牛蛙（Rana catesbeiana）皮肤活性肽Temporin-La抗肿瘤活性研究

发布时间：2018-06-18 16:00

本文选题：牛蛙皮肤活性肽 + 抗肿瘤　；参考：《吉林大学》2017年硕士论文

【摘要】：癌症已经成为全球性的公共健康问题,严重危害人类生命健康。目前癌症在我国已上升为死亡率第一的疾病。针对癌症的现有治疗方法主要有手术、化疗及放射疗法,抗癌药物主要是烷化剂类和抗代谢类药物,但由于较低的治疗指数及严重的副作用,导致这些治疗策略并不是很成功,此外,多重耐药性癌细胞的产生和进化也影响了治疗效果。因此,研究新型抗癌药物或疗法刻不容缓。抗菌肽(Antimcrobial peptide,AMPs)是哺乳动物防御体系的一个重要组成部分,作为机体天然免疫系统部分,对病原体和癌细胞具有高效的抑制活性。目前已报道了约2400多种抗菌肽,其中有166种展示出了抗癌活性,也被称作抗癌肽(Anticancer peptide,ACPs)。本课题组前期研究显示从牛蛙皮肤中提取获得的抗菌肽Temporin-La在体外具有针对多种癌细胞的抑制活性。抗菌肽与现有的化疗相比展示出许多如广谱、快速、高效等抗癌活性,且癌细胞不易产生耐受性,因此,抗菌肽具有良好的抗癌开发应用前景。本研究在实验室前期研究初步证实牛蛙皮肤活性肽Temporin-La具有抗肿瘤活性的基础上,使用化学合成抗菌肽Temporin-La,用MTT法测定了Temporin-La对6种肿瘤细胞:人肺腺癌细胞系A549、人结肠癌细胞系SW1116、人胃癌细胞系BGC-823、人宫颈癌细胞系Hela、人肝癌细胞系Hep G2和人肝癌细胞系SMMC-7721的抗肿瘤活性,其IC50分别为29.45μM、36.09μM、25.34μM、37.76μM、11.19μM、15.58μM,结果显示Temporin-La对上述6种肿瘤细胞均具有抑制增殖作用,其中对人肝癌细胞系Hep G2的抑制效果最为显著。Temporin-La对于人正常肝细胞HL7702的细胞毒性以及对于红细胞的溶血活性实验结果显示,Temporin-La在正常浓度范围内,无明显的细胞毒性和较小的溶血活性。用光学显微镜与激光共聚焦显微镜观察了Temporin-La对Hep G2肿瘤细胞形态的影响。光学显微镜观察结果显示,Temporin-La作用于癌细胞3、6、12小时后可引起明显的细胞形态学变化,且随着时间的递增,大量细胞碎片脱落,细胞核明显皱缩。激光共聚焦显微镜结果显示,Temporin-La作用于癌细胞时,首先定位于细胞膜表面,随后进入细胞浆质。用流式细胞仪观察了Temporin-La对Hep G2肿瘤细胞凋亡的影响,结果显示,Temporin-La可通过上调Caspase-3的表达,从而诱导癌细胞发生凋亡,且存在时间剂量依赖性。建立了裸鼠人肝癌细胞Hep G2皮下肿瘤模型,分析了Temporin-La的体内抗肿瘤活性。结果显示Temporin-La在体内也具有良好的抑癌活性,与对照组相比,Temporin-La治疗10天后肿瘤组织的重量和体积明显减少,肿瘤抑制率(T/C)为50%。肿瘤组织的病理学实验结果显示,与对照组相比,Temporin-La治疗组细胞核裂变较少和细胞有较大的坏死面积,表现出明显的细胞坏死特征。肿瘤组织免疫组化结果显示,Temporin-La治疗组肿瘤组织中Ki-67蛋白的表达水平明显下调,抑制了肿瘤细胞的恶性增殖;Temporin-La治疗组肿瘤组织中VEGF蛋白表达水平明显上调,抑制了肿瘤细胞的分化;Temporin-La治疗组肿瘤组织中Caspase-3蛋白表达水平明显上调,诱发肿瘤细胞凋亡。综上所述,本研究在体外,通过MTT细胞毒性、激光共聚焦及流式细胞术证实了Temporin-La在体外具有肿瘤细胞靶向杀伤活性,能够破坏肿瘤细胞完整性,并诱导肿瘤细胞发生凋亡,初步阐明Temporin-La在体外对肿瘤细胞膜的作用机制;在体内,通过对裸鼠皮下肿瘤模型的治疗实验证实Temporin-La在体内具有肿瘤组织靶向性,能够明显的抑制肿瘤细胞的恶性增殖与分化,并能够诱导实体肿瘤细胞发生凋亡。本研究在体内外证实牛蛙皮肤活性肽Temporin-La具有良好的抗肿瘤活性,特别是针对人肝癌细胞Hep G2的活性最佳,初步阐述了Temporin-La在体内外的抗肿瘤作用机制,为Temporin-La抗肿瘤制剂的开发提供数据支持,也为抗菌肽抗肿瘤作用机制的阐述奠定了基础。
[Abstract]:Cancer has become a global public health problem, seriously endangering human life and health. At present, cancer has risen to the highest mortality rate in our country. The main treatment methods for cancer are surgery, chemotherapy and radiation therapy, and anticancer drugs are mainly alkylating agents and antimetabolic drugs, but because of lower therapeutic index and Antimcrobial peptide (AMPs) is an important part of the mammalian defense system as an important component of the mammalian defense system. The immune system, which has high inhibitory activity for pathogens and cancer cells, has reported more than 2400 antimicrobial peptides, of which 166 have demonstrated anticancer activity, also called Anticancer peptide (ACPs). The previous study of our group showed that the antibacterial peptide Temporin-La extracted from the skin of the bullfrog skin was in vitro. Compared with the existing chemotherapy, antimicrobial peptides exhibit many anti-cancer activities such as broad-spectrum, rapid and efficient, and the cancer cells are not easy to produce tolerance. Therefore, antibacterial peptides have good anticancer development prospects. In this study, the preliminary study in the laboratory confirmed that the bullfrog skin active peptide Temporin-La has a preliminary study. On the basis of antitumor activity, the antitumor activity of Temporin-La against 6 tumor cells: human lung adenocarcinoma cell line A549, human colon cancer cell line SW1116, human colon cancer cell line BGC-823, human cervical cancer cell line Hela, human cancer cell line Hep G2 and human hepatoma cell line SMMC-7721, Temporin-La against 6 tumor cells were measured by MTT method. C50 was 29.45 mu M, 36.09 