TT-1多肽对TT细胞以及联合IFN-α对HepG2细胞的抗肿瘤作用及机制研究
发布时间：2018-08-13 15:14
【摘要】：癌症仍是世界上致死率最高的疾病,尽管近年来在癌症的治疗方法上多有研究,但仍无法明显降低癌症患者的死亡率。随着甲状腺癌发病率的不断提高,该类型的癌症成为最为常见的内分泌恶性疾病。据统计,在发达国家和地区每100000个女性中有9.1个人会发生甲状腺癌,每100 000个男性中有2.9个会发生甲状腺癌。传统的甲状腺癌治疗方法为手术切除全部甲状腺组织后再进行放射性碘治疗,化疗以及生化疗法也被应用于治疗晚期甲状腺肿瘤。但对于分化类型较差的甲状腺癌,上述两种治疗方案治疗效果并不理想,在临床应用中受到了极大的限制。因此,亟需为分化较差的甲状腺癌患者开发研究高效低毒的治疗药物和治疗方案。肝细胞癌(Hepatocellular carcinoma,HCC)是全球最常见的恶性肿瘤之一,是导致肝硬化患者死亡的主要原因。目前临床常用的肝癌治疗方法包括手术切除,肝移植手术以及热消融法和化疗方案,但效果均欠佳。近几年来出现的一种新型治疗方案——生物免疫治疗,具体方案包括:靶向治疗、肿瘤生物疫苗、单克隆抗体或免疫基因治疗等方法,目前这些方案已经在临床试验或者临床前试验中取得了一定的疗效,但是存在许多负面问题和弊端,治疗效果并不是十分理想。在该研究背景下,我们需为甲状腺癌和肝细胞癌的治疗寻找一种更加有效的治疗措施。近期的研究表明,抗菌肽不仅具有很好地抗菌活性,还可以选择性地杀伤肿瘤细胞,杀伤机制是肿瘤细胞表面的膜蛋白由于糖基化反应带有大量的负电荷,可以与阳离子型的两亲性抗菌肽发生特异性结合从而发挥杀伤作用。但抗菌肽对肝细胞癌和甲状腺癌的抗肿瘤效果如何,未见具体的相关报道。在本研究中,我们选取抗菌肽中已证明具有明显抗肿瘤作用的蜂毒肽(Melittin)作为原型,在其基础上利用生物信息学的方法进行改造得到新型多肽TT-1,相较其母肽极大的节约了成本。改造体TT-1不仅保留了Melittin氨基末端区域的活性位点,也增加了疏水性,并降低了净电荷,提高了TT-1的稳定性,降低了毒性。对改造后的多肽TT-1进行体内外试验,试验结果证明该多肽对甲状腺癌中分化类型较差的髓样癌以及肝细胞癌具有良好的抗肿瘤效果,具体实验结果如下:1.TT-1多肽对TT细胞具有显著的体内外抗肿瘤活性(1)细胞水平的实验表明,TT-1多肽能够在细胞水平显著抑制TT细胞的生长,而对于人正常甲状腺滤泡上皮细胞系Nthy-ori3-1细胞的生长抑制作用较低,即TT-1多肽对甲状腺癌细胞具有选择性抑制作用,并且这种生长抑制作用呈现出一定的剂量依赖和时间依赖关系。应用Annexin V-FITC/PI双染对TT细胞系进行凋亡考察,结果表明多肽TT-1能够有效地诱导TT细胞的凋亡,并且呈现一定的浓度依赖性。在TT-1多肽浓度为2、4、8μg/ml时,TT细胞的凋亡率分别为12.31%、18.62%和31.07%。机制研究显示TT-1多肽能够上调TT细胞内Bax的蛋白表达和mRNA水平,下调Bcl-2的蛋白表达和mRNA水平。此外,我们检测了TT-1多肽作用后TT细胞内Caspase-3和Caspase-9在蛋白和RNA水平的表达量,结果显示TT-1多肽的处理增强了Caspase-3和Caspase-9在蛋白质转录和翻译水平的表达。(2)动物水平实验结果表明,在TT细胞裸鼠皮下移植瘤模型中,对照组的肿瘤体积随时间的延长而增大,而TT-1多肽高中低剂量处理组(0.04 mg/kg体重、0.20 mg/kg体重、1 mg/kg体重)的肿瘤体积与其相比明显较小。并且与对照组相比,TT-1多肽给药组的肿瘤体积和重量呈现出剂量依赖性降低趋势,TT-1多肽(0.04-1 mg/kg体重)三个剂量组的抑瘤率分别为5.8%,48.6%和56.8%。不同治疗组的荷瘤鼠体重在TT-1多肽治疗过程中无明显变化。HE染色结果显示,与对照组相比,TT-1多肽治疗组裸鼠的肿瘤组织表现出明显的肿瘤细胞数目的减少,同时不论对照组与治疗组裸鼠的肝脏和脾脏均无明显疾病特征。以上结果表明,TT-1多肽能够显著抑制TT细胞裸鼠皮下移植瘤的生长,体内抗肿瘤活性明显,且毒副作用较低。2.TT-1多肽对HepG2细胞体内外生长抑制作用明显,进一步联合IFN-α时发挥出极大的协同治疗作用(1)TT-1多肽与HepG2细胞共孵育72 h后呈现出对肿瘤细胞的剂量依赖性的生长抑制作用,TT-1多肽对HepG2细胞的半数抑制浓度(IC50)为3.762±0.285μg/ml。当TT-1多肽的孵育浓度达到32μg/ml时,肿瘤细胞HepG2的细胞存活率仅为14.8%。(2)构建HepG2裸鼠异位移植瘤模型,体内实验结果表明:TT-1多肽联合IFN-α抗肿瘤效果较单纯TT-1多肽治疗组显著增强。与对照组相比,TT-1多肽联合IFN-α治疗HepG2裸鼠的效果增强了近80%。机制研究证明TT-1多肽和IFN-α联合治疗的抗肿瘤作用是由NK细胞介导的,并且通过MICA-NKG2D通路特异性介导。综上所述,本论文首创性地研究了新型多肽TT-1对TT细胞以及联合IFN-α对HepG2细胞的抗肿瘤免疫治疗活性。通过体内外实验研究,证实了TT-1多肽对TT细胞具有特异性的杀伤作用,并通过诱导细胞凋亡实现。TT-1多肽对HepG2细胞具有明显的生长抑制作用,并且联合IFN-α对HepG2裸鼠移植瘤模型具有良好的体内抗肿瘤作用,阐述了联合治疗的抗肿瘤免疫治疗机制,表明该作用由NK细胞引起,并由MICA-NKG2D介导,为甲状腺髓样癌和肝癌的治疗策略提供了新的发展方向和发展思路。
[Abstract]:Cancer is still the most lethal disease in the world. Although there have been many studies on the treatment of cancer in recent years, it still can not significantly reduce the mortality of cancer patients. 9.1 women develop thyroid cancer, and 2.9 out of 100,000 men develop thyroid cancer. Conventional thyroid cancer treatments include surgical removal of all thyroid tissues followed by radioiodine therapy. Chemotherapy and biochemical therapy are also used to treat advanced thyroid tumors. Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, which leads to poor differentiation of thyroid cancer. The main causes of death in patients with cirrhosis of the liver are surgical resection, liver transplantation, heat ablation and chemotherapy, but the results are not satisfactory. At present, these schemes have achieved certain effects in clinical trials or preclinical trials, but there are many negative problems and drawbacks, and the therapeutic effect is not very ideal. In this context, we need to find a more effective treatment for thyroid cancer and hepatocellular carcinoma. Recent studies have shown that antimicrobial peptides not only have good antimicrobial activity, but also selectively kill tumor cells. The mechanism of antimicrobial activity is that