬,利用自噬工具药检测自噬在LDH-Fu-Cur抗肿瘤中的效应。最后采取小鼠抑瘤实验检测LDH-Fu-Cur在体内的抑瘤效果。系统研究显示:我们成功制备具有高载药量且生物安全性良好的LDH共载药体系,紫外分光光度计检测纳米体系具有较高的载药量,LDH-Fu中Fu载药量为20.3%±0.4%,LDH-Fu-Cur 中 Fu 载药量为 19.7%±0.2%,Cur 的载药量 15.7%±0.6%,符合实验预期结果,能够满足实验要求。实验还提示LDH-Fu-Cur对于正常肝脏LO_2细胞没有明显毒副作用,而对于Bel 7402/Fu肿瘤细胞具有明显的抑制作用。流式细胞技术及Western blot进一实验证实LDH-Fu-Cur能够较单纯姜黄素组具有明显的促Bel 7402/Fu细胞凋亡效应。有意思的是:本课题研究发现自噬协同参与了在体系对肝癌细胞的抑制作用。虽然细胞自噬是一把"双刃剑",我们的实验还证实LDH-Fu-Cur能够启动细胞自噬,而这种自噬是一种促进细胞死亡的协同杀伤性自噬。最后,通过构建肝癌Bel 7402/Fu裸鼠异种瘤移植模型,观察LDH-Fu-Cur组、其他药物组以及PBS对照组发现,LDH-Fu-Cur能够明显抑制Bel 7402/Fu细胞在裸鼠体内生长,提示LDH-Fu-Cur可作为抑制氟尿嘧啶耐药肝癌细胞的有效药物。综上所述,通过本课题开展,我们制备了结构良好、生物安全的LDH-Fu-Cur纳米药物,实现氟尿嘧啶和姜黄素共载,新的纳米药物在体内、体外均展现处良好抑制肝癌氟尿嘧啶耐药细胞,为肝癌抗肿瘤耐药药物的研发提供新思路。
[Abstract]:Hepatocellular Carcinoma (HCC) is located in fifth of the incidence of tumor, and its mortality rate is third in the malignant tumor. At present, surgical treatment is the best treatment for the early middle HCC patients. However, the onset of HCC is more concealed, and the diagnosis is late and has lost the chance of surgical resection. The rate of postoperative recurrence is also high in some patients with early surgical resection. Therefore, the clinical chemotherapeutic and interventional therapy is of great significance for the treatment of primary liver cancer patients. However, most of HCC patients have different degrees of liver cirrhosis, their liver detoxification function is poor, chemotherapy drugs such as five fluorouracil (5-Fluorouraci, Fu), and so on. Direct exposure to tissue causes large toxic and side effects, and with the extensive development of classical chemotherapeutic drugs, Multidrug Resistance (MDR) has become a huge obstacle to the treatment of chemical drugs in cancer patients. Therefore, how to solve the drug resistance has become a key factor in the success of chemotherapy in cancer patients. The common measures include increasing the dose of the drug to achieve the effective concentration of the drug, but it brings a greater risk of toxic and side effects. In recent years, the combined therapy of chemotherapeutic drugs has been used as a drug resistant patient. It brings hope that the functions of different drugs can be combined to kill the tumor, but there are many uncertainties in the simultaneous action of various chemotherapeutic agents. The combined drug itself may also bring more toxic and side effects. Curcumin (Cur) is a traditional drug of food source, which has many advantages such as cheap, non-toxic and many other advantages. Studies show Jiang Huang It has been reported that the combination of chemotherapeutic drugs and curcumin can effectively reverse the multidrug resistance of tumor. However, curcumin has poor water solubility and fast metabolism in the body. How to fully exert the strong work energy of curcumin is the key to its inhibition of drug resistance. The development of technology provides a new direction for drug dosage forms. Nanotechnology has the following advantages: pH sensitivity of nanoscale, temperature sensitivity can be used to control drug release, so as to improve drug utilization and effectively reduce the toxic and side effects caused by direct exposure to drugs; the temperature sensitive function of nanoscale and the modification of different ligands can be realized. The nanotechnology's cocktail therapy is not only limited to the mixed use of two drugs, and can realize the timing and synergistic effect of the chemotherapeutic drugs. The layered double hydroxides (Layered hydroxide, LDH) are a class of anionic clay. It has a double layered structure, a positive charge of the outer band of the LDH plate and a large amount of hydrogen and oxygen. Root, which facilitates the formation of hydrogen bonds and carrying drugs. The interplate with negative charge, can be used for anion exchange, can be loaded into anions, and the nucleic acid.LDH of small molecules has non-toxic, high drug loading and pH sensitivity. It is a good carrier for different tumor drugs. In this subject, we use LDH interlayer load Fu to And Cur construction of nano mixture type, through Bel 7402/Fu hepatoma resistant cells as model, study the effect of nano LDH on fluorouracil resistant cancer of liver cancer. Cell level first investigated the proliferation inhibition effect of LDH-Fu-Cur on normal liver cells LO_2 and Bel 7402/Fu. Flow apoptosis was used to detect the apoptosis of LDH-Fu-Cur. The apoptosis related mechanism was detected by Western blot technique. The effect of autophagy on the anti-tumor effect of autophagy in LDH-Fu-Cur was detected by confocal microscopy, and autophagy was used to detect the effect of autophagy on the anti-tumor effect of LDH-Fu-Cur. Finally, the tumor suppressor test was used to detect the tumor inhibition effect of LDH-Fu-Cur in the body. The systematic study shows that we have successfully prepared the LDH co carrier system with high load and good biological safety. The UV spectrophotometer has high drug loading, the Fu load of Fu in LDH-Fu is 20.3% + 0.4%, the dosage of Fu in LDH-Fu-Cur is 19.7% + 0.2%, and the drug loading of Cur is 15.7% + 0.6%, which conforms to the expected result of the experiment. The experiment also showed that LDH-Fu-Cur had no obvious toxic and side effects on normal liver LO_2 cells, but had obvious inhibitory effect on Bel 7402/Fu tumor cells. Flow cytometry and Western blot experiment confirmed that LDH-Fu-Cur could significantly promote the apoptosis effect of Bel 7402/Fu cell than the simple curcumin group. It is interesting: the study found that autophagy participates in the inhibitory effect of autophagy on liver cancer cells. Although cell autophagy is a "double-edged sword", our experiment also confirms that LDH-Fu-Cur can initiate autophagy, which is a synergistic autophagy that promotes cell death. Finally, the construction of Bel 7402/Fu for liver cancer is constructed. The xenograft model of nude mice, observation group LDH-Fu-Cur, other drug groups and PBS control group, found that LDH-Fu-Cur can obviously inhibit the growth of Bel 7402/Fu cells in nude mice, suggesting that LDH-Fu-Cur can be used as an effective drug to inhibit the fluorouracil resistant liver cancer cells. The LDH-Fu-Cur nanoscale drug is safe to carry out the co loading of fluorouracil and curcumin. The new nanoscale drugs in the body show good inhibition of fluorouracil resistant cells in vitro, and provide new ideas for the research and development of anti tumor drug resistance drugs for liver cancer.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

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