摘要
背景
肿瘤是严重威胁人类健康的一类疾病,寻找有效的抗肿瘤药物与方法是世界医学界的重要研究课题。研究表明许多中药具有抗肿瘤作用,但其作用成分复杂,具有多种作用成分和通过多种作用途径产生抗肿瘤作用。弄清中药的抗肿瘤作用途径和靶位点对加速抗肿瘤中药的开发具有重要意义。近50年来尽管肿瘤治疗取得了一些进展,化疗、放疗、手术仍是治疗基本手段。但是由于现有的化疗药物和放疗的选择性不高,杀伤肿瘤细胞的同时也损害体内正常细胞,治疗中常出现较明显的毒副反应,所以研究、开发一种对肿瘤细胞选择性高、杀伤作用强但对正常组织副作用小的抗肿瘤药物非常有意义。免疫毒素是提高治疗效果的一条重要途径,是新一代的癌症治疗方法,此疗法与传统的化疗不同,其药物能在肿瘤部位有相对较高的浓度,存留较长时间,对肿瘤靶细胞有较强的杀伤活性,而对正常细胞无作用或毒性很小,免疫毒素是靶向性治疗肿瘤的有代表性的药物,也是免疫学研究的热点之一,被称为“生物导弹”,近年来利用基因工程技术构建了不同用途的免疫毒素。免疫毒素是具有导向能力的分子(载体)和具有细胞毒性的分子(毒素)构成的具有特异性细胞杀伤能力的杂合分子。载体一般由抗体、细胞因子和激素等组成,毒素一般为来自于细菌或植物。载体可以把毒素定向带到病灶部位,将癌细胞或病变细胞杀死,而很少伤害正常细胞。研究表明,免疫毒素在体内具有靶向性,使毒素更易于与肿瘤细胞结合,大大提高了毒素对肿瘤细胞的杀伤率,降低了其对正常细胞的毒副作用。
天花粉蛋白(trichosanthin,TCS)是从葫芦科植物栝蒌的根块中提取出来的一种碱性蛋白,属于Ⅰ型核糖体失活蛋白(ribosome inactivating proteins,RIPs)。天花粉蛋白临床主要用于引产及宫外孕、葡萄胎、绒癌等的治疗,另外使核糖体不可逆失活,从而抑制蛋白质的合成,其应用范围已涉及抗病毒、抗肿瘤等方面,同时可抑制艾滋病病毒在感染的免疫失活细胞内复制繁衍,此外还发现在培养的细胞系统中,TCS对乙脑、乙肝、麻疹、单纯疱疹及水泡性口腔炎等病毒均有明显抑制作用,并且这种抑制作用与TCS的浓度呈正相关。但是由于天花粉蛋白在临床上使用所产生的副作用,限制了它进一步的推广和应用,我们可以考虑如何在TCS不引发过敏反应的剂量内,最大限度的发挥其抗肿瘤作用,这就需要我们找到一种能与TCS一起最大限度发挥协同杀伤肿瘤细胞的物质。
表皮生长因子受体(epidermal growth factor receptor,EGFR)的异常高表达可见于多种恶性肿瘤,并与肿瘤细胞的恶性生物学行为以及肿瘤患者的不良预后密切相关。EGFR已成为阳性表达肿瘤的重要治疗靶标。以EGFR为靶点的治疗主要有单克隆抗体以及小分子化合物激酶拮抗剂两种方式,目前已进行了广泛的实验与临床研究,其抗肿瘤的主要作用机制包括:细胞周期阻滞、促凋亡、抗肿瘤浸润与转移、抗新生血管生成、放化疗细胞毒增敏等。此外,以人EGFR为免疫原在小鼠体内可以激发特异的免疫反应,该反应能交叉反应于鼠同源EGFR,进而达到治疗EGFR阳性表达肿瘤的目的。迄今,EGFR靶向治疗均很少观察到显著的EGFR相关的毒副作用,这可能与正常细胞EGFR显著的调控性低表达以及对EGFR通路信号的相对不依赖性相关。
为此,本研究以传统中药天花粉蛋白为毒素,人表皮生长因子为载体,利用基因工程技术,制备重组免疫毒EGF-Linker-TCS,并对其靶向性抗肿瘤作用给予评价,为中医药在抗肿瘤方面的应用进行了有益的探索,对促进中药开发、中药现代化具有一定的意义。
目的
1、因天花粉蛋白成分复杂,纯化步骤繁琐,得率低,目前构建的携带有天花粉蛋白基因的表达载体其表达水平较低,并且费用昂贵,为后续的研究带来困难。本实验拟通过基因工程技术,摸索出可以高效、稳定、简单、廉价的制备高纯度融合蛋白TCS、EGF-TCS、EGF-Linker-TCS的方法。
2、对正常人肝LO-2细胞、肝癌BEL-7402细胞、乳腺癌MCF-7细胞和胃腺癌BGC-823细胞表面EGF受体表达量进行检测,探讨EGF作为靶向抗肿瘤免疫毒素载体的可能性。
3、通过结晶紫法,计算TCS、EGF-TCS、EGF-Linker-TCS对正常人肝LO-2细胞、肝癌BEL-7402细胞、乳腺癌MCF-7细胞和胃腺癌BGC-823细胞的半数抑制有效浓度(IC_(50))及其选择指数、应用流式细胞仪对TCS、EGF-TCS、EGF-Linker-TCS选择性诱导细胞凋亡的研究,对TCS、EGF-TCS、EGF-Linker-TCS的体外靶向抗肿瘤作用给予评价。
4、研究免疫毒素EGF-Linker-TCS对建立荷人肝癌BEL-7402细胞的裸鼠模型,研究肿瘤生长抑制及移植肿瘤的肝转移情况,对EGF-Linker-TCS的体内靶向抗肿瘤作用给予评价。
方法
1、克隆、表达、纯化TCS、EGF-TCS、EGF-Linker-TCS融合蛋白
