ABD蛋白的表达及对K562，HL60细胞的作用研究
摘要
研究背景慢性粒细胞白血病(chronic myeloid leukemia, CML)是发生于造血干细胞的克隆性恶性肿瘤,1845年Craigie[1]和Bennet [2]在爱丁堡发现。在1960年,Nowell和Hunger-ford首先在费城发现CML病人具有获得性的染色体异常,将这个异常的染色体命名为费城染色体(Philadelphia,Ph[3, 4])。1973年,Rowley发现它是9号和22号染色体长臂间交互易位的结果,即t(9,22) (q34;ql1)[ 5]。1983年以后,证实了9号染色体上c-abl与22号染色体上bcr基因交互易位,在22号染色体上形成了BCR—ABL融合基因,编码BCR—ABL融合蛋白,表达P190,P210,P230融合蛋白,具有很强的酪氨酸激酶活性,可使一系列信号蛋白发生持续性的磷酸化,导致白血病的发生[6]。
CML病人经历慢性期、加速期、急变期三个阶段,各自的中位数时间分别为3～4年;6～9个月;3～6个月[7]。目前,CML的治疗方法主要有化疗、干扰素治疗、STi571(甲磺酸伊马替尼)、干细胞移植等。传统的化疗手段虽能改善症状,缓解病情,但不能阻止疾病的转化,不能从根本上消除恶性克隆;干扰素治疗血液学缓解率70～80%,存活期延长72个月;异基因骨髓移植(allo-BMT)是目前最有希望治愈慢粒的方法,但有30%～35%患者移植后出现与移植物抗宿主病(GVHD)相关的致命性并发症,另外,患者本身的年龄要求以及不到1/3的患者可找到配型相一致的同胞骨髓供者等因素限制了其临床应用;自体骨髓移植(ABMT)相关并发症发生率较低,不受年龄限制,但缺乏有效的体外骨髓净化措施以预防残留白血病细胞导致的复发。STi571是一种bcr-abl融合蛋白酪氨酸激酶抑制剂,STi571单独或者联合干扰素治疗有效的CML患者,血液学缓解率可以达到90%,已经肯定能延长CML患者生存期,这为CML的靶向治疗开辟了一个新的领域,但是STi571的耐药性问题至今无法解决。目前,众多的临床及实验研究表明,bcr-abl融合基因及其表达产物bcr-abl融合蛋白是慢性粒细胞性白血病治疗上的特异性靶位[8, 9]。随着对bcr-abl癌蛋白的结构和功能的不断研究,人们逐渐认识到在不同的结构域水平对该蛋白的结构和功能进行干预,同样也可以抑制bcr-abl蛋白酪氨酸激酶的活性,这也提示人们继续探索更好的慢粒的治疗方法。ABD(肌动蛋白结合域,actin binding domain)区是bcr-abl融合蛋白上C末端的一个结构域[10],研究表明,ABD在BCR/ABL PTK介导的白血病生成中起重要作用[11-13],白血病的发生依赖于碳末端的ABD存在,ABD变异可以降低体外肿瘤细胞的生存活力[11],而在体内则延迟肿瘤的发病时间,因此如果能够有效调控ABD区,就有可能控制bcr-abl融合蛋白的转化活性,从而达到治疗CML的目的。本实验通过分子生物学方法得到ABD片断,进行融合表达,对ABD的功能进行研究,从而为白血病的靶向治疗奠定新的分子基础。
实验目的体外获得bcr-abl融合基因的ABD区结构域,进行原核表达,并将融合表达蛋白作用于白血病细胞系,进一步探讨其对白血病的细胞周期和增殖的影响,为探索CML新的药物作用靶点和新的治疗药物奠定了基础。
实验方法根据GenBank中人ABD结构域基因的碱基序列,按照引物设计原则,针对ABD基因设计引物。以带有人类慢性粒细胞白血病bcr-abl基因全长的PGD210质粒[14]为模板,应用PCR技术扩增出ABD基因的编码序列,测序结果证实与GenBank中完全一致;将ABD基因的插入到融合有PTD(蛋白转导结构域,protein tansductong domain)基因片段的原核表达载体pET32a中,以获得PTD-ABD融合基因。将构建重组原核表达载体pET32a-PTD-ABD转化感受态细胞大肠杆菌BL21后,予以IPTG诱导,改变诱导条件,使目的蛋白PTD-ABD在上清中有较大量表达。大量诱导细菌,用次氮基三乙酸镍琼脂糖(Ni-NTA agarose)亲和层析珠对可溶性目的蛋白进行纯化。将目的蛋白PTD-ABD与K562细胞和Hela,HL60细胞共培养,用免疫荧光和Western blot的方法观察和验证目的蛋白的跨膜转运情况;用光学显微镜和细胞计数研究细胞形态和生长曲线的变化; MTT、流式细胞仪技术研究PTD-ABD融合蛋白对K562细胞和HL60细胞的增殖和凋亡的作用。
实验结果ABD结构域基因被有效的扩增,DNA序列分析表明所构建的PTD -ABD融合基因的表达质粒与预期设计相同,ABD、PTD、PTD-ABD融合蛋白得到正确的表达。且PTD-ABD目的蛋白成功导入K562细胞和HL60中,能显著抑制HL60细胞的生长,并且对HL60细胞的增殖具有明显的抑制作用。
实验讨论本研究成功地构建了原核表达载体pET32a-PTD-ABD,并顺利表达PTD-ABD融合蛋白于上清中。在功能实验中,研究了目的蛋白对K562,HL60细胞的作用。这一实验结果为下一步研究目的蛋白的作用机制,以及为探索CML新的治疗药物和新的药物作用靶点奠定了基础。
Background: Chronic myeloid leukaemia (CML) is a rare disease with an incidence of 1 or 2 cases per 105 people every year. It is classified into 3 stages: the chronic stage, the accelerate stage, the blast crisis. The median survival duration is 3-4 years, 6-9 years, 3-6 years respectively.
