【作者】 茹琴；

【导师】 甄永苏； 李电东； 邵荣光；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2010， 博士

【摘要】 力达霉素(Lidamycin, LDM, C-1027)是由球孢链霉菌(Streptomyces globisporus)产生的烯二炔类抗生素,是迄今报道过的对肿瘤细胞杀伤作用最强的大分子肽类抗肿瘤抗生素,具有很强的细胞毒性、抗血管生成活性及抑制实验肿瘤生长的作用。LDM的分子由烯二炔结构的发色团(AE)和辅基蛋白(LDP)构成,两者可进行拆分和重建,以往的研究表明发色团是力达霉素分子的活性部分,辅基蛋白具有稳定和保护发色团的功能。辅基蛋白是否具有抗肿瘤活性,目前还没有定论。神经胶质瘤是最常见的原发性脑肿瘤,是高度血管化的血管生成依赖性生长的人类肿瘤。目前的报道表明神经胶质瘤的血管微环境在神经胶质瘤发生与发展、侵袭、临床诊断及治疗效果中起关健性作用。由于血脑屏障的存在,限制了大部分抗血管生成药物在神经胶质瘤治疗方面的应用。细胞穿透肽(cell penetrating peptides, CPPs)是一类以非受体依赖方式、非经典内吞方式直接穿过细胞膜进入细胞的多肽,它们的长度一般不超过30个氨基酸且富含碱性氨基酸,能够介导与其相连的各种本不能通过细胞膜的生物活性物质进入细胞。迄今的研究提示CPPs能在体外或体内介导一系列生物活性分子如蛋白质、肽类、寡聚核苷酸、质粒及脂质体等进入各种不同组织和细胞,这些重要发现为将亲水性蛋白、寡肽及寡核苷酸应用于药学、基因治疗、细胞生物学等研究领域开辟了一条新的道路。本研究利用本室构建表达的力达霉素重组辅基蛋白rLDP,研究其对多种肿瘤细胞的体外亲和活性及其体内外抗肿瘤活性,通过分子重建制备重组力达霉素(rLDM),研究其与正常力达霉素的结构和活性异同；利用基因工程方法构建表达辅基蛋白与细胞穿透肽(Arg)9的融合蛋白(Arg)9-LDP,对其穿膜能力和抗肿瘤活性进行研究,再通过分子强化技术组装发色团AE,得到强化融合蛋白(Arg)9-LDP-AE,研究其对神经胶质瘤细胞的体外杀伤作用以及体内抑瘤活性。1.重组力达霉素的制备及活性分析利用本室保存的含有pET30-sldp质粒的E.coli BL21(DE3)starTM菌株,IPTG诱导带有组氨酸标签肽(His-tag)的重组辅基蛋白的表达。重组辅基蛋白rLDP分子量约为12 kDa,除存在于周质腔中外,还分泌到培养液上清中。Ni2+亲和层析对培养液上清的蛋白进行纯化,得到的目的蛋白经SDS-PAGE电泳检测纯度在98%以上,每升发酵液得到23 mg活性蛋白。细胞免疫荧光和基于流式细胞术的荧光实验结果显示rLDP蛋白对肿瘤细胞,如人肺癌A549细胞、人卵巢癌OVCAR3细胞、人肝癌Bel-7402细胞、人神经胶质瘤U87细胞、人非小细胞肺癌H460细胞均有很强的结合活性,而对正常肝组织来源的L02细胞则无亲和活性。细胞增殖实验证明rLDP蛋白可抑制Bel-7402细胞的增殖。流式细胞仪检测结果表明rLDP蛋白可引起Bel-7402细胞周期的G2／M期阻滞,且存在浓度依赖性。体内抑瘤实验表明rLDP蛋白能够显著抑制小鼠肝癌H22移植瘤的生长,且毒副作用较小。rLDP蛋白静脉注射15 mg/kg、30 mg/kg和60 mg/kg 3个剂量组11天的抑瘤率分别为44.6%、57.1%和50.1%,与对照组相比有显著差异；rLDP蛋白腹腔注射30mg/kg、60mg/kg和120 mg/kg 3个剂量组11天的抑瘤率分别为52.1%、49.3%和59.5%,与对照组相比有显著差异；rLDP蛋白灌胃给药30 mg/kg、60 mg/kg和120 mg/kg 3个剂量组11天的抑瘤率分别为34.3%、30.2%和60.3%,与对照组相比有显著差异。将AE分子与rLDP蛋白在体外进行分子组装后,经HPLC检测在350 nm处出现了特定的吸收峰,表明AE分子与rLDP蛋白进行了成功组装,得到了重组力达霉素rLDM。MTT试验结果显示重组力达霉素rLDM对不同的肿瘤细胞系均表现出了强烈的杀伤作用,其对U87细胞、MCF-7细胞、SKOV3细胞和Bel-7402细胞的IC50值与力达霉素LDM接近。流式细胞仪检测结果表明rLDM和LDM在浓度为1 nM时引起U87细胞和SKOV3细胞周期的S期阻滞和G2／M期阻滞,在浓度为0.001 nM时SKOV3细胞出现G2／M期阻滞,U87细胞出现G1期阻滞。2.细胞穿透肽(Arg)9与力达霉素融合蛋白(Arg)9-LDP的构建及活性分析利用基因重组技术扩增含有细胞穿透肽(Arg)9和力达霉素辅基蛋白LDP基因的(arg)9ldp片段,连接到质粒pET30a(+)上,构建重组质粒pET30-(arg)9ldp,转化到E.coli BL21(DE3)starTM中,IPTG诱导带有组氨酸标签肽(His-tag)的融合蛋白的表达。融合蛋白分子量约为15 kDa,主要以包涵体形式存在。Ni2+亲和柱纯化融合蛋白,得到的目的蛋白经SDS-PAGE电泳检测纯度在95%以上,每升发酵液得到15 mg活性蛋白。细胞荧光检测和基于流式细胞术的荧光实验结果显示带有穿透肽的融合蛋白可以穿透细胞膜进入细胞内部,与重组辅基蛋白和牛血清白蛋白相比有明显差异,其在37℃的穿透能力优于4℃,说明穿透过程与能量有关。细胞增殖实验证明融合蛋白可抑制肿瘤细胞的增殖,其对U87细胞的IC50值为5.25×10-5mol／L。流式细胞仪检测结果表明20μM的融合蛋白可引起U87细胞周期的G1期阻滞,浓度为2001μM时引起U87细胞周期的G2／M期阻滞和S期阻滞。3.细胞穿透肽(Arg)9与力达霉素强化融合蛋白(Arg)9-LDP-AE的制备及活性分析将含有细胞穿透肽和力达霉素辅基蛋白的融合蛋白(Arg)9-LDP与力达霉素活性发色团组装,经HPLC检测在350 nm处出现了特定的吸收峰,表明强化融合蛋白(Arg)9-LDP-AE组装成功。细胞增殖实验证明强化融合蛋白对肿瘤细胞具有强杀伤作用,强化融合蛋白和力达霉素对U87细胞和MCF-7细胞的IC50值相近,强化融合蛋白对OVCAR3细胞和Bel-7402细胞的IC50值分别是力达霉素的1／20和1／10。流式细胞仪检测结果表明强化融合蛋白可引起U87细胞周期的G2／M期阻滞和S期阻滞,Annexin V-FITC/PI双染结合流式细胞仪结果表明强化融合蛋白以剂量依赖方式诱导细胞凋亡。体内抑瘤实验表明强化融合蛋白(Arg)9-LDP-AE对肝癌H22小鼠移植瘤具有明显的抑制作用,且毒副作用较LDM小。强化融合蛋白0.2 mg/kg和0.3 mg/kg剂量组抑瘤率分别为85.3%和89.2%,与LDM0.05 mg/kg组相比有明显提高。(Arg)9-LDP-AE对人神经胶质瘤U87细胞形成的实体瘤有明显的抑制作用,28天时0.1 mg/kg和0.2mg/kg 2个剂量组抑瘤率分别为79.2%和88.8%,与LDM组存在显著差异(P<0.01)。更多还原