membrane proteins on the surface of tumor cells can specifically bind to cationic amphiphilic antimicrobial peptides because of their negative charges due to glycosylation reaction. In this study, we selected Melittin, which has been proved to have significant antitumor effect, as the prototype. On the basis of this, we used bioinformatics methods to modify TT-1, a novel peptide, which is more powerful than its mother peptide. The modified TT-1 not only retains the active sites in the amino terminal region of Melittin, but also increases the hydrophobicity, decreases the net charge, improves the stability and reduces the toxicity of TT-1. The modified TT-1 was tested in vitro and in vivo, and the results showed that the modified TT-1 had a poor differentiation in medullary carcinoma. The specific experimental results are as follows: 1. TT-1 polypeptide has significant antitumor activity on TT cells in vitro and in vivo (1) Cell level experiments show that TT-1 polypeptide can significantly inhibit the growth of TT cells at the cellular level, while it can inhibit the growth of human normal thyroid follicular epithelial cell line Nthy-ori3-1 cells. The results showed that TT-1 polypeptide could effectively induce the apoptosis of TT cells by Annexin V-FITC/PI double staining. The apoptotic rates of TT cells were 12.31%, 18.62% and 31.07% at TT-1 polypeptide concentration of 2,4,8 ug/ml. Mechanisms showed that TT-1 polypeptide could up-regulate the expression of Bax protein and mRNA, down-regulate the expression of Bcl-2 protein and mRNA in TT cells. The expression of Caspase-3 and Caspase-9 at protein and RNA levels showed that the treatment of TT-1 peptide enhanced the expression of Caspase-3 and Caspase-9 at protein transcription and translation levels. (2) Animal level experiments showed that the tumor volume of the control group increased with time, while the TT-1 peptide was high in the TT cell subcutaneous transplanted tumor model in nude mice. The tumor volume of the low dose group (0.04 mg/kg body weight, 0.20 mg/kg body weight, 1 mg/kg body weight) was significantly smaller than that of the control group. Compared with the control group, the tumor volume and weight of the TT-1 polypeptide group showed a dose-dependent reduction trend, and the tumor inhibition rates of the TT-1 polypeptide group (0.04-1 mg/kg body weight) were 5.8%, 48.6% and 56.8% respectively. The results of HE staining showed that compared with the control group, the number of tumor cells in the tumor tissues of the TT-1 polypeptide treatment group was significantly decreased, and there was no significant disease characteristic in the liver and spleen of the control group and the treatment group. The results showed that TT-1 polypeptide could inhibit the growth of subcutaneous transplanted tumor of TT cells in nude mice, and its antitumor activity was obvious in vivo, and its toxicity was low. 2. TT-1 polypeptide could inhibit the growth of HepG2 cells in vitro and in vivo. Further combination with IFN-alpha, TT-1 polypeptide exerted a great synergistic effect on the growth of HepG2 cells. (1) TT-1 polypeptide showed a pairing effect after incubating with HepG2 cells for 72 The half inhibitory concentration (IC50) of TT-1 polypeptide on HepG2 cells was 3.762 (+ 0.285 ug/ml). When the incubation concentration of TT-1 polypeptide reached 32 ug/ml, the survival rate of HepG2 cells was only 14.8%. (2) HepG2 heterotopic xenograft tumor model was established in nude mice. The results in vivo showed that TT-1 polypeptide conjugated with TT-1 polypeptide. The anti-tumor effect of TT-1 polypeptide combined with IFN-alpha was significantly