CML is a malignant haemopoietic stem cell disorder. 1845, CML was identified by Craigie and Bennet.1960, Nowell and Hunger-ford found the abnormal chromosome in the CML patients in Philadelphia, the Ph chromosome (Ph) was named then. 1973, Rowley conformed that Ph was characterized by the t (9; 22) (q34; q11) reciprocal chromosomal translocation. 1983 ,researchers confirmed that Ph is cytogenetic hall- mark ,a shortened chromosome 22 that arises from a reciprocal translocation, t(9;22) (q34;q11), which gives origin to a hybrid bcr-abl oncogene, encoding a BCR-ABL(p210) fusion protein with elevated tyrosine kinase activity, and its functional consequence is the Bcr-Abl oncoprotein. Most clinic and laboratary researches provided direct evidences that BCR-ABL is probabaly the main cause of CML, and this fusion gene and BCR–ABL (p210) fusion protein are the main target in the therapy for CML.
Currently, there are many methods to treat CML, including chemotherapy, interferon, STi571, stem cell transplant and so on. Conventional chemotherapy can usually control the overall tumor burden during the chronic phase, with resolution of symptoms. However, such treatment could not prevent disease progression or kill the malignant cells. Interferon-αhas been used in CML therapy for many years, however the hematology remission rate is only 70-80 percent . Survival time of cases only can be extended for 72 months. Allogeneic bone marrow transplatantion (allo-BMT) is the only treatment which has been shown to be curative potential in CML. However, some fatal complications often develop, such as graft -versus-host disease (GVHD) in 30%-35% percent of patients. In addition, the requirements for patient’s age and histocompatibility (only 30 percent patients can find a matched sibling donor) limit its applicability. The majority of patients are not eligible for this treatment. Autologous bone marrow transplantation (ABMT) has a lower accidence of complications than allo-BMT, and may become a standard treatment for those patients ineligible for sibling domor allogeneic BMT.
With the advancement of the construction and function of bcr-abl fusion protein reseaches, scientists have found that blocking some domain’s fuction of the BCR-ABL protein may be an effective method for inhibiting CML cell growth. Maybe this method is hopeful to cure CML in the future.
ABD (actin binding domain), is a C-terminal domain of BCR-ABL protein. It is required for CML pathogenesis. Therefore, inhibition of ABD represents a potential strategy for inhibiting BCR-ABL oncogencity. It also can helpful to find a new clinical therapeutic medicine or a new target of CML.
Aim: To study the ABD’s fouctions of BCR-ABL protein on cell cycle and apoptosis of K562 cell and HL60 cell, and to provide the experimental basis for new therapy of CML.
Methods: The ABD gene was amplified from PGD210 plasmid. In order to obtain PTD-ABD fusion gene, the ABD and PTD gene were successively inserted into pET32a vector. The recombination plasmids were transformed into E.coli BL21 and the expression of the fusion protein was induced by IPTG. Fusion protein was purificated from the surpernatant of cell lysates via Ni-NTA affinity chromatography. The purified fusion protein was loaded to cultured K562 and HL60 cells. Whether the fusion protein entered into K562 cells and Hela cells was detected by western blot and immunofluorescence. Growth curve of K562 and HL60 cells treated by PTD-ABD fusion protein was determined by MTT assay. The apoptosis of PTD-ABD fusion protein on K562 and HL60 cells was investigated by flow cytometry.
Results: The ABD gene was effectively amplified by the method of PCR. The DNA sequencing result showed that the constructed plasmid containing PTD-ABD fusion gene was the same as that designed. The fusion protein was successfully expressed in prokaryotic plasmid, PTD-ABD protein was shifted into both adherent cell and suspension cell successfully.
