【作者】 黄世峰；

【导师】 冯文莉；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2009， 博士

【摘要】 慢性粒细胞白血病(chronic myeloid leukemia,CML)以具有强烈酪氨酸激酶活性的BCR-ABL融合蛋白的持续表达为重要特征。BCR-ABL癌蛋白能激活多条癌性信号通路,通过抑制细胞分化和凋亡,促进增殖和异常黏附而导致细胞恶性转化,在CML的发生发展过程中起到关键性作用。针对BCR-ABL酪氨酸激酶活性的特异性小分子抑制剂如格列卫、达沙替尼和尼洛替尼等的临床应用使CML治疗获得了很大进展,但最终因不能清除CML恶性克隆而导致部分患者出现耐药或复发,因此,急需进一步探讨CML治疗新靶点。BCR-ABL融合蛋白N端寡聚化区域(oligomerization domam,OD域)所介导的BCR-ABL同源寡聚化是BCR-ABL自身磷酸化导致分子构型改变,并最终异常激活Abl酪氨酸激酶活性的关键因素。我们推测:通过转导外源性OD域蛋白入CML细胞后干扰BCR-ABL寡聚化来抑制BCR-ABL酪氨酸激酶活化可成为CML治疗的新切入点。但由于生物大分子无法自动穿越细胞膜和需要精确定位到特定亚细胞池中才能发挥功能等原因而使蛋白类药物为基础的细胞治疗策略受到极大限制。胞浆转导肽(Cytoplasmic transduction peptide,CTP)是一种新近报道的能携带蛋白类大分子穿越细胞膜并定位于细胞浆中的新型转导肽,非常适合于作为携带胞浆蛋白治疗肽的穿膜系统。鉴于本研究中设计的OD域短肽拟在胞浆区发挥竞争性抑制胞浆蛋白BCR-ABL自身寡聚化的作用,CTP可成为合适的OD域转运载体。本实验在构建、表达并纯化具有胞浆定位活性的CTP-OD-HA融合肽(为便于后期的免疫学检测而引入HA标签)的基础上,以含有BCR-ABL融合基因的K562、KU812、BP210和DP210等各种CML细胞株为体外模型,以BP210-Babl/C CML小鼠为体内模型,探讨外源性CTP-OD-HA融合蛋白作用CML细胞及模型动物后是否能通过抑制BCR-ABL同源寡聚化进而抑制其酪氨酸激酶活性而对CML细胞生长、增殖和凋亡产生影响,旨在探索一种新的CML分子靶向治疗策略。采用的主要实验方法如下:1.CTP-OD-HA融合蛋白的克隆构建、原核表达、纯化及生物学活性分析:通过次序严密的分步克隆,分别将OD、HA和CTP基因片段顺次克隆入原核表达载体pET32a(+)中。进行酶切与测序鉴定后,将构建正确的pCTP-OD-HA重组质粒转化大肠杆菌BL-21(DE3)菌株,在IPTG诱导下表达CTP-OD-HA融合蛋白,表达产物用SDS-PAGE和western blot进行鉴定;采用组氨酸亲和层析法纯化目的蛋白;经脱盐定量后进行肠激酶切割、回收、FITC标记,荧光显微镜检测FITC-CTP-OD-HA融合蛋白在CML细胞株中的转导活性;免疫细胞化学染色和激光共聚焦显微镜观察融合蛋白转导入CML细胞后的亚细胞定位情况;Western blot进一步分析CTP-OD-HA在各种CML细胞内的转导活性;以期为进一步探讨CTP-OD-HA融合蛋白治疗肽对CML的治疗作用奠定实验基础。2.观察CTP-OD-HA融合蛋白转导在CML细胞株中的特异性抑增殖、促凋亡等生物学效应,并对其作用机制进行探讨。3.构建BCR-ABL转化的BP210造血细胞株在等基因Balb/C小鼠体内定殖的CML动物模型,并对其血液学、脏器浸润特征以及建模可重复性等方面进行鉴定和验证后,观察CTP-OD-HA融合蛋白预转导对CML模型动物的保护作用。通过以上实验,获得如下主要结果和结论:1.CTP-OD-HA融合蛋白在37℃、1mM IPTG诱导4h的条件下获得高效、可溶性表达;经Ni-NTA亲和纯化获得了高纯度CTP-OD-HA融合蛋白;经脱盐、肠激酶切割和FITC标记后证实了CTP-OD-HA融合蛋白在CML细胞中的高效转导效率;免疫细胞化学染色和激光共聚焦显微镜观察证实了CTP-OD-HA明显的胞浆定位偏性;Western blot实验进一步证实了CTP-OD-HA在各种CML模型细胞中的高转导活性。由此可见,CTP-OD-HA融合基因能在PET表达系统中以可溶性形式高效表达,重组蛋白CTP-OD-HA具有良好的生物学活性,为下一步研究CTP-OD-HA的生物学功能奠定了基础。2.纯化的CTP-OD-HA融合蛋白转导后在各种CML细胞株中表现出BCR-ABL特异性增殖抑制和凋亡诱导效应;进一步的机制研究证实:CTP-OD-HA转导入CML细胞后能通过竞争性结合BCR-ABL癌蛋白而抑制其自身寡聚化和后续酪氨酸磷酸化,阻断癌性酪氨酸激酶信号通路,并诱导促凋亡基因的表达,不改变BCR-ABL癌蛋白的胞浆定位。以上结果提示通过外源性转导OD域抑制BCR-ABL同源寡聚化有可能成为一种有效的抗CML治疗策略。3.成功构建BP210转化细胞株在Babl/C小鼠体内定殖的CML模型,并对其进行了鉴定,进一步实验证实CTP-OD-HA融合蛋白预转导能显著抑制CML细胞在受体小鼠的致白血病潜能,明显延长模型动物的生存期。更多还原

【Abstract】 Chronic myeloid leukemia (CML) is characterized by the persistentexpression of the BCR-ABL fusion protein with constitutive tyrosine kinase activity. The BCR-ABL oncoprotein can activate multiple oncogenic signal transduction pathways and plays a critical role in the pathogenesis and progression of CML through inhibition of cellular differentiation and apoptosis, as well as promotion of proliferation and deregulated adhesion that eventually lead to malignant transformation. In the recent years, with the advent of the era of small molecular inhibitors, Imatinb, Dasatinib and Nilotnib have greatly advanced the therapeutic effects on CML, however, drug resistance and disease relapse inevitablly emerged due to their inability to kill the malignant clones in CML. As a result, further exploration for new targeted therapeutic targets in CML is urgently