EBV体外转染人B淋巴细胞方法的优化及B淋巴母细胞样细胞系的体外建立
摘要
B淋巴细胞是机体体液免疫的主要效应细胞,以表达和分泌免疫球蛋白为特征。从某种意义上讲,机体的体液免疫应答过程就是B细胞针对特异抗原而活化、增殖和分化过程。检测B细胞特性及免疫球蛋白,了解机体体液免疫状况,对于探讨各种疾病的免疫发病机理及药物治疗效应均有重要的临床意义;分析B细胞发育不同阶段的表面标记,了解B细胞发育和分化阶段的特性与疾病的关系,可为临床提供诊治相关疾病的有用资料。
EB病毒(Epstein-Barr Virus,EBV)是一种γ亚科疱疹病毒,它与人类很多恶性疾病有关,特别是上皮和淋巴起源的肿瘤。EBV在体外能感染正常静止的B细胞,使它们变成永生化的淋巴细胞系(LCL)。由于EBV的这一特性,目前已被广泛应用于多种细胞的永生化,转化的细胞株染色体稳定,保留了原有的遗传性状。病毒转化后的B淋巴母细胞样细胞系(B-LCLs)依然保存了B细胞的分化特征和免疫表型,作为抗原提呈细胞,它可以摄取加工抗原,并将其载体部分表达于细胞表面,供T细胞受体识别;也可作为抗体基因的来源,将其与骨髓瘤细胞融合,制备分泌相应的单克隆抗体的杂交瘤细胞株。近年尤其值得关注的是,利用B-LCLs生物学特性作为人源化抗体的生产体系。
CpG基序(motif)可以活化免疫细胞,激发机体产生Th1型为主的免疫应答。目前的研究已证明:CpG寡核苷酸能够活化人类B淋巴细胞,诱导多种细胞因子和细胞表面免疫相关分子的表达。B细胞上高表达重要的免疫分子CD40,CD40mAb与B细胞上的CD40分子发生交联,而使B细胞免于凋亡和增加生存能力。因此本实验旨在探讨CpG基序和CD40激发型抗体(5C11)对EBV转化的作用,从而优化B-LCLs建系的方法,对于体外利用和研究B淋巴细胞株具有重要的理论价值和潜在的运用意义。
第一部分EBV体外转染人B细胞方法的优化
目的探讨体外建立健康人外周血的B淋巴母细胞样细胞系(LCLs)的优化方法及其影响因素。方法用EB病毒感染健康人外周血中分离的单个核细胞(PBMC),加入CD40激发型抗体(5C11)、CpG DNA免疫调节基序以诱导B淋巴细胞增殖,环胞菌素A(CysA)抑制T淋巴细胞的活性。光学显微镜下观察LCLs的形态特征,利用流式细胞术分析LCLs膜表面分子CD19的表达水平。结果PBMC经EBV感染3周后转化成永生化B淋巴母细胞系。转化后的B淋巴母细胞体积增大积聚成团,可进一步分裂增殖并长期传代培养。CD40激发型抗体(5C11)及CpG免疫调节基序联合运用,明显地提高了EBV转化效率,转化后的LCLs保持了成熟B淋巴细胞的生物学特征。结论CD40信号激发和CpG免疫调节基序联合运用有效地促进EBV对人B淋巴细胞的转化及体外建系。
第二部分人B淋巴母细胞系的体外建立及生物学活性鉴定
目的体外建立健康人外周血的B淋巴母细胞样细胞系(LCLs)并鉴定其生物学活性。方法光学显微镜下观察,并做Giemsa染色,观察LCLs的形态特征;提取LCLs的总RNA,检测EB病毒潜伏膜蛋白基因LMP1的存在;利用流式细胞术分析LCLs膜表面分子CD19、CD40的表达水平。结果PBMC经EBV感染3周后转化成永生化B淋巴母细胞系。转化后的B淋巴母细胞体积增大积聚成团,可进一步分裂增殖并长期传代培养。转化后的LCLs含有LMP1基因,并保持了成熟B淋巴细胞的生物学特征。结论用EBV体外转染方法成功建立B淋巴母细胞样细胞系,转化后的B淋巴母细胞能持续分裂,并保持了成熟B淋巴细胞的生物学特性。
B cells are lymphocytes that play a critical role in the humoral immune response(HIR). In this case, the HIR is a process that B cells recognition of antigen undergo activation, proliferation and differentiation. The study of B cell characters and immunoglobulin secretion has significant clinical value for immune diseases pathogenesis and medical therapy. To detect the cell surface markers in different stages of B cell activation and proliferation, could be helpful for the comprehension of correlations between B cells and diseases and clinical treatments for deseases.
Epstein-Barr virus (EBV), a prominent member ofγ-herpesviruses, causes many human malignant diseases, especially tumors derived from epithelial or lymphocytic origin. It could infect primary resting B lymphocytes and transform B cells into EBV-immortalized LCLs. As a result, EBV is thought to be an effective method for cell immortalization with stable chromosome and original genetic character. B-LCLs are convenient sources of antibody-secreting cells and antigen-presenting cells. In recent studies, scientists used B-LCLs as an important mean to produce humanized antibodies. CpG motifs could activate human immune cells and induce Th1 type immune response. It has been reported that CpG motif-containing oligodeoxynucleotides could activeate human B lymphocytes, lead to cytokine secretion and cell surface molecules expression. B cells can avoid apoptosis and improve its living ability for the important moleculear CD40 could binds with CD40mAb. We use CpG DNA motifs and agonist CD40mAb(5C11) to raise EBV transfection efficiency. Optimization of transfected human B lymphoblastoid cell lines by EBV makes great sense to study B cells and its application in vitro.
