TT-1多肽對TT細胞以及聯(lián)合IFN-α對HepG2細胞的抗腫瘤作用及機制研究
發(fā)布時間：2018-08-13 15:14
【摘要】：癌癥仍是世界上致死率最高的疾病,盡管近年來在癌癥的治療方法上多有研究,但仍無法明顯降低癌癥患者的死亡率。隨著甲狀腺癌發(fā)病率的不斷提高,該類型的癌癥成為最為常見的內分泌惡性疾病。據(jù)統(tǒng)計,在發(fā)達國家和地區(qū)每100000個女性中有9.1個人會發(fā)生甲狀腺癌,每100 000個男性中有2.9個會發(fā)生甲狀腺癌。傳統(tǒng)的甲狀腺癌治療方法為手術切除全部甲狀腺組織后再進行放射性碘治療,化療以及生化療法也被應用于治療晚期甲狀腺腫瘤。但對于分化類型較差的甲狀腺癌,上述兩種治療方案治療效果并不理想,在臨床應用中受到了極大的限制。因此,亟需為分化較差的甲狀腺癌患者開發(fā)研究高效低毒的治療藥物和治療方案。肝細胞癌(Hepatocellular carcinoma,HCC)是全球最常見的惡性腫瘤之一,是導致肝硬化患者死亡的主要原因。目前臨床常用的肝癌治療方法包括手術切除,肝移植手術以及熱消融法和化療方案,但效果均欠佳。近幾年來出現(xiàn)的一種新型治療方案——生物免疫治療,具體方案包括:靶向治療、腫瘤生物疫苗、單克隆抗體或免疫基因治療等方法,目前這些方案已經(jīng)在臨床試驗或者臨床前試驗中取得了一定的療效,但是存在許多負面問題和弊端,治療效果并不是十分理想。在該研究背景下,我們需為甲狀腺癌和肝細胞癌的治療尋找一種更加有效的治療措施。近期的研究表明,抗菌肽不僅具有很好地抗菌活性,還可以選擇性地殺傷腫瘤細胞,殺傷機制是腫瘤細胞表面的膜蛋白由于糖基化反應帶有大量的負電荷,可以與陽離子型的兩親性抗菌肽發(fā)生特異性結合從而發(fā)揮殺傷作用。但抗菌肽對肝細胞癌和甲狀腺癌的抗腫瘤效果如何,未見具體的相關報道。在本研究中,我們選取抗菌肽中已證明具有明顯抗腫瘤作用的蜂毒肽(Melittin)作為原型,在其基礎上利用生物信息學的方法進行改造得到新型多肽TT-1,相較其母肽極大的節(jié)約了成本。改造體TT-1不僅保留了Melittin氨基末端區(qū)域的活性位點,也增加了疏水性,并降低了凈電荷,提高了TT-1的穩(wěn)定性,降低了毒性。對改造后的多肽TT-1進行體內外試驗,試驗結果證明該多肽對甲狀腺癌中分化類型較差的髓樣癌以及肝細胞癌具有良好的抗腫瘤效果,具體實驗結果如下:1.TT-1多肽對TT細胞具有顯著的體內外抗腫瘤活性(1)細胞水平的實驗表明,TT-1多肽能夠在細胞水平顯著抑制TT細胞的生長,而對于人正常甲狀腺濾泡上皮細胞系Nthy-ori3-1細胞的生長抑制作用較低,即TT-1多肽對甲狀腺癌細胞具有選擇性抑制作用,并且這種生長抑制作用呈現(xiàn)出一定的劑量依賴和時間依賴關系。應用Annexin V-FITC/PI雙染對TT細胞系進行凋亡考察,結果表明多肽TT-1能夠有效地誘導TT細胞的凋亡,并且呈現(xiàn)一定的濃度依賴性。在TT-1多肽濃度為2、4、8μg/ml時,TT細胞的凋亡率分別為12.31%、18.62%和31.07%。機制研究顯示TT-1多肽能夠上調TT細胞內Bax的蛋白表達和mRNA水平,下調Bcl-2的蛋白表達和mRNA水平。此外,我們檢測了TT-1多肽作用后TT細胞內Caspase-3和Caspase-9在蛋白和RNA水平的表達量,結果顯示TT-1多肽的處理增強了Caspase-3和Caspase-9在蛋白質轉錄和翻譯水平的表達。