needed.The BCR-ABL homodimerization mediated by its N-terminal oligomerization (OD) domain is the critical factor for both its tyrosine autophosphorylation, molecular configulation transformation, as well as the deregulated Abl tyrosine kinase activity. We thus speculated that transduction of an exogenous OD domain into the CML cells may be a novel CML treatment strategy through inhibition of BCR-ABL tyrosine kinase by intefering with the its homodimerization. Recently, protein drugs have been finding its way into clinical practice, however, protein drug-based cellular therapeutic strategy is being limited by the fact that macromolecules can not penetrate cellular membranes and that many proteins need accurate subcelluar localization to exert their functions.Cytoplasmic transduction peptide (CTP) is a recently reported novel transduction peptide that can carry macromolecules to penetrate the cellular membranes with a preference to localize into the cytoplasmic compartment, and is therfore applicable for the transduction of cytoplasmic protein peptides. Given that the designed OD peptide in the present study is aimed to competitively inhibit the homodimerization of the cytoplasmic BCR-ABL oncoprotein, the CTP system fulfills an appropriate transduction system for the OD domian.The present study was launched to construct, express and purify the cytoplasmic localizing CTP-OD-HA fusion peptide (HA tag was introduced to further facilitate immunoblot detection), and to further investigate its potential effects on cellular growth, proliferation and apoptosis in both the various in vitro cultured BCR-ABL positive CML cells and the in vivo BP210-Babl/C CML model mice. It is hoped that the CTP-OD-HA fusion peptide will exert its anti-CML effect through its inhibition of both the homodimerization of the BCR-ABL oncoprotein and the resultant inhibition of tyrosine kinase activity ’and that it can be exploited as a novel targeted molecular therapeutic strategy for CML. The main experiments arepresented as follows:1. The cloning, prokaryotic expression, purification and biological activity analysis for the CTP-OD-HA fusion protein: The OD, HA and CTPfragments were sequentially cloned into the prokaryotic expression vector pET32a(+) followed by double enzyme digestions and bi-directional sequencing. The correctly constructed pCTP-OD-HA recombinant plasmid was further transduced into the E.coli. BL-21(DE3) expression bacteria and IPTG was added subsequently to induce the expression of the CTP-OD-HA fusion protein. The expression products were further identified by bothSDS-PAGE and western blot; The histidine affinity chromatography wasemployed to purify the target protein; The transduction potential of the FITC-CTP-OD-HA in CML cells was detected under the fluorescence microscopy after it was desalted, cleaved by enterokinase, reclaimed by asecond affinity purification process and labelled by FITC ;Immunocytochemical staining and confocal microscopy were performed to observe the subcellular localization of the transduced fusion