【作者】 刘树雷；

【导师】 何威；

【作者基本信息】 第三军医大学 ， 皮肤性病学， 2008， 博士

【摘要】 背景:皮肤T细胞淋巴瘤(cutaneous T cell lymphomas, CTCL)和急性T细胞白血病(acute T cell leukemia, ATCL)均为来源于T细胞的恶性肿瘤,其发生率和病死率均呈上升趋势,严重威胁人类的健康。通常采用的治疗方法是放疗或化疗,但这些治疗措施抗肿瘤能力低并有明显的毒副作用。长期使用非特异免疫抑制剂可使患者免疫功能严重受损,导致许多并发症。免疫毒素被称为“生物导弹”,它是由具有靶向能力的分子(载体)和具有细胞毒性的分子组合而成的具有特异性杀伤能力的杂合分子。它通过载体的靶向作用,使其所携带的毒素分子到达靶细胞,发挥特异性杀伤作用。免疫毒素作为一种新型的治疗手段主要有两个特性优于其它治疗方案:其一是免疫毒素对肿瘤细胞的杀伤作用不依赖于宿主的免疫系统,而是依赖靶向载体所携带的毒素起作用;其二是免疫毒素对正常细胞无杀伤作用,但能被有效地内吞入靶细胞,具有特异性杀伤靶细胞的特性。因此,采用免疫毒素治疗肿瘤具有突出的优势。美国FDA首次批准免疫毒素药物DAB389IL-2,也称为地尼白介素2(denileukin diftitox)治疗皮肤T细胞淋巴瘤的临床研究,虽已进入Ⅲ期临床试验但仍存在一些问题。目前免疫毒素存在的主要问题是:一方面,是由于现有的免疫毒素多是由异源性的抗体为靶向载体,免疫原性强;另一方面,由于通常的毒素分子量较大,对组织的渗透性差。要想提高免疫毒素对靶细胞的杀伤作用,主要通过以下途径改善:①寻找靶细胞上特异的靶点以及通过调节细胞表面靶点的表达促进免疫毒素的作用。②降低免疫毒素的抗原性,包括对载体及毒素的改造,降低它们的分子量,以及选择人源化的载体及毒素分子。因此,免疫毒素分子的小型化和人源化是免疫毒素研究的发展趋势。T淋巴细胞相关的肿瘤及部分B淋巴细胞相关的肿瘤高表达CD25分子,CD25是IL-2受体(IL-2R)的α链,它与IL-2Rβ、IL-2Rγ链共同构成高亲和力的IL-2受体。在理论上可利用低浓度的人IL-2融合蛋白选择性作用于这些淋巴瘤细胞,介导对瘤细胞的抑制或杀伤作用。用人IL-2作为载体具有靶向性好、免疫原性低,半衰期长、可在动物中应用的优势。植物来源的核糖体灭活蛋白(RIP)是一类作用很强的毒素,它们直接作用于核糖体,灭活其60S大亚基,具有很强的抑制蛋白质合成的活性以及诱导细胞凋亡的作用。Luffin家族是一类从丝瓜种子中提取的RIP,而Luffin P1是新近发现的Luffin家族成员,是迄今已发现的活性最强、分子量最小(5.2 kD)单链核糖体失活蛋白。由于它具有分子量小的特点,不仅便于靶向载体携带进入靶细胞,还有利于降低免疫毒素的免疫原性,减少毒副作用,提高疗效。维A酸类药物具有调节上皮细胞分化与生长、维持上皮组织正常角化过程、抗肿瘤等作用,已用于多种肿瘤的诱导分化治疗。维A酸类药物还可以上调IL-2R的α、β链的表达,通过调节细胞表面靶点的表达而促进免疫毒素的作用。在初步临床试验中免疫毒素与维A酸联合应用治疗CTCL,使病情缓解率提高,并且没有出现毒副作用的增加。基于以上理由,我们设想利用基因工程方法构建一种新的免疫毒素hIL-2-Luffin P1,使其hIL-2片段特异性识别表达IL-2受体的肿瘤靶细胞,从而把毒素分子带入靶细胞内,发挥抗肿瘤的作用。另一方面,希望凭借芳维A酸乙酯能上调IL-2受体的表达而与上述免疫毒素联合使用,以提高免疫毒素的靶向结合能力,增强hIL-2-Luffin P1对靶细胞的杀伤作用。目的:构建、表达及纯化能特异性杀伤皮肤T细胞淋巴瘤和急性T细胞白血病的重组免疫毒素hIL-2-Luffin P1及作为对照的毒素分子蛋白Luffin P1。观察hIL2-Luffin P1、芳维A酸乙酯、hIL2-Luffin P1联合芳维A酸乙酯、Luffin P1对Hut-78细胞和Jurkat细胞增殖、凋亡及细胞周期的影响。方法:(1)应用PCR扩增目的基因(hIL2-Luffin P1、Luffin P1)片段,然后将它们分别克隆到表达载体pET32a(+)中,并对重组质粒进行酶切鉴定及基因测序。测序正确的重组质粒转化到表达菌BL21中,进行诱导表达。分别用鼠抗人的His单克隆抗体、兔抗人IL-2多克隆抗体进行Western blot鉴定。蛋白大量诱导表达后经过蛋白的纯化、复性、脱盐、肠激酶酶切、再纯化,最后用兔抗人IL-2多克隆抗体进行Western blotting鉴定目的蛋白hIL2-Luffin P1中IL-2成分的表达。(2)应用免疫细胞化学技术检测Hut-78细胞、Jurkat细胞表面CD25分子的表达。然后分为hIL-2-Luffin P1组、芳维A酸乙酯组、hIL-2-Luffin P1联合芳维A酸乙酯组、Luffin P1组,分别于不同浓度和不同时相点分别作用于Hut-78细胞和Jurkat细胞。应用MTT法观察细胞的生长抑制率,流式细胞仪检测细胞的早期凋亡及细胞周期。结果:(1)成功构建了带有KDEL结构的原核表达质粒PET32a(+)-hIL-2-Luffin P1和PET32a(+)-Luffin P1。(2)经过蛋白的诱导表达,超声破菌后,分离结果显示,hIL-2-Luffin P1蛋白以包涵体的形式存在,而Luffin P1蛋白为部分可溶性表达。经过蛋白的纯化、肠激酶酶切及鉴定,最后得到了具有较好活性的hIL2-Luffin P1蛋白及作为对照的毒素分子蛋白Luffin P1(。3)免疫细胞化学结果显示:IL-2受体α链(CD25)在Hut-78细胞和Jurkat细胞表面高表达,其表达阳性细胞比例分别为79.32%和54.47%。(4)MTT结果显示:hIL-2-Luffin P1组、芳维A酸乙酯组、hIL-2-Luffin P1联合芳维A酸乙酯组均可抑制Hut-78细胞和Jurkat细胞增殖,且呈剂量和时间依赖性效应。其中联合组的作用明显强于单一用药组,而Luffin P1组对两种细胞增殖均没有明显的抑制作用。hIL-2-Luffin P1组和hIL-2-Luffin P1联合芳维A酸乙酯组对Hut-78细胞的抑制率明显高于Jurkat细胞。hIL-2-Luffin P1对Hut-78细胞和Jurkat细胞的IC50分别为27.361μg/ml和39.634μg/ml。芳维A酸乙酯组对Hut-78细胞和Jurkat细胞均有明显的抑制作用,但对两种细胞抑制率的差异不明显。(5)Annexin V/PI双标法流式细胞术检测显示,hIL-2-Luffin P1、芳维A酸乙酯、hIL-2-Luffin P1联合芳维A酸乙酯均可以诱导Hut-78细胞和Jurkat细胞发生早期凋亡,其中联合组的作用明显强于单一用药组。而Luffin P1无明显诱导细胞凋亡的作用。(6)流试细胞术检测细胞周期显示:hIL-2-Luffin P1组及hIL-2-Luffin P1联合芳维A酸乙酯组均可使Hut-78和Jurkat细胞停滞在G1期,提示hIL-2-Luffin P1或hIL-2-Luffin P1联合芳维A酸乙酯均能阻止两种细胞由G1期向S期转化。