【作者】 王凤霞；

【导师】 吉爱国； 林秀坤；

【作者基本信息】 山东大学 ， 微生物与生化药学， 2013， 博士

【摘要】 中华真地鳖(Eupolyphaga sinensis Walker),俗称土鳖、土元等,属于蜚蠊目(Blattodea),鳖蠊科(Corydiidae),地鳖亚科(Polyphaginae),真地鳖属(Eupolyphaga)昆虫,目前已实现大规模人工养殖。中华真地鳖作为一种传统中药,在我国具有悠久的药用历史。现代研究表明,中华真地鳖具有溶解血栓、抗凝血、抗肿瘤、促进骨折愈合、调节血脂等十分广泛的药理作用。对于中华真地鳖的研究,国外学者很少涉及,其研究近乎空白。近年来,国内学者研究表明中华真地鳖蛋白提取物能够抑制血管生成和肿瘤生长。可见,蛋白组分被认为是中华真地鳖的抗肿瘤有效组分。但是,到目前为止,尚未见中华真地鳖抗肿瘤蛋白成分的报导。我们利用现代生化分离技术,从中华真地鳖新鲜雌虫体中分离纯化得到一分子质量约为72kDa的抗肿瘤蛋白,命名为EPS72。该蛋白对人肝癌细胞株Bel-7402、肺癌细胞株A549等多种人癌细胞株表现出较强的增殖抑制作用。以A549细胞作为体外肿瘤模型,研究了EPS72对细胞形态、增殖、凋亡、粘附、伸展、迁移以及侵袭的影响,发现EPS72具有体外抗肿瘤转移活性,并初步探讨了EPS72体外抗肿瘤转移可能的分子机制。期望为EPS72进一步开发利用提供一定的线索,同时为抗肿瘤蛋白类药物的研究提供一些新的思路。本课题的主要研究内容及结果有以下几个方面：1中华真地鳖抗肿瘤蛋白的提取、纯化及鉴定首先采用硫酸铵分级沉淀(50%-80%饱和度)、超滤(截留分子质量为10kDa)和冷冻干燥技术获得蛋白粗提物(组分Ⅰ)。由组分Ⅰ得到抗肿瘤活性蛋白的纯化方案由捕获、中度纯化和精制三个步骤组成：(1)捕获：分别采用CM-Sepharose FF阳离子交换色谱和DEAE-Sepharose FF阴离子交换色谱对抗肿瘤有效活性组分进行捕获；(2)中度纯化：分别采用Q-Sepharose HP阴离子交换色谱和Butyl Sepharose HP疏水色谱进行中度纯化；(3)精制：采用Superdex75凝胶过滤色谱进行精制。整个纯化过程中采用280nm检测蛋白质洗脱情况,分别收集各洗脱峰,采用MTT法追踪抗肿瘤活性组分,最后得到的活性组分进行冷冻干燥即为纯化所得抗肿瘤蛋白(组分Ⅵ)。每1kg新鲜活虫材料大约可得到电泳纯的抗肿瘤蛋白56mg,蛋白得率0.0056%。活性组分Ⅵ在SDS-PAGE上显示为单一条带,表观分子质量约为72kDa,表明其达到了电泳纯;MALDI-TOF质谱结果表明其分子质量为71.737kDa,与SDS-PAGE结果基本吻合,说明纯化得到的抗肿瘤蛋白为单链蛋白,分子量约为72kDa,故命名为EPS72。2EPS72体外抗肿瘤活性研究MTT存活分析表明EPS72对肝癌Bel-7402细胞、肺癌A549细胞等多种人癌细胞株表现出较强的增殖抑制作用。接着以A549细胞作为体外肿瘤模型,研究了EPS72对细胞形态、凋亡、粘附、伸展、迁移以及侵袭的影响。相差显微镜下细胞形态学研究、台盼蓝染色计数、AO/EB染色以及JC-1检测细胞膜电位(△Ψm)结果表明,EPS72作用早期可诱导已贴壁生长的肿瘤细胞脱粘附,但是刚脱粘附细胞为胞膜完整的活细胞,撤药后能正常生长,而脱粘附细胞持续受药物作用后则会进一步诱导细胞凋亡性死亡。可见,EPS72诱导肿瘤细胞脱粘附的作用是直接的,而诱导细胞凋亡的作用是间接的。细胞粘附实验进一步表明,EPS72能够显著抑制肿瘤细胞向细胞外基质(ECM)蛋白成分纤粘连蛋白(fibronectin,FN)和Ⅳ型胶原蛋白(collagen Ⅳ,Col Ⅳ)的粘附,但对细胞向非ECM成分多聚赖氨酸(poly-L-lysine, PL)的粘附却没有影响。癌细胞体外伸展实验结果表明,EPS72能够抑制肿瘤细胞在ECM表面上伸展。创伤愈合实验表明EPS72能够阻止肿瘤细胞向创伤面迁移。Transwell实验进一步证明EPS72能够影响肿瘤细胞向Matrigel的侵袭能力。可见,EPS72通过影响肿瘤细胞的粘附、伸展、转移、侵袭及增殖、存活能力发挥体外抗肿瘤作用,EPS72具有体外抗肿瘤转移活性。3EPS72体外抗肿瘤作用的分子机制为了进一步探讨EPS72体外抗肿瘤转移的分子机制,利用荧光显微镜采用免疫荧光法研究了EPS72对细胞肌动蛋白骨架成分F-actin的影响；采用流式细胞仪和Western blotting技术研究了EPS72作用后肿瘤细胞β1-整合素和整合素关联蛋白激酶(ILK)表达情况。结果表明,EPS72作用后F-actin的聚合受到抑制,肿瘤细胞p1-整合素和ILK的表达受到抑制,推测EPS72可能通过直接靶向肿瘤细胞表面的β1-整合素,进而影响整合素—细胞骨架信号通路发挥体外抗肿瘤转移作用。本研究取得的成果和结论主要有：(1)采用现代生化分离技术,首次从中华真地鳖新鲜雌虫体中分离纯化得到一种分子量约为72kDa的抗肿瘤蛋白,命名为EPS72。该分离纯化工艺条件温和,操作简单,并易于规模化放大生产。(2)体外研究表明,EPS72对人肝癌Bel-7402细胞、肺癌A549细胞等人癌细胞株表现出较强的胞毒活性,而对非癌细胞株MRC5的细胞毒性较低,显示该活性蛋白具有较强的广谱抗肿瘤活性,且对肿瘤细胞显示出良好的选择性。(3)确定了EPS72通过影响肿瘤细胞的粘附、伸展、转移、侵袭及增殖、存活能力发挥体外抗肿瘤作用,EPS72具有体外抗肿瘤转移能力。(4)初步推测EPS72可能通过直接靶向肿瘤细胞表面的β1-整合素,进而影响整合素—细胞骨架信号通路发挥体外抗肿瘤转移作用。更多还原

【Abstract】 Eupolyphaga sinensis Walker (Blattaria:Corydiidae), popularly known as’Tubie’, is a well-known edible and medicinal insect in China and now has been artificially propagated on a large scale. In Chinese folk and traditional medicine, it is widely used as a natural healthy product to prevent and treat many diseases, including bone injury, cancer and immune-related diseases, etc. In recent years, it has been reported that E. sinensis has antithrombotic, antitumor, immune-protective and antioxidant activities. The antitumor and thrombolytic activities are the most attractive among these bioactivities. Previous