發(fā)布時(shí)間：2019-05-28 11:32

【摘要】：蜂毒肽,作為蜂毒的主要成分,是一種具有α螺旋結(jié)構(gòu)的兩親性多肽。蜂毒肽在疾病治療上有廣泛的應(yīng)用,具有抗炎、鎮(zhèn)痛、抗菌、抗病毒、抗輻射和降血壓等作用。蜂毒肽應(yīng)用于腫瘤治療具有獨(dú)特優(yōu)勢(shì),能夠在質(zhì)膜上打孔造成細(xì)胞裂解死亡,從而避開了腫瘤的耐藥性問(wèn)題。然而,蜂毒肽自身存在的溶血性問(wèn)題、非特異性作用以及易被代謝降解等缺點(diǎn),嚴(yán)重阻礙了其在抗腫瘤方面的臨床應(yīng)用。如何在活體內(nèi)安全有效地運(yùn)輸蜂毒肽抵達(dá)腫瘤組織而不產(chǎn)生毒副作用,仍是極具挑戰(zhàn)的難題。本文采用以仿HDL多肽脂質(zhì)納米顆粒為基礎(chǔ)的運(yùn)載方法,有效地解決了蜂毒肽在活體腫瘤治療中存在的溶血問(wèn)題,并延長(zhǎng)了其在體內(nèi)的循環(huán)周期,而且實(shí)現(xiàn)了在腫瘤區(qū)域特異性地釋放,主要取得了以下研究結(jié)果： (1)發(fā)明了一種新型的含蜂毒肽的功能性雜化多肽α-melittin。它是由蜂毒肽與一條α螺旋結(jié)構(gòu)的載脂蛋白模擬肽(α-peptide)通過(guò)共價(jià)連接而成。α-melittin在保留了蜂毒肽的自發(fā)形成多聚體和裂解細(xì)胞等屬性的基礎(chǔ)上,具有更強(qiáng)的磷脂親和力以及α螺旋結(jié)構(gòu)形成能力。與α-peptide相比,α-melittin具有更強(qiáng)大的粒徑控制力,能夠?qū)⒘字纬傻募{米顆粒粒徑控制在10nm左右甚至更小,同時(shí)具有很高的包封率(80%)。 (2)利用α-melittin合成的脂質(zhì)納米顆粒α-melittin-NP,實(shí)現(xiàn)了活體內(nèi)蜂毒肽的安全有效遞送。α-melittin-NPs是一種粒徑在14nm左右,分散性良好的球形納米顆粒,其核心可以同時(shí)運(yùn)載疏水性分子。α-melittin-NPs性質(zhì)穩(wěn)定,在血清和血漿等生理溶液中仍然保持穩(wěn)定的納米結(jié)構(gòu)。憑借蜂毒肽與α-peptide羧基端疏水性氨基酸的連接設(shè)計(jì)以及α-melittin與磷脂層的強(qiáng)力結(jié)合,α-melittin-NPs有效地隱藏了蜂毒肽的陽(yáng)離子電荷(21.33±1.64mV),表現(xiàn)為近中性納米顆粒(2.45±0.56mV)。α-melittin-NPs明顯降低了蜂毒肽的溶血性和非特異性破壞作用。體外溶血實(shí)驗(yàn)表明,α-melittin-NPs對(duì)血細(xì)胞的裂解作用顯著降低,即使在501μM的高濃度下也只有8%左右的溶血率。細(xì)胞增殖實(shí)驗(yàn)顯示,α-melittin-NPs對(duì)細(xì)胞生長(zhǎng)抑制的IC50值(11.26±1.37μM)較蜂毒肽(1.71±0.04μM)有顯著的提升。激光共聚焦連續(xù)成像的結(jié)果表明,α-melittin-NPs與黑色素瘤細(xì)胞接觸后造成細(xì)胞膜通透性改變,誘導(dǎo)細(xì)胞內(nèi)容物泄漏、細(xì)胞核分解,最終導(dǎo)致細(xì)胞死亡。Annexin V-FITC/PI的雙色標(biāo)記和流式分析結(jié)果表明,α-melittin-NPs導(dǎo)致腫瘤細(xì)胞死亡的主要途徑是發(fā)生壞死,也能誘導(dǎo)少量細(xì)胞發(fā)生凋亡。最后,α-melittin-NPs通過(guò)尾靜脈注射至皮下荷瘤鼠體內(nèi)。經(jīng)過(guò)4次給藥治療,黑色素瘤的生長(zhǎng)得到了明顯抑制,與對(duì)照組相比其抑制率高達(dá)82.8%。與此同時(shí),通過(guò)對(duì)治療小鼠血液的生理生化指標(biāo)的檢測(cè),以及對(duì)臟器的病理切片分析,并未顯示α-melittin-NPs對(duì)機(jī)體產(chǎn)生明顯的毒副作用。 (3)研制同步運(yùn)輸紫杉醇和蜂毒肽的超小納米顆粒(PTX-OL)α-melittin-NPs,具有協(xié)同抗黑色素瘤的作用。利用激光共聚焦顯微鏡觀察到α-melittin-NPs在與腫瘤細(xì)胞相互作用時(shí),會(huì)將蜂毒肽釋放至包括質(zhì)膜在內(nèi)的膜系統(tǒng)上,而核心內(nèi)的染料分子則被直接釋放至胞漿中。(PTX-OL)α-melittin-NPs在兩個(gè)治療機(jī)制上充分發(fā)揮了紫杉醇和蜂毒肽的抗腫瘤功能。腫瘤細(xì)胞增殖實(shí)驗(yàn)的結(jié)果顯示,(PTX-OL)α-melittin-NPs對(duì)黑色素瘤殺傷效率明顯增強(qiáng),IC50值是α-melittin-NPs的44.8%。Annexin V-FITC/PI雙色標(biāo)記和流式分析結(jié)果表明,相比于α-melittin-NPs,(PTX-OL)α-melittin-NPs能夠誘導(dǎo)更多細(xì)胞發(fā)生壞死,提升幅度為10-20%。而且,在活體黑色素瘤治療上,(PTX-OL)α-melittin-NPs同樣展現(xiàn)出增強(qiáng)的腫瘤生長(zhǎng)抑制效果。 綜上所述,本研究利用仿HDL多肽脂質(zhì)納米顆粒作為蜂毒肽的運(yùn)載體,通過(guò)將蜂毒肽隱藏在單層磷脂分子層內(nèi),有效地解決了其在體內(nèi)運(yùn)輸中存在的溶血性和非特異性作用的問(wèn)題,提高了蜂毒肽在體內(nèi)的循環(huán)半衰期,為在活體內(nèi)使用蜂毒肽治療腫瘤提供了有效的工具。采用蜂毒肽與化療藥物協(xié)同配合的方法,將會(huì)突破單一靶標(biāo)在腫瘤治療上的限制,為腫瘤的多靶點(diǎn)治療帶來(lái)了新的機(jī)遇。