提取栝蒌基因组,使用PCR方法扩增TCS基因,将TCS基因插入到pQE30载体中,构建TCS的原核表达质粒pQE30/TCS;使用PCR方法扩增EGF基因,将EGF基因插pQE30/TCS中,构建原核表达质粒pQE30/EGF-TCS;考虑到融合蛋白中间加Linker可能会使两个蛋白更好的保持各自的构象,更好的保持各自的生物学活性,使用PCR方法扩增了EGF-Linker基因,将EGF-Linker基因插入pQE30/TCS中,构建了原核表达质粒pQE30/EGF-Linker-TCS,分别进行基因测序。将这三个重组质粒转化至M15工程菌中诱导表达,SDS-PAGE重组蛋白在M15工程菌中的表达,检测表达量占菌体蛋白总量。将鉴定的重组质粒pQE30/TCS、pQE30/EGF-TCS、pQE30/EGF-Linker-TCS转化至M15工程菌中,挑单菌落接种于含Amp50μg/ml LB培养基中,摇床培养,至OD值为0.6时,分别加入IPTG诱导表达,取样做SDS-PAGE分析。IPTG诱导大肠杆菌表达TCS、EGF-TCS、EGF-Linker-TCS 3h,超声波破碎菌体,离心后取上清液,用Ni-NTA Agrose亲和纯化,检测样品的纯度。
2、正常细胞和肿瘤细胞的EGF受体表达量的检测
EGF和TCS构成的融合蛋白靶向性抗肿瘤的靶点是EGF受体,使用流式细胞仪对正常细胞和肿瘤细胞表面的EGF受体表达量进行检测。
3、TCS、EGF-TCS、EGF-Linker-TCS对正常细胞及肿瘤细胞的半数抑制有效浓度(IC_(50))及其选择指数的确定
采用结晶紫法检测纯化的TCS、EGF-TCS、EGF-Linker-TCS对肿瘤细胞和正常细胞的细胞毒作用,测定其对细胞的半数抑制有效浓度(IC_(50))及其选择指数。按以下公式计算杀伤率:杀伤率(%)=[(对照孔A值-实验孔A值)/对照孔A值]×100%,通过计算细胞生长的抑制百分率,用origin 6.0软件计算出IC_(50),并根据IC_(50)值计算出选择指数:选择指数=IC_(50)(正常细胞)/IC_(50)(肿瘤细胞)。
4、流式细胞仪检测EGF-TCS、EGF-Linker-TCS选择性诱导细胞凋亡情况
EGF-TCS和EGF-Linker-TCS可以通过EGF受体结合肿瘤细胞,TCS可以使肿瘤细胞的核糖体失活,蛋白合成抑制而死亡。TCS除了核糖体失活,有可能还有其它机制导致真核细胞死亡,我们使用流式细胞仪观察EGF-TCS、EGF-Linker-TCS诱导LO-2、BEL-7402、MCF-7和BGC-823细胞的凋亡作用。
5、电镜观察EGF-Linker-TCS对肝癌BEL-7402细胞凋亡的影响
使用电镜观察EGF-Linker-TCS对肝癌BEL-7402细胞的影响,进一步研究EGF-Linker-TCS诱导细胞凋亡的机制。
6、EGF-Linker-TCS对裸鼠的肿瘤模型的体内抑瘤实验
分别给裸鼠右腋皮下接种人肝癌BEL-7402细胞,接种后第6天,尾静脉注射100μg/kg、50μg/kg和25μg/kg EGF-Linker-TCS进行治疗,设空白对照组,停药后第2天处死小鼠,称量瘤重,计算抑瘤率,取出肝脏计算肿瘤转移灶。肿瘤组织用10%甲醛固定做免疫组织化学实验,从而研究其抑制肿瘤生长、转移的途径。
结果
1、TCS、EGF-TCS、EGF-Linker-TCS融合蛋白的制备
对pQE30/TCS、pQE30/EGF-TCS和pQE30/EGF-Linker-TCS三个质粒分别进行基因测序,测序结果表明与设计一致;将这三个重组质粒转化至M15工程菌中,诱导表达,SDS-PAGE结果表明重组蛋白在M15工程菌中以可溶性形式表达,表达量均占菌体蛋白总量的30%。免疫印迹分析结果显示,重组蛋白可与特异性抗体发生特异性免疫反应。纯化的样品纯度均超过95%。
2、正常肝细胞和肿瘤细胞的EGF受体检测
使用流式细胞仪对正常肝细胞和肿瘤细胞的EGF受体进行了检测,检测结果显示正常肝LO-2细胞EGF受体阳性表达量为5.51%,肝癌BEL-7402细胞EGF受体阳性表达量为72.33%,乳腺癌MCF-7细胞EGF受体阳性表达量为63.92%,胃腺癌BGC-823细胞EGF受体阳性表达量为70.41%。肿瘤细胞的EGF受体数量明显高于正常细胞。
3、TCS、EGF-TCS、EGF-Linker-TCS对正常细胞及肿瘤细胞的半数抑制有效浓度(IC_(50))及其选择指数结果
