發(fā)布時(shí)間：2018-06-18 16:00

  本文選題：牛蛙皮膚活性肽 + 抗腫瘤��； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：癌癥已經(jīng)成為全球性的公共健康問(wèn)題,嚴(yán)重危害人類生命健康。目前癌癥在我國(guó)已上升為死亡率第一的疾病。針對(duì)癌癥的現(xiàn)有治療方法主要有手術(shù)、化療及放射療法,抗癌藥物主要是烷化劑類和抗代謝類藥物,但由于較低的治療指數(shù)及嚴(yán)重的副作用,導(dǎo)致這些治療策略并不是很成功,此外,多重耐藥性癌細(xì)胞的產(chǎn)生和進(jìn)化也影響了治療效果。因此,研究新型抗癌藥物或療法刻不容緩。抗菌肽(Antimcrobial peptide,AMPs)是哺乳動(dòng)物防御體系的一個(gè)重要組成部分,作為機(jī)體天然免疫系統(tǒng)部分,對(duì)病原體和癌細(xì)胞具有高效的抑制活性。目前已報(bào)道了約2400多種抗菌肽,其中有166種展示出了抗癌活性,也被稱作抗癌肽(Anticancer peptide,ACPs)。本課題組前期研究顯示從牛蛙皮膚中提取獲得的抗菌肽Temporin-La在體外具有針對(duì)多種癌細(xì)胞的抑制活性。抗菌肽與現(xiàn)有的化療相比展示出許多如廣譜、快速、高效等抗癌活性,且癌細(xì)胞不易產(chǎn)生耐受性,因此,抗菌肽具有良好的抗癌開發(fā)應(yīng)用前景。本研究在實(shí)驗(yàn)室前期研究初步證實(shí)牛蛙皮膚活性肽Temporin-La具有抗腫瘤活性的基礎(chǔ)上,使用化學(xué)合成抗菌肽Temporin-La,用MTT法測(cè)定了Temporin-La對(duì)6種腫瘤細(xì)胞:人肺腺癌細(xì)胞系A(chǔ)549、人結(jié)腸癌細(xì)胞系SW1116、人胃癌細(xì)胞系BGC-823、人宮頸癌細(xì)胞系Hela、人肝癌細(xì)胞系Hep G2和人肝癌細(xì)胞系SMMC-7721的抗腫瘤活性,其IC50分別為29.45μM、36.09μM、25.34μM、37.76μM、11.19μM、15.58μM,結(jié)果顯示Temporin-La對(duì)上述6種腫瘤細(xì)胞均具有抑制增殖作用,其中對(duì)人肝癌細(xì)胞系Hep G2的抑制效果最為顯著。Temporin-La對(duì)于人正常肝細(xì)胞HL7702的細(xì)胞毒性以及對(duì)于紅細(xì)胞的溶血活性實(shí)驗(yàn)結(jié)果顯示,Temporin-La在正常濃度范圍內(nèi),無(wú)明顯的細(xì)胞毒性和較小的溶血活性。用光學(xué)顯微鏡與激光共聚焦顯微鏡觀察了Temporin-La對(duì)Hep G2腫瘤細(xì)胞形態(tài)的影響。光學(xué)顯微鏡觀察結(jié)果顯示,Temporin-La作用于癌細(xì)胞3、6、12小時(shí)后可引起明顯的細(xì)胞形態(tài)學(xué)變化,且隨著時(shí)間的遞增,大量細(xì)胞碎片脫落,細(xì)胞核明顯皺縮。激光共聚焦顯微鏡結(jié)果顯示,Temporin-La作用于癌細(xì)胞時(shí),首先定位于細(xì)胞膜表面,隨后進(jìn)入細(xì)胞漿質(zhì)。用流式細(xì)胞儀觀察了Temporin-La對(duì)Hep G2腫瘤細(xì)胞凋亡的影響,結(jié)果顯示,Temporin-La可通過(guò)上調(diào)Caspase-3的表達(dá),從而誘導(dǎo)癌細(xì)胞發(fā)生凋亡,且存在時(shí)間劑量依賴性。建立了裸鼠人肝癌細(xì)胞Hep G2皮下腫瘤模型,分析了Temporin-La的體內(nèi)抗腫瘤活性。結(jié)果顯示Temporin-La在體內(nèi)也具有良好的抑癌活性,與對(duì)照組相比,Temporin-La治療10天后腫瘤組織的重量和體積明顯減少,腫瘤抑制率(T/C)為50%。腫瘤組織的病理學(xué)實(shí)驗(yàn)結(jié)果顯示,與對(duì)照組相比,Temporin-La治療組細(xì)胞核裂變較少和細(xì)胞有較大的壞死面積,表現(xiàn)出明顯的細(xì)胞壞死特征。腫瘤組織免疫組化結(jié)果顯示,Temporin-La治療組腫瘤組織中Ki-67蛋白的表達(dá)水平明顯下調(diào),抑制了腫瘤細(xì)胞的惡性增殖;Temporin-La治療組腫瘤組織中VEGF蛋白表達(dá)水平明顯上調(diào),抑制了腫瘤細(xì)胞的分化;Temporin-La治療組腫瘤組織中Caspase-3蛋白表達(dá)水平明顯上調(diào),誘發(fā)腫瘤細(xì)胞凋亡。綜上所述,本研究在體外,通過(guò)MTT細(xì)胞毒性、激光共聚焦及流式細(xì)胞術(shù)證實(shí)了Temporin-La在體外具有腫瘤細(xì)胞靶向殺傷活性,能夠破壞腫瘤細(xì)胞完整性,并誘導(dǎo)腫瘤細(xì)胞發(fā)生凋亡,初步闡明Temporin-La在體外對(duì)腫瘤細(xì)胞膜的作用機(jī)制;在體內(nèi),通過(guò)對(duì)裸鼠皮下腫瘤模型的治療實(shí)驗(yàn)證實(shí)Temporin-La在體內(nèi)具有腫瘤組織靶向性,能夠明顯的抑制腫瘤細(xì)胞的惡性增殖與分化,并能夠誘導(dǎo)實(shí)體腫瘤細(xì)胞發(fā)生凋亡。本研究在體內(nèi)外證實(shí)牛蛙皮膚活性肽Temporin-La具有良好的抗腫瘤活性,特別是針對(duì)人肝癌細(xì)胞Hep G2的活性最佳,初步闡述了Temporin-La在體內(nèi)外的抗腫瘤作用機(jī)制,為Temporin-La抗腫瘤制劑的開發(fā)提供數(shù)據(jù)支持,也為抗菌肽抗腫瘤作用機(jī)制的闡述奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96

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