studies on the antitumor activities showed that the crude protein extracts from E. sinensis could inhibit angiogenesis and tumor growth in vivo. However, the components responsible for its activity have not been addressed, and no purified active protein fraction has been obtained from this species.In this study, we purified a72kDa anticancer protein, designated as EPS72, from the fresh female E. sinensis using a line of purification techniques. EPS72showed potent cytotoxicity against the human liver cancer Bel-7402, lung cancer A549cell lines, etc. To gain insight into the potential antitcancer mechanism of this protein, we further investigated its effects on cellular morphology, proliferation, apoptosis, adhesion, spreading, migration and invasion of A549cells. We found that EPS72showed an antimetastatic activity in vitro. The probable molecular mechanism underlining its effects on A549cancer cells was also studied. All the results of the anticancer protein were summarized as follows:1Isolation, purification and identification of the E. sinensis anticancer proteinThe crude extracts (Fraction I) were obtained by ammonium sulfate precipitation (50%～80%saturation), ultra-filtration (10kDa cut-off) and lyophilization. The anticancer protein fraction(s) was/were purified using three phases Capture, Intermediate Purification and Polishing:(1) Capture:CM-Sepharose FF cation-exchange chromatography (CIEX) and DEAE-Sepharose FF anion-exchange chromatography (AIEX) were used;(2) Intermediate Purification:Q-Sepharose HP anion-exchange chromatography (AIEX) and Butyl Sepharose HP hydrophobic chromatography (HIC) were used;(3) Polishing:Superdex75gel filtration chromatography (GF) was used. During the separation and purification process, the protein elution profile was monitored at280nm and MTT assay was performed to monitor the cytotoxicity of each elution fraction. The final active fraction (Fraction Ⅵ) was lyophilized to obtain a purified anticancer protein. The recovery of the purified anticancer protein was about0.0056%.The Fraction VI appeared as a single band on SDS-PAGE, with an apparent molecular mass of about72kDa. Its accurate molecular mass was71.737kDa as determined by MALDI-TOF mass spectrometry analysis. The results suggested that after performing several steps of purification, a pure72-kDa single-stranded protein with anticancer activity in vitro, named EPS72, was obtained.2Antitumor activities in vitro of EPS72The results of MTT assay indicated that EPS72displayed potent cytotoxicity against the human liver cancer Bel-7402, lung cancer A549cell lines, etc. We further investigated its effects on cellular morphology, proliferation, apoptosis, adhesion, spreading, migration and invasion using the human lung cancer A549cell line as a tumor model in vitro. The results of microscopic morphological observation, re-culture when withdrawn EPS72, trypan blue staining, JC-1staining to detect mitochondrial membrane potential (ΔΨm) and adridine orange/ethidium