结果显示:TCS对LO-2、BEL-7402、MCF-7和BGC-823细胞的IC_(50)分别为39.70μg/ml、33.37μg/ml、36.53μg/ml、41.52μg/ml,对BEL-7402、MCF-7和BGC-823细胞选择指数分别为1.19、1.09、0.96;EGF-TCS对LO-2、BEL-7402、MCF-7和BGC-823细胞的IC_(50)分别为115.34μg/ml、8.90μg/ml、13.75μg/ml、15.36μg/ml,对BEL-7402、MCF-7和BGC-823细胞选择指数分别为12.96、8.39、7.51;EGF-Linker-TCS对LO-2、BEL-7402、MCF-7和BGC-823细胞的IC_(50)分别为113.99μg/ml、6.98μg/ml、11.97μg/ml、13.53μg/ml,对BEL-7402、MCF-7和BGC-823细胞选择指数分别为16.33、9.52、8.43。实验的结果符合我们的设计,EGF-TCS和EGF-Linker-TCS对肿瘤细胞的IC_(50)要小于TCS,而对正常细胞的IC_(50)与TCS接近,EGF-TCS和EGF-Linker-TCS的选择指数高于TCS,均大于2,符合靶向药物的要求,EGF-Linker-TCS对肿瘤细胞的IC_(50)低于EGF-TCS,选择指数高于EGF-TCS。
4、EGF-TCS、EGF-Linker-TCS诱导肿瘤细胞凋亡作用
流式细胞仪检测结果显示:EGF-TCS对乳腺癌MCF-7细胞、胃腺癌BGC-823、肝癌BEL-7402细胞的凋亡率分别为35.61%、39.59%、50.57%,同样的剂量只能引起正常肝LO-2细胞少量的凋亡,凋亡率仅为8.99%,结果表明EGF-TCS(5μg/ml)具有诱导肿瘤细胞产生凋亡的作用;EGF-Linker-TCS(5μg/ml)也同样具有诱肿瘤细胞乳腺癌MCF-7细胞、胃腺癌BGC-823细胞、肝癌BEL-7402细胞凋亡的作用,凋亡率分别为:36.67%、42.56%、58.56%,同样的剂量只能引起正常肝LO-2细胞少量的凋亡,凋亡率仅为8.08%。
5、电镜观察EGF-Linker-TCS对BEL-7402细胞的影响
电镜下可见对照组细胞核清晰完整,有丰富正常的线粒体及内质网;EGF-Linker-TCS蛋白组出现细胞凋亡不同时期特征,染色质浓集,核不规则,可见空泡状内质网,凋亡小体。
6、EGF-Linker-TCS对荷人肝癌裸鼠肿瘤模型体内的抑瘤实验
为了进一步观察重组毒素的抗肿瘤效果,我们使用EGF-Linker-TCS重组蛋白对裸鼠的肿瘤模型进行了体内的抑瘤实验,结果表明EGF-Linker-TCS能明显的抑制裸鼠实体瘤的生长。高、中和低剂量组的抑制率分别为71.3%、60.87%和45.22%,对治疗结束后各组平均瘤重进行统计学分析,说明EGF-Linker-TCS能明显的抑制裸鼠实体瘤的生长,结果有显著性差异(F=8.712P=0.006);同时我们也观察了药物对移植肿瘤肝转移的抑制情况,解剖裸鼠检查肝脏,可见移植瘤转移到肝脏后呈现单个的白色病灶,对每只裸鼠的肝转移病灶计数后进行统计学分析,结果表明EGF-Linker-TCS能显著的抑制裸鼠移植瘤肝脏转移,结果有显著性差异(F=8.744 P=0.002);免疫组化结果显示EGF-Linker-TCS可以明显的抑制肿瘤血管的形成,高剂量组肿瘤组织未有可见血管,中低剂量组肿瘤组织血管明显少于模型组,其抑制肿瘤生长、转移的途径可能是通过抑制肿瘤血管生长来抑制肿瘤生长及转移。
结论
经上述研究,可以得出以下结论:
1、通过基因工程技术,摸索出了可以高效、稳定、简单、廉价的制备高纯度融合蛋白TCS、EGF-TCS、EGF-Linker-TCS的方法,为下一步研究其生物学功能奠定了基础。
2、在很多肿瘤细胞表面EGF受体过度表达,EGF可以特异地结合肿瘤细胞过度表达的EGF受体,可以用于免疫毒素的导向。
3、通过TCS、EGF-TCS、EGF-Linker-TCS对肿瘤细胞体外靶向杀伤作用的研究,证明EGF-TCS、EGF-Linker-TCS对肿瘤细胞具有良好的选择性,选择指数大于2,并且具有较高的细胞毒性,对正常细胞的细胞毒性要小于TCS。
4、免疫毒素EGF-Linker-TCS能明显抑制肝癌动物模型肿瘤生长及肿瘤的肝转移,说明EGF-Linker-TCS对实体瘤作用明显。
Background
Tumor is such a kind of diseases that seriously threaten human health. To find effective anti-tumor drugs and methods is an important research project in world medicine field. Research indicates that many Chinese traditional medicines have anti-tumor effects, but the components of them are complicated and their anti-tumor effects are caused by many components and many action pathways. So fully understanding the anti-tumor mechanism and tumor targeted site is important to accelerate the development of anti-tumor Chinese traditional