[Abstract]:The bee venom peptide, as the main component of bee venom, is an amphiphilic polypeptide with a polar helix structure. The bee venom peptide has wide application in the treatment of diseases, and has the effects of resisting inflammation, relieving pain, resisting bacteria, resisting virus, resisting radiation and lowering blood pressure, and the like. The bee venom peptide has the unique advantages of being applied to the treatment of the tumor, and the cell lysis and death can be caused by punching on the plasma membrane, so that the drug resistance problem of the tumor is avoided. However, the problems of haemolytic, non-specific and easy to be metabolically degraded by the bee venom peptide have seriously hindered their clinical application in the field of anti-tumor. It is still a challenge to how to safely and effectively transport the bee venom peptide to the tumor tissue without any toxic and side effect. The method has the advantages of effectively solving the hemolysis problem of the bee venom peptide in the treatment of the in-vivo tumor and prolonging the cycle period in the body, and realizing the specific release in the tumor region, The following results were mainly obtained: (1) The invention provides a novel functional hybrid polypeptide-melitti containing a bee venom peptide, N. It is by covalent attachment of a bee venom peptide to an apoprotein mimic peptide of a polar helix. The method has the advantages that, on the basis of the properties such as the spontaneous formation of the multimer and the lysing cell of the bee venom peptide, the antigen-melittin has stronger phospholipid affinity and the formation energy of the dendritic spiral structure, L-melittin has a more powerful particle size control, and the particle size of the nano-particles formed by the phospholipid can be controlled to be about 10 nm or less, and has a very high encapsulation rate (80%, compared to the P-peptide. and (2) using the lipid nano-particles I-melittin-NP synthesized by the chitosan-melittin to realize the safety of the bee venom peptide in the living body, It is a spherical nano-particle with a particle size of about 14 nm and a good dispersivity, and its core can carry water at the same time. Stable, stable in physiological solutions, such as serum and plasma. The cationic charge (21.33-1.64 mV) of the bee venom peptide (21.33-1.64 mV) was effectively hidden by the connection design of the hydrophobic amino acid and the strong binding of the antigen-melittin with the phospholipid layer. MV). The antigen-melittin-NPs significantly reduce the hemolytic and non-specific rupture of the bee venom peptide. The results of in vitro hemolysis showed that the lysis of the blood cells was significantly reduced, even at a high concentration of 501. m u.M, even at a high concentration of 501. m The results of cell proliferation showed that the IC50 value of the inhibition of cell growth (11.26-1.37. mu.M) was significantly higher than that of the melittin (1.71-0.04. mu.M). The