结论:用基因工程方法成功构建、表达、纯化了免疫毒素hIL-2-Luffin P1蛋白及毒素分子Luffin P1蛋白。hIL-2-Luffin P1能够抑制Hut-78细胞和Jurkat细胞增殖,促进Hut-78细胞和Jurkat细胞凋亡,使细胞周期停滞在G1期,并且其抑制增殖和诱导凋亡的作用呈剂量和时间依赖性效应。而毒素Luffin P1蛋白对上述两种细胞的增殖、凋亡及细胞周期均无明显作用。这提示免疫毒素hIL-2-Luffin P1能够针对表达IL-2R的Hut-78细胞和Jurkat细胞产生定向杀伤作用。hIL-2-Luffin P1联合芳维A酸乙酯也能抑制Hut-78细胞和Jurkat细胞的增殖,促进Hut-78细胞和Jurkat细胞凋亡,并可使细胞周期停滞在G1期,二者合用时抑制增殖和诱导凋亡的作用也呈剂量和时间依赖性效应。hIL-2-Luffin P1联合芳维A酸乙酯的作用明显比单用hIL-2-Luffin P1或单用芳维A酸乙酯时强。由此提示免疫毒素hIL-2-Luffin P1联合芳维A酸乙酯对Hut-78细胞和Jurkat细胞具有协同作用。本课题的研究结果对皮肤T细胞淋巴瘤和急性T细胞白血病的治疗具有探索、启示和借鉴意义。更多还原

【Abstract】 BackgroundCutaneous T cell lymphomas (CTCL) and acute T cell leukemia originate from T cells. The morbidity and mortality of CTCL and acute T cell leukemia have increasing trend, which greatly threatening human health. Conventional radiotherapy or chemotherapy show poor anti-tumor and significant toxicity and side effects. Long-term use of nonspecific immunosuppressants obviously impair the immunity and results in many complications.Immunotoxins, also called biological missiles, comprise a targeting molecule (vehicle) and a cytotoxic component. They specificly kill target cells Immunotoxins have two major advantages:①. the killing effects of immunotoxins on tumor cells does not depend on the host’s immune system, but depend on toxins carried by targeting vectors;②. immunotoxins do not kill normal cells, but they can specifically kill target cells after enter the target tumor cells. Hence, immunotoxins are ideal agent for tumor therapy. DAB389IL-2, i.e, denileukin diftitox, was the first immunotoxin approved by FDA for the treatment of CTCL, and is now under phase III clinical study. However, there are some problems about DAB389IL-2. The major problems about immunotoxins include: high immunogenicity of heterologous antibodies which are used as targeting vectors in current immunotoxins, and poor tissue permeability due to large molecular weight of immunotoxin. To enhance the killing effect of immunotoxins on target cells, the following should be considered:①to seek specific targets on target cells and enhance the effect of immunotoxins through up regulating the expression of cell surface targets;②to reduce immunotoxin antigenicity by modifying vectors and toxins, reduce their molecular weight or select human-derived vectors and toxin molecules. Accordingly, To miniaturize immunotoxins and choses human-derived immunotoxins are the two major solutions in the field of constructing immunotoxins.T cell associated tumors and a small number of B cell associated tumors highly express CD25. CD25 (αchain of IL-2 receptor) and IL-2 receptorβandγchains constitute high-affinity IL-2 receptor. In theory, low concentrations of human IL-2 fusion protein can selectively act on these lymphoma cells. Hence, IL-2 R can be an ideal therapeutic target on these tumor cells, which mediates T lymphoma cell suppression or killing. Human IL-2 is characterized by good targeting performance, low immunogenicity, long half-life time, and applicability in animals.Plant-derived ribosomes inactivate proteins (RIPs) are a kind