medicines. Although some progress has been achieved since recent 50 years, chemotherapy、radiotherapy and surgical operation are still the main method for tumor therapy. There are obvious reactions of toxicity in treatment because the selectivity of existing chemotherapy and radiotherapy drugs is low and these drugs injure normal cells when they kill tumor cells. As a new generation of tumor therapy method , immunotoxin therapy is different with traditional chemotherapy. There is relatively high concentration of immunotoxin therapy drugs in tumor location and the retention time of these drugs is longer than that of chemotherapy drugs. Because the killing activity of immunotoxin drugs on tumor cells is strong and the toxicity of these drugs on normal cells is none or little, immunotoxin therapy is an important way to improve curing effect on tumor. Called "biomissile", immunotoxins are representative drugs in tumor-targeted therapy, and are one of hotspots in immunology research. Diverse uses of immunotoxins are constructed these years. Immunotoxins are special cell-killed molecular, composed of molecular of target and molecular of toxicity. The molecular of target is composed of antibody、cytokines and hormones, and toxins are derived from bacteria or plants. The molecular of target can take toxins to tumor focus and toxins can kill tumor cells but few normal cells. Research indicates that the target character of immunotoxins makes toxins easier to bind tumor cells, and to improve rate of killing tumor cells by toxins, and to lower toxicity effects on normal cells.
Trichosanthin( TCS ) is such a kind of alkaline protein that is extracted from a plant of bryony, and is type I ribosome inactivating protein. TCS can be mainly used in the treatment of odinopoeia, extrauterine pregnancy, hydatidiform mole and choriocarcinoma. TCS can also inreversiblely inactivate ribosome and inhibit protein synthesis, and its applications include anti-virus and in anti-tumor and other fields. TCS can inhibit HIV proliferation in HIV-infected immunity inactivated cells. It is also found that in cultured cells TCS