(2)動物水平實驗結果表明,在TT細胞裸鼠皮下移植瘤模型中,對照組的腫瘤體積隨時間的延長而增大,而TT-1多肽高中低劑量處理組(0.04 mg/kg體重、0.20 mg/kg體重、1 mg/kg體重)的腫瘤體積與其相比明顯較小。并且與對照組相比,TT-1多肽給藥組的腫瘤體積和重量呈現(xiàn)出劑量依賴性降低趨勢,TT-1多肽(0.04-1 mg/kg體重)三個劑量組的抑瘤率分別為5.8%,48.6%和56.8%。不同治療組的荷瘤鼠體重在TT-1多肽治療過程中無明顯變化。HE染色結果顯示,與對照組相比,TT-1多肽治療組裸鼠的腫瘤組織表現(xiàn)出明顯的腫瘤細胞數(shù)目的減少,同時不論對照組與治療組裸鼠的肝臟和脾臟均無明顯疾病特征。以上結果表明,TT-1多肽能夠顯著抑制TT細胞裸鼠皮下移植瘤的生長,體內抗腫瘤活性明顯,且毒副作用較低。2.TT-1多肽對HepG2細胞體內外生長抑制作用明顯,進一步聯(lián)合IFN-α時發(fā)揮出極大的協(xié)同治療作用(1)TT-1多肽與HepG2細胞共孵育72 h后呈現(xiàn)出對腫瘤細胞的劑量依賴性的生長抑制作用,TT-1多肽對HepG2細胞的半數(shù)抑制濃度(IC50)為3.762±0.285μg/ml。當TT-1多肽的孵育濃度達到32μg/ml時,腫瘤細胞HepG2的細胞存活率僅為14.8%。(2)構建HepG2裸鼠異位移植瘤模型,體內實驗結果表明:TT-1多肽聯(lián)合IFN-α抗腫瘤效果較單純TT-1多肽治療組顯著增強。與對照組相比,TT-1多肽聯(lián)合IFN-α治療HepG2裸鼠的效果增強了近80%。機制研究證明TT-1多肽和IFN-α聯(lián)合治療的抗腫瘤作用是由NK細胞介導的,并且通過MICA-NKG2D通路特異性介導。綜上所述,本論文首創(chuàng)性地研究了新型多肽TT-1對TT細胞以及聯(lián)合IFN-α對HepG2細胞的抗腫瘤免疫治療活性。通過體內外實驗研究,證實了TT-1多肽對TT細胞具有特異性的殺傷作用,并通過誘導細胞凋亡實現(xiàn)。TT-1多肽對HepG2細胞具有明顯的生長抑制作用,并且聯(lián)合IFN-α對HepG2裸鼠移植瘤模型具有良好的體內抗腫瘤作用,闡述了聯(lián)合治療的抗腫瘤免疫治療機制,表明該作用由NK細胞引起,并由MICA-NKG2D介導,為甲狀腺髓樣癌和肝癌的治療策略提供了新的發(fā)展方向和發(fā)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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R96
本文編號：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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R96
【相似文獻】
相關博士學位論文 前2條
1 張大奇;BMAP-28、TT-1對人甲狀腺髓樣癌細胞系TT的抗腫瘤作用及其機理研究[D];吉林大學;2016年
2 萬蘭蘭;TT-1多肽對TT細胞以及聯(lián)合IFN-α對HepG2細胞的抗腫瘤作用及機制研究[D];吉林大學;2016年
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