【Abstract】 Lidamycin (LDM, C-1027), a macromolecular peptide antibiotic produced by Streptomyces globisporus, showed extremely potent cytotoxicity against tumor cell lines, anti-angiogenic activity and marked growth inhibition of transplantable tumors in mice. LDM consists of an active enedinye chomophore (AE) and an apoprotein (LDP), which can be separated and reconstituted without losing its activity. The primary role of LDP is as a packing carrier protein for the chemically unstable chromophore, however, the antitumor activity of LDP remains unidentified.Glioma, the most common primary brain tumor, is highly vascularized and angiogenesis-dependent. Current reports show that the vascular microenvironment of glioma plays a central role in glioma progression, invasion, diagnosis and the effectiveness of clinic treatment. Because of blood-brain barrier, the application of antiangiogenesis drugs in glioma therapy is limited.Cell-penetrating peptides (CPPs) are short peptides of less than 30 amino acids that are able to penetrate cell membranes and translocate different cargoes into cells. The mechanism of cell translocation is not known but it is apparently receptor and energy independent, although, in certain cases, translocation can be partially mediated by endocytosis. Cargoes that are successfully internalized by CPPs range from small molecules, oligonucleotide to proteins, bangosome and supramolecular particles. CPPs are novel vehicles for the translocation of cargo into cells, their properties make them potential drug delivery agents of interest for future use.In this study, recombinant lidamycin apoprotein rLDP was prepared and recombinant lidamycin rLDM was reconstituted, the affinity activity of rLDP to tumor cells and the antitumor activity of rLDP and rLDM was observed. Fusion protein (Arg)9-LDP was prepared and energized fusion protein (Arg)9-LDP-AE was reconstituted. The membrane translocation ability of fusion protein and the potent cytotoxicity of fusion protein and its energized protein were observed.1. Preparation of recombinant lidamycin and the study of its activityE.coli BL21 (DE3) starTM cells which has the recombinant plasmid pET30-sldp were preserved in our laboratory. The recombinant lidamycin apoprotein rLDP with His-tag was successfully secreted into the culture medium and periplasmic space of E.coli after IPTG inducing. rLDP was purified by Ni2+ affinity chromatography and the purity of fusion proteins was all over 98%as determined by SDS-PAGE. Finally about 23 mg of purified protein was obtained from 1 L of culture medium. The immunofluorescent cytochemical staining and FACS-based fluorescence assay showed that rLDP has strong binding activity to cancer cell lines, such as human lung carcinoma A549 cells and H460 cells, human ovarian carcinoma OVCAR3 cells, human glioma U87 cells, and human hepatoma Bel-7402 cells. However, rLDP has no binding activity to L02 cells came from normal hepatic tissue. The results of MTT assay proved that rLDP displayed cytotoxicity to Bel-7402 cells with IC50 value of 7.05×10-5 mol/L. FACS analysis of cell cycle showed that the cells were arrested in G2/M phase after rLDP treatment, and the degree of arrest was concentration dependent. Transplantable hepatoma 22 (H22) in Kunming mice was used to investigate the inhibitory effects of rLDP. Evidently, rLDP suppressed H22 tumor growth. The inhibition rates of rLDP at dose of 15 mg/kg,30 mg/kg and 60 mg/kg