can obviously inhibit many viruses such as encephalitis B virus, hepatitis B virus, measles virus, herpes simplex virus and vesicular stomatitis virus and this inhibition effect is positively related to TCS concentration. However, the side-effect and toxicity of TCS in clinic limit its further popularizing and application, so we may consider how to make the most of the anti-tumor effects of TCS on the concentration with the anaphylactic reaction not induced, and we should find a kind of material which can kill tumor cells mostly cooperated with TCS.
Exceptional high expressions of epidermal growth factor receptors( EGFR ) are observed in many malignant tumors, and are closely-related with malignant biology behaviors in tumor cells and poor prognosis in tumor patients. EGFR has been the important therapy target for EGFR-positive tumors. The EGFR-targeted therapies are mainly composed of two methods, monoclonal antibody and small molecular kinase antagonist. The main mechanisms of anti-tumor effects, which is undergoing widely experiments and clinical tests, mainly include cell cycle retardation effect, apoptosis promotion effect, anti tumor infiltration and migration effect, anti angiogenesis, more sensitive to cytotoxicity of chemotherapy and radiotherapy. On the other hand, as antigen, human EGFR can excite special immunity reaction in mouse, and there are also cross reactions between human EGFR antibodies and mouse homologous EGFR, through which we can cure EGFR-positive tumors, So far, little obvious EGFR-related toxicity in EGFR-targeted therapy has been observed, and it is perhaps because remarkable controlled low EGFR expression in normal cells and relative independence of EGFR pathway signal in normal cells.
In this respect, we used Chinese traditional medicine TCS as toxin, and used EGF as carrier, to prepare recombinant immunotoxin EGF-TCS, and evaluate its targeted anti-tumor effects. In these works we carried useful explorations in anti-tumor therapy using Chinese traditional medicines, and it is important for promoting development and modernization of Chinese traditional medicines.