enhanced compared with that of TT-1 polypeptide alone. Compared with the control group, the anti-tumor effect of TT-1 polypeptide combined with IFN-alpha in HepG2 nude mice was enhanced by nearly 80%. Mechanisms study showed that the anti-tumor effect of TT-1 polypeptide combined with IFN-alpha was mediated by NK cells and specifically mediated by MICA-NKG2D pathway. The anti-tumor immunotherapeutic activity of TT-1 on TT cells and combined with IFN-alpha on HepG2 cells was studied. The specific killing effect of TT-1 polypeptide on TT cells was confirmed in vitro and in vivo, and apoptosis was induced. The combination of IFN-alpha and NKG2D has a good anti-tumor effect in vivo on HepG2 xenograft tumor model in nude mice. The anti-tumor immunotherapy mechanism of the combination therapy is expounded. The results show that the effect is caused by NK cells and mediated by MICA-NKG2D. It provides a new development direction and thinking for the treatment strategy of medullary thyroid carcinoma and liver cancer.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96
本文编号：2181362
[Abstract]:Cancer is still the most lethal disease in the world. Although there have been many studies on the treatment of cancer in recent years, it still can not significantly reduce the mortality of cancer patients. 9.1 women develop thyroid cancer, and 2.9 out of 100,000 men develop thyroid cancer. Conventional thyroid cancer treatments include surgical removal of all thyroid tissues followed by radioiodine therapy. Chemotherapy and biochemical therapy are also used to treat advanced thyroid tumors. Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, which leads to poor differentiation of thyroid cancer. The main causes of death in patients with cirrhosis of the liver are surgical resection, liver transplantation, heat ablation and chemotherapy, but the results are not satisfactory. At present, these schemes have achieved certain effects in clinical trials or preclinical trials, but there are many negative problems and drawbacks, and the therapeutic effect is not very ideal. In this context, we need to find a more effective treatment for thyroid cancer and hepatocellular carcinoma. Recent studies have shown that antimicrobial peptides not only have good antimicrobial activity, but also selectively kill tumor cells. The mechanism of antimicrobial activity is that membrane proteins on the surface of tumor cells can specifically bind to cationic amphiphilic antimicrobial peptides because of their negative charges due to glycosylation reaction. In this study, we selected Melittin, which has been proved to have significant antitumor effect, as the prototype. On the basis of this, we used bioinformatics methods to modify TT-1, a novel peptide, which is more powerful than its mother peptide. The modified TT-1 not only retains the active sites in the amino terminal region of Melittin, but also increases the hydrophobicity, decreases the net charge, improves the stability and reduces the toxicity of TT-1. The modified TT-1 was tested in vitro and in vivo, and the results showed that the modified TT-1 had a poor differentiation in medullary carcinoma. The specific experimental results are as follows: 1. TT-1 polypeptide has significant antitumor activity on TT cells in vitro and in vivo (1) Cell level experiments show that TT-1 polypeptide can significantly inhibit the growth of TT cells at the cellular level, while it can inhibit the growth of human normal thyroid follicular epithelial cell line Nthy-ori3-1 cells. The results showed that TT-1 polypeptide could effectively induce the apoptosis of TT cells by Annexin V-FITC/PI