of toxins with strong toxicity, and they directly act on ribosomes to inactivate 60S subunit, thus strongly suppressing protein synthesis activity and inducing apoptosis. The Luffin family consists of RIPs extracted from the seeds of Luffa cylindrica. Luffin P1, a recently discovered member of the Luffin family, is the smallest RIP yet known (molecular weight, 5.2kD), and it exhibits high activity on suppressing protein synthesis. Due to its low molecular weight, Luffin P1 can enter target cells easily, and help to reduce immunogenicity and side effect of immunotoxins, as well as increases therapeutic effect.Retinoic acid (RA) regulates epithelial cell differentiation and growth, maintains normal keratinization of epithelial tissue, and prevents tumorigenesis; hence, retinoic acid has been used to induce the differentiation of various tumors. In addition, retinoic acid upregulates the expression of IL-2 Rα,βchains, so it can regulate the expression of IL-2 receptor on target cell surface and promote the action of immunotoxins. In preliminary clinical studies, the combination of retinoic acid and immunotoxin increases the remission rate of CTCL, and does not increase toxicity and side effects.Accordingly, we constructed a new immunotoxin hIL-2-Luffin P1 by gene engineering, so as to (1) allow human IL-2 to specifically recognize IL-2 receptors on tumor cells and toxin molecules to enter target cells to exert their anti-tumor effect; (2) upregulate IL-2 receptor expression by retinoic acid to increase the targeting performance of the immunotoxin and enhance the killing effect of hIL-2-Luffin P1 on target cells.Objectives: To construct, express and purify hIL-2-Luffin P1 immunotoxin and Luffin P1 protein and observe the effects of hIL2-Luffin P1 and hIL2-Luffin P1 plus arotinoid ethylester on the proliferation and apoptosis of Hut-78 cells and Jurkat cells. Methods: (1) Target gene fragments (hIL2-Luffin P1, Luffin P1) were amplified by PCR and cloned into the expression vector pET32a (+). The recombinant plasmids were subjected to identification by enzymatic digestion and gene sequencing. The correctly constructed recombinant plasmids were used to transform BL21 bacteria, followed by induced expression. Western blotting was carried out to identify the expressed protein using mouse anti-human His and rabbit anti-human IL-2 polyclonal antibodies. The expressed protein was subjected to purification, renaturation, desalting, digestion with enterokinase, and re-purification, and Western blotting analysis of hIL2-Luffin P1 protein was performed using rabbit anti-human IL-2 polyclonal antibody. (2) CD25 expression on Hut-78 cells and Jurkat cells was analyzed immunocytochemically. Hut-78 cells and Jurkat cells were treated with hIL-2-Luffin P1, arotinoid ethylester, hIL-2-Luffin P1 plus arotinoid ethyl ester and Luffin P1 at different concentrations for different time periods. The cell growth inhibition rate was determined by MTT assay. Early apoptosis and cell cycle distribution were analyzed by flow cytometry.Results: (1) Prokaryotic expression plasmids PET32a(+)-hIL-2-Luffin P1 and PET32a(+)-Luffin P1 were constructed successfully. (2) hIL-2-Luffin P1 protein was mainly detected in inclusion bodies, and Luffin P1 protein was expressed in partial soluble form. After protein purification, digestion with enterokinase and identification, highly active hIL-2-Luffin P1 and Luffin P1 proteins were obtained. (3) Immunocytochemical analysis demonstrated high expression of CD25 on Hut-78 cells and Jurkat cells, with an expression rate of 79.32% and 54.47%, respectively. (4) MTT assay demonstrated that hIL-2-Luffin P1 protein, retinoic acid, or both suppressed the proliferation of Hut-78 cells and Jurkat cells in a dose and time-dependent manner. The suppression effect of hIL-2-Luffin P1 protein and arotinoid ethylester was significantly higher than that of hIL-2-Luffin P1 protein or arotinoid ethylester, and Luffin P1 protein had no obvious inhibitory effect on the proliferation of the two cell types. After treatment with hIL-2-Luffin P1 or hIL-2-Luffin P1 and arotinoid ethyl ester, the inhibition rate of Hut-78 cells were significantly higher than that of Jurkat cells. IC50 of hIL-2-Luffin P1 for Hut-78 cells and Jurkat cells were 27.361μg/ml and 39.634μg/ml, respectively. arotinoid ethyl ester significantly and similarly suppressed Hut-78 cells and Jurkat cells. (5) Flow cytometry demonstrated that hIL-2-Luffin P1, arotinoid ethyl ester or hIL-2-Luffin P1 and retinoic acid induced early apoptosis of Hut-78 cells and Jurkat cells. The apoptosis rate was significantly higher in cells treated with hIL-2-Luffin P1 and arotinoid ethyl ester than in those treated with arotinoid ethyl ester or hIL-2-Luffin P1. Luffin P1 did not show effect of inducting apoptosis. (6) Flow cytometry demonstrate that after treatment with hIL-2-Luffin P1 or hIL-2-Luffin P1 and arotinoid ethyl ester, Hut-78 cells and Jurkat cells were retained at G1 phase, suggesting that both hIL-2 -Luffin P1 and hIL-2-Luffin P1 plus arotinoid ethylester prevent cell cycle transition from G1 phase to S phase in both cell types.Conclusions: Recombinant hIL-2-Luffin P1 immunotoxin and Luffin P1 protein were successful constructed and expressed by gene engineering. hIL-2-Luffin P1 suppresses the proliferation of Hut-78 cells and Jurkat cells, promotes apoptosis of these two cell types in a dose-and time-dependent manner, and makes these cells arrested at G1 phase. In contrast, Luffin P1 protein does not influence the proliferation, apoptosis and cell cycle of these two cells types. It is suggested that hIL-2-Luffin P1 protein shows specific targeting killing effect on IL-2R expressing Hut-78 cells and Jurkat cells. The effect of hIL-2-Luffin P1 plus arotinoid ethylester is similar with that of hIL-2-Luffin P1 alone, but is stronger than that of hIL-2-Luffin P1 or arotinoid ethyl ester, suggesting the synergism of hIL-2-Luffin P1 and arotinoid ethylester in influencing Hut-78 cells and Jurkat cells. This study may be helpful for treating CTCL and acute T cell leukemia.更多还原