results of laser confocal continuous imaging show that the cell membrane permeability changes after contact with melanoma cells, inducing the leakage of the cell contents and the decomposition of the nucleus, leading to the fine formation of the cell membrane. The results of two-color labeling and flow analysis of Annexin V-FITC/ PI showed that the main route of tumor cell death was necrosis, and a small number of cells could be induced by the two-color labeling and flow analysis of Annexin V-FITC/ PI. Apoptosis. Finally, the antigen-melittin-NPs was injected into the subcutaneous tumor by the tail vein. The growth of melanoma was significantly inhibited after 4 administration, and the inhibition rate was as high as 82 in the control group. 8%. At the same time, by the detection of the physiological and biochemical indexes of the blood of the treated mice, and the analysis of the pathological sections of the organs, it is not shown that the antigen-melittin-NPs is toxic to the body. Side effects. (3) Preparation of ultra-small nanoparticles (PTX-OL)1-melittin-NPs for simultaneous transport of paclitaxel and bee venom, with synergistic anti-black The role of the antigen-melittin-NPs in the interaction with the tumor cells is observed by a laser confocal microscope, and the bee venom peptide is released to the membrane system, including the plasma membrane, while the dye molecules in the core are directly released. (PTX-OL)-Melittin-NPs played the role of paclitaxel and melittin in two treatment mechanisms. The results of the experiment of tumor cell proliferation showed that (PTX-OL)-melittin-NPs increased the killing efficiency of melanoma and the IC50 value was 44.8% of the antigen-melittin-NPs. The Annexin V-FITC/ PI two-color marker and the flow analysis showed that, compared with the P-melittin-NPs, (PTX-OL)-melittin-NPs can induce more fine. Necrosis of the cell, the lifting amplitude is 1 0-20%. Also, in the treatment of live melanoma, (PTX-OL)--melittin-NPs also exhibit enhanced tumor growth To sum up, the present study makes use of the HDL-polypeptide lipid nanoparticles as the carrier of the bee venom peptide, and effectively solves the hemolytic and non-special effects of the bee venom peptide in the in-vivo transportation by hiding the bee venom peptide in the single-layer phospholipid molecule layer. The problem of the opposite sex effect improves the circulating half-life of the bee venom peptide in the body, and is a method for treating the tumor by using the bee venom peptide in the living body. The method of the invention can break through the limitation of the single target in the treatment of the tumor, and is a multi-target therapy for the tumor,
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R943;R96

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