double staining. The apoptotic rates of TT cells were 12.31%, 18.62% and 31.07% at TT-1 polypeptide concentration of 2,4,8 ug/ml. Mechanisms showed that TT-1 polypeptide could up-regulate the expression of Bax protein and mRNA, down-regulate the expression of Bcl-2 protein and mRNA in TT cells. The expression of Caspase-3 and Caspase-9 at protein and RNA levels showed that the treatment of TT-1 peptide enhanced the expression of Caspase-3 and Caspase-9 at protein transcription and translation levels. (2) Animal level experiments showed that the tumor volume of the control group increased with time, while the TT-1 peptide was high in the TT cell subcutaneous transplanted tumor model in nude mice. The tumor volume of the low dose group (0.04 mg/kg body weight, 0.20 mg/kg body weight, 1 mg/kg body weight) was significantly smaller than that of the control group. Compared with the control group, the tumor volume and weight of the TT-1 polypeptide group showed a dose-dependent reduction trend, and the tumor inhibition rates of the TT-1 polypeptide group (0.04-1 mg/kg body weight) were 5.8%, 48.6% and 56.8% respectively. The results of HE staining showed that compared with the control group, the number of tumor cells in the tumor tissues of the TT-1 polypeptide treatment group was significantly decreased, and there was no significant disease characteristic in the liver and spleen of the control group and the treatment group. The results showed that TT-1 polypeptide could inhibit the growth of subcutaneous transplanted tumor of TT cells in nude mice, and its antitumor activity was obvious in vivo, and its toxicity was low. 2. TT-1 polypeptide could inhibit the growth of HepG2 cells in vitro and in vivo. Further combination with IFN-alpha, TT-1 polypeptide exerted a great synergistic effect on the growth of HepG2 cells. (1) TT-1 polypeptide showed a pairing effect after incubating with HepG2 cells for 72 The half inhibitory concentration (IC50) of TT-1 polypeptide on HepG2 cells was 3.762 (+ 0.285 ug/ml). When the incubation concentration of TT-1 polypeptide reached 32 ug/ml, the survival rate of HepG2 cells was only 14.8%. (2) HepG2 heterotopic xenograft tumor model was established in nude mice. The results in vivo showed that TT-1 polypeptide conjugated with TT-1 polypeptide. The anti-tumor effect of TT-1 polypeptide combined with IFN-alpha was significantly enhanced compared with that of TT-1 polypeptide alone. Compared with the control group, the anti-tumor effect of TT-1 polypeptide combined with IFN-alpha in HepG2 nude mice was enhanced by nearly 80%. Mechanisms study showed that the anti-tumor effect of TT-1 polypeptide combined with IFN-alpha was mediated by NK cells and specifically mediated by MICA-NKG2D pathway. The anti-tumor immunotherapeutic activity of TT-1 on TT cells and combined with IFN-alpha on HepG2 cells was studied. The specific killing effect of TT-1 polypeptide on TT cells was confirmed in vitro and in vivo, and apoptosis was induced. The combination of IFN-alpha and NKG2D has a good anti-tumor effect in vivo on HepG2 xenograft tumor model in nude mice. The anti-tumor immunotherapy mechanism of the combination therapy is expounded. The results show that the effect is caused by NK cells and mediated by MICA-NKG2D. It provides a new development direction and thinking for the treatment strategy of medullary thyroid carcinoma and liver cancer.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96
【相似文献】
相关博士学位论文 前2条
1 张大奇;BMAP-28、TT-1对人甲状腺髓样癌细胞系TT的抗肿瘤作用及其机理研究[D];吉林大学;2016年
2 万兰兰;TT-1多肽对TT细胞以及联合IFN-α对HepG2细胞的抗肿瘤作用及机制研究[D];吉林大学;2016年
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