Objective
1. The ingredient of TCS was complicated, and the steps of purification were complicated and the recovery rate was low. Currently TCS in expression vectors constructed was low expressed and expensive, which imposes difficulty for further research. We hope to obtain a method, which is effectively, stably, simply and cheaply, to prepare protein TCS, EGF-TCS and EGF-Linker-TCS with high purity by gene engineering.
2. We detected the EGF receptors on normal human liver cell LO-2, breast cancer cell MCF-7 and stomach adenocarcinoma cell BCG-823, and studied the feasibility of EGF as carrier of targeted anti-tumor Immunotoxins.
3. We evaluated targeted anti-tumor effects of TCS, EGF-TCS and EGF-Linker-TCS anti-tumor effects in vitro through calculating half inhibition concentration( IC_(50)) and selection index of TCS, EGF-TCS and EGF-Linker-TCS for normal human liver cell LO-2, hepatoma cell BEL-7402, breast tumor cell MCF-7 and stomach adenocarcinoma cell BCG-823 and researching on cell apoptosis selectively induced by TCS, EGF-TCS and EGF-Linker-TCS.
4. We carried research on tumor growth inhibition rate and liver migration in constructed hepatoma animal model. And we evaluated anti-tumor effects of EGF-Linker-TCS in vivo.
Methods
1. Cloning, Expression and Purification of recombinant fusion protein TCS、EGF-TCS、EGF-linker-TCS
The genome of Kuowei was extracted, and PCR was used to amplify TCS gene. The plasmid pQE30/TCS was constructed. The EGF gene was amplified by PCR , and was inserted into pQE30/TCS, and the plasmid pQE30/EGF-TCS was constructed. We considered that adding linker to the middle of fusion protein perhaps could make each of two proteins keep its own conformation better and keep its own activity better. Then EGF-Linker gene was amplified and was inserted to pQE30/TCS and the prokaryotic expression plasmid pQE30/EGF-Linker-TCS was constructed. The plasmid pQE30/TCS, plasmid pQE30/EGF-TCS and plasmid pQE30/EGF-Linker-TCS were sequenced .The three recombinant plasmids was transformed into bacteria M15. After induced, recombinant protein TCS,EGF-TCS and EGF-Linker-TCS were expressed in engineering bacteria M15, the expression rate of total bacteria protein which was analyzed by SDS-PAGE were examined. After identified the recombinant plasmid pQE30/TCS、pQE30/EGF-TCS、pQE30/EGF-Linker-TCS were transformed into M15. Single bacteria was selected and inoculated into LB culture medium which contained 50ug/ml ampicillin. The bacteria was cultured in the shaker and when the OD reached 0.6, IPTG was added to induce recombinant protein expression for 3 hours and the bacteria sample was analyzed by SDS-PAGE. Then the bacteria were broken by ultrasonic and the supernatants were recovered after centrifuged. The purity of recombinant TCS、EGF-TCS、EGF-Linker-TCS was examined by Ni-NTA Agrose affinity chromatography.
2. Detection of EGF receptor on normal cells and tumor cells
Because the targeted point of the fusion protein composed of EGF and TCS is EGF receptors, we used flow cytometry to carry EGF receptor detection on normal cells and tumor cells.
3. Detection of the 50% inhibitory concentration ( IC_(50)) and the selectivity index of purified TCS, EGF-TCS and EGF-Linker-TCS on tumor cells and normal cell
We detected the effects of purified TCS, EGF-TCS and EGF-linker-TCS on tumor cells and normal cell by crystal violet method. We also detected the 50% inhibitory concentration ( IC_(50) ) and the selectivity index. The killing rate was calculated with the following formula: The killing rate (%)=[(A value in control- A value in test)/A value in control]×100%.The inhibitory rate was calculated and through which IC50 was calculated by origin 6.0. Then the selectivity index was calculated on basis of IC_(50):The selectivity index=IC_(50)(normal cell)/IC_(50)(tumor cell).