bromide(AO/EB) staining of A549cells indicated that EPS72could induce cell detachment in the early stage, which further led to cell apoptosis in the late stage. But the initially detached cell clusters induced by EPS72remained viable, suggesting that EPS72had a direct role on cell adhesion and indirect role on cell apoptosis.5μg/ml of EPS72remarkably inhibited A549cells attachment to fibronectin (FN) and collagen IV (Col IV), but could not inhibit A549cells adhesion to poly-L-lysine (PL) in a cell adhesion assay, could affect spreading ability of A549cells on ECM gel in a time-dependent mode in an spreading assay in vitro, and could inhibit A549cells migration into the wounded areas in a wound healing assay. In a Boyden chamber assay, EPS72was found to inhibit the invasive activities of A549cancer cells in a dose-dependent mode. These results suggested that EPS72exerted its antitumor activities in vitro by affecting adhesion, spreading, migration, invasion, proliferation and survival of cancer cells and EPS72has an antimetastatic activity in vitro.3Possible molecular mechanism underlining its antitumor effects in vitro of EPS72In order to further investigate the possible molecular mechanism underlining the antimetastatic effects in vitro of EPS72, we examined the effect of EPS72on F-actin with an Actin-tracker Green fluorescent probe under a fluorescence microscope and performed flow cytometry and Western blot analysis to detect the expression of β1-integrin and/or integrin-linked kinase (ILK) in A549cells. The results indicated that EPS72could induce depolymerization of F-actin and down-regulate the expression of β1-integrin and ILK in A549cells, suggesting that EPS72exerted the antimetastatic effects in vitro probably by directly targeting β1-integrin, then further influencing the integrin-cytoskeleton signaling pathway.Major achievements and conclusions of the article are as follows:(1) We purified a72-kDa anticancer protein (EPS72) from E. sinensis by using ammonium-sulfate-precipitation, ultra-filtration, CIEX-AIEX-HIC-GF isolation and purification protocol, which is mild, easily operational and easy-to-scaling-up. The purified protein, to our knowledge, is the first purified anticancer protein from this species.(2) In an in vitro study, EPS72was found to display potent cytotoxicity against the human liver cancer Bel-7402, lung cancer A549cell lines, etc., but the normal cell line human fibroblast cell MRC5was not very sensitive to the treatment of EPS72, suggesting EPS72exhibited relatively broad antitumor activities in vitro and showed some specificity to human cancer cells.(3) We found that EPS72exerted its antitumor effects in vitro by affecting adhesion, spreading, migration, invasion, proliferation and apoptosis of cancer cells, and EPS72has an antimetastatic activity in vitro.(4) We tentativly concluded that EPS72exerted the antimetastatic effects in vitro probably by directly targeting β1-integrin, then further influencing the integrin-cytoskeleton signaling pathway.更多还原