PTD-ABD fusion protein can significantly inhibit the growth of HL60 cell,and could accelerate the apoptosis of HL60 cell.
Conclusion: The ABD gene has been successfully amplified and expressed, and stuy to the effects of PTD-ABD fusion protein on K562 and HL60 cells was done. The results of the experiment provide a basis to find a new therapeutic target of CML.
CML病人经历慢性期、加速期、急变期三个阶段,各自的中位数时间分别为3～4年;6～9个月;3～6个月[7]。目前,CML的治疗方法主要有化疗、干扰素治疗、STi571(甲磺酸伊马替尼)、干细胞移植等。传统的化疗手段虽能改善症状,缓解病情,但不能阻止疾病的转化,不能从根本上消除恶性克隆;干扰素治疗血液学缓解率70～80%,存活期延长72个月;异基因骨髓移植(allo-BMT)是目前最有希望治愈慢粒的方法,但有30%～35%患者移植后出现与移植物抗宿主病(GVHD)相关的致命性并发症,另外,患者本身的年龄要求以及不到1/3的患者可找到配型相一致的同胞骨髓供者等因素限制了其临床应用;自体骨髓移植(ABMT)相关并发症发生率较低,不受年龄限制,但缺乏有效的体外骨髓净化措施以预防残留白血病细胞导致的复发。STi571是一种bcr-abl融合蛋白酪氨酸激酶抑制剂,STi571单独或者联合干扰素治疗有效的CML患者,血液学缓解率可以达到90%,已经肯定能延长CML患者生存期,这为CML的靶向治疗开辟了一个新的领域,但是STi571的耐药性问题至今无法解决。目前,众多的临床及实验研究表明,bcr-abl融合基因及其表达产物bcr-abl融合蛋白是慢性粒细胞性白血病治疗上的特异性靶位[8, 9]。随着对bcr-abl癌蛋白的结构和功能的不断研究,人们逐渐认识到在不同的结构域水平对该蛋白的结构和功能进行干预,同样也可以抑制bcr-abl蛋白酪氨酸激酶的活性,这也提示人们继续探索更好的慢粒的治疗方法。ABD(肌动蛋白结合域,actin binding domain)区是bcr-abl融合蛋白上C末端的一个结构域[10],研究表明,ABD在BCR/ABL PTK介导的白血病生成中起重要作用[11-13],白血病的发生依赖于碳末端的ABD存在,ABD变异可以降低体外肿瘤细胞的生存活力[11],而在体内则延迟肿瘤的发病时间,因此如果能够有效调控ABD区,就有可能控制bcr-abl融合蛋白的转化活性,从而达到治疗CML的目的。本实验通过分子生物学方法得到ABD片断,进行融合表达,对ABD的功能进行研究,从而为白血病的靶向治疗奠定新的分子基础。
实验目的体外获得bcr-abl融合基因的ABD区结构域,进行原核表达,并将融合表达蛋白作用于白血病细胞系,进一步探讨其对白血病的细胞周期和增殖的影响,为探索CML新的药物作用靶点和新的治疗药物奠定了基础。
实验方法根据GenBank中人ABD结构域基因的碱基序列,按照引物设计原则,针对ABD基因设计引物。以带有人类慢性粒细胞白血病bcr-abl基因全长的PGD210质粒[14]为模板,应用PCR技术扩增出ABD基因的编码序列,测序结果证实与GenBank中完全一致;将ABD基因的插入到融合有PTD(蛋白转导结构域,protein tansductong domain)基因片段的原核表达载体pET32a中,以获得PTD-ABD融合基因。将构建重组原核表达载体pET32a-PTD-ABD转化感受态细胞大肠杆菌BL21后,予以IPTG诱导,改变诱导条件,使目的蛋白PTD-ABD在上清中有较大量表达。大量诱导细菌,用次氮基三乙酸镍琼脂糖(Ni-NTA agarose)亲和层析珠对可溶性目的蛋白进行纯化。将目的蛋白PTD-ABD与K562细胞和Hela,HL60细胞共培养,用免疫荧光和Western blot的方法观察和验证目的蛋白的跨膜转运情况;用光学显微镜和细胞计数研究细胞形态和生长曲线的变化; MTT、流式细胞仪技术研究PTD-ABD融合蛋白对K562细胞和HL60细胞的增殖和凋亡的作用。
实验结果ABD结构域基因被有效的扩增,DNA序列分析表明所构建的PTD -ABD融合基因的表达质粒与预期设计相同,ABD、PTD、PTD-ABD融合蛋白得到正确的表达。且PTD-ABD目的蛋白成功导入K562细胞和HL60中,能显著抑制HL60细胞的生长,并且对HL60细胞的增殖具有明显的抑制作用。
实验讨论本研究成功地构建了原核表达载体pET32a-PTD-ABD,并顺利表达PTD-ABD融合蛋白于上清中。在功能实验中,研究了目的蛋白对K562,HL60细胞的作用。这一实验结果为下一步研究目的蛋白的作用机制,以及为探索CML新的治疗药物和新的药物作用靶点奠定了基础。
Background: Chronic myeloid leukaemia (CML) is a rare disease with an incidence of 1 or 2 cases per 105 people every year. It is classified into 3 stages: the chronic stage, the accelerate stage, the blast crisis. The median survival duration is 3-4 years, 6-9 years, 3-6 years respectively.