protein in CML cells; Western blot was employed to further evaluate the transduction potential of the CTP-OD-HA fusion protein in CML cells in service of the goal of exploration of the therapeutic effect of the CTP-OD-HA fusion protein in CML.2. To observe the potential specific biological effects of CTP-OD-HA, such as proliferation inhibiting and apotosis inducing effects on CML cells and to further probe into its molecular mechanisms.3. To construc the Balb/C CML model mice transplanted by the BCR-ABL transformed BP210 hematological cells, and to further evaluate and validate its features in respect of its hemetological and organ infiltration characteristics as well as the repeatability of model construction followed by the observation of the protective effect of its transduction on CML model mice.All the following results and conclusions were reached after the aboveexperiments:1. CTP-OD-HA fusion protein can be induced to express in a soluble form at a high efficency by 1mM IPTG at 37℃for 4h; High purity CTP-OD-HA fusion protein was acquired after Ni-NTA affinity purification; CTP-OD-HA fusion protein was proved to have potently transduced into CML cells after it was desalted, enterokinase cleaved and FITC labelled ; The cytoplasmic localization preference of the CTP-OD-HA fusion protein was validated by Immunocytochemical staining and confocal microscopy; It’s potent transduction potential in various CMLcells were further confirmed by Western blot. All the above results indicated that the CTP-OD-HA fusion gene can be induced to express in a soluble form at high efficiency in the pET expression systems and the resultant fusion protein showed excellent biological activities, thus laying a sound foundation for further evaluation of the biological functions of the CTP-OD-HA fusion peptide.2. The purified CTP-OD-HA fusion protein was demonstrated to have BCR-ABL specific proliferation inhibiting and apotosis inducing effects onCML cells after its transduction; Further exploration into the underlyingmechanisms revealed that CTP-OD-HA inhibited both the homodimerization, tyrosine phosphorylation and oncogenic tyrosine kinase pathway triggered by the BCR-ABL oncoprotein through its competitive binding to the BCR-ABL oncoprotein after it was transduced into CML cells. The transduced CTP-OD-HA peptide was also shown to induce the expression of proapototic genes without altering the cytoplasmic localization of the BCR-ABL oncoprotein. All the above results indicated that the inhibition of homodimerization of the BCR-ABL oncoprotein through exogenous transduction of the OD domain can be developed as a potential anti-CML therapeutic strategy. 3. The Balb/C CML model mice transplanted by the BCR-ABL transformed BP210 hematological cells were successfully constructed and validated, and further in vivo experiments showed that the pre-transduction of CTP-OD-HA fusion protein could not only significantly inhibit the leukemogenic potential of CML cells in the recipient mice, but also prolong the survival time of the CML model mice.更多还原