4. Observation of apoptosis of cells induced by EGF-TCS and EGF-linker-TCS through flow cytometry
EGF-TCS and EGF-Linker-TCS could bind tumor cells through EGF receptors. TCS could inactivate ribosome and inhibit protein synthesis to make tumor cells die. Perhaps there were other mechanisms to lead eukaryotic cells death. We used flow cytometry to observe apoptosis of LO-2、BEL-7402、MCF-7 and BGC-823 induced by EGF-TCS and EGF-Llinker-TCS.
5. Observation of the effects of EGF-Linker-TCS on BEL-7402 by electric microscope
To make further observation on mechanism of cell apoptosis induced by fusion protein composed of EGF and TCS, we used electric microscope to observe the effects of EGF-Linker-TCS on hepatoma cell BEL-7402.
6. Anti-tumor experiment in naked mice tumor model by EGF-Linker-TCS
The hepatoma cell BEL-7402 was inoculated subcutaneously on right armpit in naked mice and EGF-linker-TCS was injected intravenously (100ug/kg,50ug/kg,25ug/kg) to mice for therapy after 6 days of inocluation. There was control group and mice were killed after 2 days of drug withdrawal, and the tumors were weighed and the tumor inhibitory rate was calculated. The livers were taken to calculate tumor migration rate. Tumor tissues were fixed by 10% formaldehyde for histology and vWA immunohistochemistry experiments.
Results
1. Construction of recombinant fusion protein TCS、EGF-TCS、EGF-Linker-TCS
The plasmid pQE30/TCS, plasmid pQE30/EGF-TCS and plasmid pQE30/EGF-Linker-TCS were sequenced,and the determined sequence was identical with the designed one. The three recombinant plasmids was transformed into M15. After induced, recombinant protein TCS, EGF-TCS and EGF-Linker-TCS were expressed in engineering bacteria M15 with soluble form and the expression rate reached 30% of total bacteria protein which was analyzed by SDS-PAGE. The recombinant TCS could occur immune response with special antibody specially which was analyzed by western blot. The purity of recombinant TCS、EGF-TCS、EGF-Linker-TCS was more than 95% after purified by Ni-NTA Agrose affinity chromatography.
2. Detection of EGF receptor on normal liver cells and tumor cells
we used flow cytometry to carry EGF receptor detection on normal liver cells and tumor cells. The results: the EGF receptor expression rate was 5.51% in normal liver cell LO-2, and was 72.33% in hepatoma cell BEL-7402, and was 63.92% in breast tumor cell MCF-7, and was 70.41% in stomach adenocarcinoma cell BGC-823. The quantity of EGF receptors are much more than that of normal cells.
3. Detection of the 50% inhibitory concentration ( IC_(50)) and the selectivity index of purified TCS, EGF-TCS and EGF-linker-TCS on tumor cells and normal cell
The results: IC_(50) of TCS was 39.70 ug/ml、33.37 ug/ml、36.53 ug/ml、41.52 ug/ml for LO-2、BEL-7402、MCF-7 and BGC-823 respectively, and the selectivity index of TCS was 1.19、1.09、0.96 for BEL-7402、MCF-7 and BGC-823 respectively;IC_(50) of EGF-TCS was 115.34 ug/ml、8.90 ug/ml、13.75 ug/ml、15.36 ug/ml for LO-2、BEL-7402、MCF-7 and BGC-823 respectively, and the selectivity index of EGF-TCS was 12.96、8.39、7.51 for BEL-7402、MCF-7 and BGC-823 respectively;IC_(50) of EGF-Linker-TCS was 113.99 ug/ml、6.98 ug/ml、11.97 ug/ml、 13.53 ug/ml for LO-2、BEL-7402、MCF-7 and BGC-823 respectively, and the selectivity index was 16.33、9.52、8.43 for BEL-7402、MCF-7 and BGC-823 respectively. The results of experiment accorded with our design. The IC_(50) of EGF-TCS and EGF-linker-TCS for tumor cells was much less than that of TCS, and the IC_(50) of EGF-TCS and EGF-linker-TCS for normal cells was very close to that of TCS. The selectivity index of EGF-TCS and EGF-Linker-TCS was obviously higher than that of TCS, and was both greater than 2 and these two proteins were fit for the demand of targeted drugs. The IC_(50) of EGF-linker-TCS for tumor cells was a little less than that of EGF-TCS and the selectivity index of EGF-Linker-TCS was a little higher than that of EGF-TCS.