CML is a malignant haemopoietic stem cell disorder. 1845, CML was identified by Craigie and Bennet.1960, Nowell and Hunger-ford found the abnormal chromosome in the CML patients in Philadelphia, the Ph chromosome (Ph) was named then. 1973, Rowley conformed that Ph was characterized by the t (9; 22) (q34; q11) reciprocal chromosomal translocation. 1983 ,researchers confirmed that Ph is cytogenetic hall- mark ,a shortened chromosome 22 that arises from a reciprocal translocation, t(9;22) (q34;q11), which gives origin to a hybrid bcr-abl oncogene, encoding a BCR-ABL(p210) fusion protein with elevated tyrosine kinase activity, and its functional consequence is the Bcr-Abl oncoprotein. Most clinic and laboratary researches provided direct evidences that BCR-ABL is probabaly the main cause of CML, and this fusion gene and BCR–ABL (p210) fusion protein are the main target in the therapy for CML.
Currently, there are many methods to treat CML, including chemotherapy, interferon, STi571, stem cell transplant and so on. Conventional chemotherapy can usually control the overall tumor burden during the chronic phase, with resolution of symptoms. However, such treatment could not prevent disease progression or kill the malignant cells. Interferon-αhas been used in CML therapy for many years, however the hematology remission rate is only 70-80 percent . Survival time of cases only can be extended for 72 months. Allogeneic bone marrow transplatantion (allo-BMT) is the only treatment which has been shown to be curative potential in CML. However, some fatal complications often develop, such as graft -versus-host disease (GVHD) in 30%-35% percent of patients. In addition, the requirements for patient’s age and histocompatibility (only 30 percent patients can find a matched sibling donor) limit its applicability. The majority of patients are not eligible for this treatment. Autologous bone marrow transplantation (ABMT) has a lower accidence of complications than allo-BMT, and may become a standard treatment for those patients ineligible for sibling domor allogeneic BMT.
With the advancement of the construction and function of bcr-abl fusion protein reseaches, scientists have found that blocking some domain’s fuction of the BCR-ABL protein may be an effective method for inhibiting CML cell growth. Maybe this method is hopeful to cure CML in the future.
ABD (actin binding domain), is a C-terminal domain of BCR-ABL protein. It is required for CML pathogenesis. Therefore, inhibition of ABD represents a potential strategy for inhibiting BCR-ABL oncogencity. It also can helpful to find a new clinical therapeutic medicine or a new target of CML.
Aim: To study the ABD’s fouctions of BCR-ABL protein on cell cycle and apoptosis of K562 cell and HL60 cell, and to provide the experimental basis for new therapy of CML.
Methods: The ABD gene was amplified from PGD210 plasmid. In order to obtain PTD-ABD fusion gene, the ABD and PTD gene were successively inserted into pET32a vector. The recombination plasmids were transformed into E.coli BL21 and the expression of the fusion protein was induced by IPTG. Fusion protein was purificated from the surpernatant of cell lysates via Ni-NTA affinity chromatography. The purified fusion protein was loaded to cultured K562 and HL60 cells. Whether the fusion protein entered into K562 cells and Hela cells was detected by western blot and immunofluorescence. Growth curve of K562 and HL60 cells treated by PTD-ABD fusion protein was determined by MTT assay. The apoptosis of PTD-ABD fusion protein on K562 and HL60 cells was investigated by flow cytometry.
Results: The ABD gene was effectively amplified by the method of PCR. The DNA sequencing result showed that the constructed plasmid containing PTD-ABD fusion gene was the same as that designed. The fusion protein was successfully expressed in prokaryotic plasmid, PTD-ABD protein was shifted into both adherent cell and suspension cell successfully.
PTD-ABD fusion protein can significantly inhibit the growth of HL60 cell,and could accelerate the apoptosis of HL60 cell.
Conclusion: The ABD gene has been successfully amplified and expressed, and stuy to the effects of PTD-ABD fusion protein on K562 and HL60 cells was done. The results of the experiment provide a basis to find a new therapeutic target of CML.
引文
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