mu M, 25.34 mu M, 37.76 mu M, 11.19 micron M, 15.58 mu M. The results showed that Temporin-La could inhibit the proliferation of the 6 tumor cells, and the inhibition effect of Hep G2 on human hepatocellular carcinoma cell line was the most significant for the cytotoxicity of human normal hepatocyte HL7702 and the hemolysis activity for red cells. The results showed that Temporin-La had no obvious cytotoxicity and small hemolytic activity in the normal concentration range. The effects of Temporin-La on the morphology of Hep G2 tumor cells were observed with optical microscopy and laser confocal microscopy. The results of optical microscopy showed that Temporin-La could cause obvious effect on cancer cells after 3,6,12 hours. The cell morphology changes, and with the increase of time, a large number of cell fragments fall off, and the nucleus shrinks. The laser confocal microscope results show that when Temporin-La acts on the cancer cells, it first locates on the surface of the cell membrane and then enters the cytoplasm. The effect of Temporin-La on the apoptosis of Hep G2 tumor cells is observed by flow cytometry. The results showed that Temporin-La could induce the apoptosis of the cancer cells by up regulation of the expression of Caspase-3, and there was a time and dose dependence. The Hep G2 subcutaneous tumor model of human hepatoma cells in nude mice was established and the anti-tumor activity of Temporin-La in vivo was analyzed. The results showed that Temporin-La had good anti-cancer activity in the body and was in contrast with the control group. The weight and volume of tumor tissue decreased significantly after 10 days of Temporin-La treatment, and the tumor inhibition rate (T/C) was the pathological test of 50%. tumor tissue. Compared with the control group, the nuclear fission of the Temporin-La group was less and the cell had a larger necrotic area. The results showed that the expression level of Ki-67 protein in the tumor tissue of the Temporin-La treatment group was obviously down, and the malignant proliferation of the tumor cells was inhibited. The expression of VEGF protein in the tumor tissue of the Temporin-La treatment group was obviously up-regulated, and the differentiation of the tumor cells was inhibited. The expression of Caspase-3 protein in the tumor tissue of the Temporin-La treatment group was obviously up-regulated. To sum up the apoptosis of tumor cells. In summary, this study demonstrated that Temporin-La has the targeting activity of tumor cells in vitro by MTT cytotoxicity, laser confocal and flow cytometry, which can destroy the integrity of tumor cells and induce tumor cells to wither and fall, and preliminarily elucidate the membrane of Temporin-La in vitro to the tumor cell membrane. In vivo, in vivo, through the treatment of tumor model in nude mice, it is proved that Temporin-La has the targeting of tumor tissue in vivo, can obviously inhibit the malignant proliferation and differentiation of tumor cells, and can induce the apoptosis of solid tumor cells. This study has confirmed that the bullfrog skin active peptide Temporin-La is in vitro and in vivo. Good antitumor activity, especially the activity of Hep G2 for human hepatoma cells is the best. The anti tumor mechanism of Temporin-La in vivo and in vivo is preliminarily expounded. It provides data support for the development of Temporin-La antitumor agents, and lays a foundation for the exposition of anti-tumor mechanism of antimicrobial peptides.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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