4. The apoptosis effect on tumor cells induced by EGF-TCS and EGF-Linker-TCS
The apoptosis effect on breast tumor cell MCF-7 , stomach adenocarcinoma cell BCG-823 and hepatoma cell BEL-7402 induced by EGF-TCS in the concentration of 5μg/ml was detected by flow cytometry, and the apoptosis rate was 35.61%、39.59% and 50.57% respectively. EGF-TCS in the same concentration could induce little apoptosis on normal liver cell LO-2 and the apoptosis rate was 8.99%;The apoptosis effect on breast tumor cell MCF-7,stomach adenocarcinoma cell BCG-823 and hepatoma cell BEL-7402 induced by EGF-Linker-TCS in the concentration of 5μg/ml was detected by flowcytomy, and the apoptosis rate was 36.67%, 42.56%, and 58.56%, respectively. EGF-Linker-TCS in the same concentration could induce little apoptosis on normal liver cell LO-2 and the apoptosis rate was 8.08%.
5. Observation of the effects of EGF-Linker-TCS on hepatoma cell BEL-7402 by electric microscope
It was observed by electric microscope that the cell nucleolus was clear and intact, and there were abundant mitochondria and endoplasmic reticulum in control group; some characters of cell apoptosis in various phase appeared such as chromatin concentration, non-regular nucleolus, vacuolar endoplasmic reticulum and apoptosis body in EGF-Linker-TCS group.
6. Tumor inhibition test by EGF-Linker-TCS in naked mouse tumor model
To further observe the anti-tumor effect of recombinant toxin, we carried tumor inhibition test in vivo in naked mouse tumor model. The result illustrated that EGF-Linker-TCS could inhibit tumor growth markedly. The inhibition rate was 71.3%、60.87% and 45.22%, for high dosage group, middle dosage group and low dosage group respectively. Variance analysis illustrated that EGF-Linker-TCS could markedly inhibit the growth of naked mouse tumors,variance analysis had significant difference( F=8.712 P=0.006 );and We also observed the inhibition effect on tumor migration by EGF-Linker-TCS. After anatomizing naked mouse and checking liver, we found single white focus caused by migration of tumor transplant to liver. The number of liver migration focus in each naked mice was calculated, revealed that EGF-Linker-TCS could markedly inhibit liver migration of tumor transplant in naked mouse,Variance analysis showed significant difference( F=8.744 P=0.002 ) Immunohistochemistry experiment showed that EGF-Linker-TCS could makedly inhibit tumor angiogenesis. There were no visible blood vessels in tumor tissues in high dosage group and there were fewer blood vessels in tumor tissues in middle and low dosage group than those in control group, which revealed that perhaps EGF-Linker-TCS inhibited tumor growth and migration by inhibiting tumor angiogenesis.
Conclusions
After the study, we can make some conclusions as below:
1. We find a method, which is effectively, stably, simply and cheaply to prepare fusion protein TCS, EGF-TCS and EGF-Linker-TCS with high purity by gene engineering. The development of TCS lays a foundation of further study on the relationship between structure and the function of it.
2. EGF receptors are over-expressed in many tumor cells. EGF can bind to these over-expressed EGF receptors specially and can be used as the target part of immunotoxins.
3. We carried the research about targeted toxicity of TCS, EGF-TCS and EGF-Linker-TCS on tumor cells in vitro and proved that EGF-TCS and EGF-Linker-TCS had good selectivity and relatively higher cyto-toxicity on tumor cells with no selectivity on normal cells. The selectivity index of EGF-TCS and EGF-Linker-TCS for tumor cells is more than 2, and the cyto-toxicity of the two proteins on normal cells was less than that of TCS.
4. Immunotoxin EGF-Linker-TCS inhibited tumor growth and liver migration markedly, which illustrated that the anti-tumor effect of EGF-Linker-TCS was evident in naked mouse tumor model.
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