were 44.6%,57.1%and 50.1%respectively for intravenous injection. The inhibition rates of rLDP at dose of 30 mg/kg,60 mg/kg and 120 mg/kg were 52.1%,49.3%and 59.5%respectively for intraperitoneal injection. The inhibition rates of rLDP at dose of 30 mg/kg,60 mg/kg and 120 mg/kg were 34.3%, 30.2%and 60.3%respectively for intragastric administration. The recombinant lidamycin rLDM was prepared by the reconstitution of rLDP and AE. The results of MTT assay showed that the IC50 values of rLDM for SKOV3 cells, U87 cells, MCF-7 cells and Bel-7402 cells were close to those of LDM. FACS analysis of cell cycle showed that rLDM and LDM induced similar cell cycle arrest in SKOV3 cells and U87cells. Cells were both arrested in G2/M phase and S phase after rLDM and LDM treatment at 1 nM, SKOV3 cells were arrested in G2/M phase and U87cells were arrested in G1 phase after treatment at 0.001 nM.2. Construction of fusion protein (Arg)9LDP and the study of its activity(arg)9ldp gene was ligated into the plasmid pET-30a(+), then the recombinant plasmid pET30-(arg)yldp was transformed into competent E.coli BL21 (DE3) starTM cells. Fusion protein (Arg)9-LDP with His-tag was produced in the form of inclusion after IPTG inducing. (Arg)9-LDP was purified by Ni2+ affinity chromatography and the purity of fusion proteins was all over 95%as determined by SDS-PAGE. Finally about 15 mg of purified protein was obtained from 1 L of culture medium. The result of fluorescent cytochemical staining showed that (Arg)9-LDP could be transferred into tumor cells and distributed in cytoplasm for 2 h incubation. The FACS-based fluorescence assay revealed that the translocation of (Arg)9-LDP was influenced by temperature. The results of MTT assay proved that (Arg)9-LDP had moderate cytotoxicity to U87 cells with IC50 value of 5.25×10-5 mol/L. FACS ananlysis of cell cycle showed that the cells were arrested in G2/M phase and S phase after (Arg)9-LDP treatment at 200μM and G1 phase at 20μM. 3. Preparation of energized fusion protein (Arg)9-LDP-AE and the study of its antitumor activityThe energized fusion protein (Arg)9-LDP-AE was prepared by the reconstitution of (Arg)9-LDP and AE. The results of MTT assay proved that (Arg)9-LDP-AE had stronger cytotoxicity than LDM. The IC50 values of (Arg)9-LDP-AE for U87 cells and MCF-7 cells were close to those of LDM, the IC50 values of (Arg)9-LDP-AE was only 1/20 of that of LDM for OVCAR3 cells and 1/10 for Bel-7402 cells respectively. FACS ananlysis of cell cycle showed that the U87 cells were arrested in G2/M phase and S phase after (Arg)9-LDP-AE treatment, and the degree of arrest was concentration dependent. The results of Annexin V-FITC/PI assay indicated the potent and dose-dependent proapoptotic effect of (Arg)9-LDP-AE on U87 cells. In vivo, the energized fusion protein (Arg)9-LDP-AE showed stronger antitumor activity than LDM. The inhibition rates of (Arg)9-LDP-AE against murine hepatoma H22 at dose of 0.2 mg/kg and 0.3 mg/kg were 85.3%and 89.2%respectively, while that of LDM at 0.05 mg/kg was 74.6%. (Arg)9-LDP-AE at the dose of 0.1 mg/kg and 0.2 mg/kg inhibited the growth of human glioma U87 xenografts by 79.2%and 88.8%, while that of LDM at 0.05 mg/kg was 62.9%.更多还原