The optimization of transfected human B lymphoblastoid cell lines by EBV in vitro
AIM: Investigation of optimized methods and the influencing factors for the establishment of B lymphoblastoid cell lines (LCLs) derived from human PBMC in vitro. METHODS: The peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and incubated with EB virus (EBV) while agonist CD40mAb(5C11), CpG DNA motifs for stimulating B lymphocytes and cyclospofin A (CyA) for inhibiting T lymphocytes were added at the same time. Morphological characteristics of immortalized B-lymphocytes were observed under optical microscope. Flow cytometry analysis was used to identify the expression of surface molecule CD19 on LCLs. RESULTS: PBMC incubated with EBV for 3 weeks were transformed into immortalized B lymphoblastoid cell lines. These immortalized cells accumulated into groups and were able to be cultured in the long term proliferation. The agonist CD40mAb and CpG DNA motifs markedly promoted the EBV transformation process. EBV-immortalized LCLs maintained the biological features of mature B lymphocytes. CONCLUSION: The combination of CD40 signal activation and CpG DNA motifs could effectively promote the EB virus transformation into human B lymphocytes and cell line establishment.
The establishment of human B lymphoblastoid cell lines in vitro and its biological function
AIM: Establish B lymphoblastoid cell lines (LCLs) derived from healthy human PBMC in vitro and study its biological function. METHODS: Optical microscope was used to observe B lymphoblastoid cells stained by Giemsa technique. We extracted total RNA from LCLs and detected the expression of EpsteinBarr virus (EBV) encoded latent membrane protein 1(LMP1) gene. Flow cytometry analysis identified the expression of surface molecule CD19 and CD40 on LCLs. RESULTS: PBMC incubated with EBV for 3 weeks were transformed into immortalized B lymphoblastoid cell lines. These immortalized cells accumulated into groups and were able to be cultured in the long term proliferation. EBV-immortalized LCLs expressing LMP1 maintained the biological features of mature B lymphocytes. CONCLUSION: LCLs was successfully established by EBV transformation method. The transfected B lymphoblastoid cells not only continue to proliferate but also keep the biological features as mature B cells.
EB病毒(Epstein-Barr Virus,EBV)是一种γ亚科疱疹病毒,它与人类很多恶性疾病有关,特别是上皮和淋巴起源的肿瘤。EBV在体外能感染正常静止的B细胞,使它们变成永生化的淋巴细胞系(LCL)。由于EBV的这一特性,目前已被广泛应用于多种细胞的永生化,转化的细胞株染色体稳定,保留了原有的遗传性状。病毒转化后的B淋巴母细胞样细胞系(B-LCLs)依然保存了B细胞的分化特征和免疫表型,作为抗原提呈细胞,它可以摄取加工抗原,并将其载体部分表达于细胞表面,供T细胞受体识别;也可作为抗体基因的来源,将其与骨髓瘤细胞融合,制备分泌相应的单克隆抗体的杂交瘤细胞株。近年尤其值得关注的是,利用B-LCLs生物学特性作为人源化抗体的生产体系。
CpG基序(motif)可以活化免疫细胞,激发机体产生Th1型为主的免疫应答。目前的研究已证明:CpG寡核苷酸能够活化人类B淋巴细胞,诱导多种细胞因子和细胞表面免疫相关分子的表达。B细胞上高表达重要的免疫分子CD40,CD40mAb与B细胞上的CD40分子发生交联,而使B细胞免于凋亡和增加生存能力。因此本实验旨在探讨CpG基序和CD40激发型抗体(5C11)对EBV转化的作用,从而优化B-LCLs建系的方法,对于体外利用和研究B淋巴细胞株具有重要的理论价值和潜在的运用意义。
第一部分EBV体外转染人B细胞方法的优化
目的探讨体外建立健康人外周血的B淋巴母细胞样细胞系(LCLs)的优化方法及其影响因素。方法用EB病毒感染健康人外周血中分离的单个核细胞(PBMC),加入CD40激发型抗体(5C11)、CpG DNA免疫调节基序以诱导B淋巴细胞增殖,环胞菌素A(CysA)抑制T淋巴细胞的活性。光学显微镜下观察LCLs的形态特征,利用流式细胞术分析LCLs膜表面分子CD19的表达水平。结果PBMC经EBV感染3周后转化成永生化B淋巴母细胞系。转化后的B淋巴母细胞体积增大积聚成团,可进一步分裂增殖并长期传代培养。CD40激发型抗体(5C11)及CpG免疫调节基序联合运用,明显地提高了EBV转化效率,转化后的LCLs保持了成熟B淋巴细胞的生物学特征。结论CD40信号激发和CpG免疫调节基序联合运用有效地促进EBV对人B淋巴细胞的转化及体外建系。
第二部分人B淋巴母细胞系的体外建立及生物学活性鉴定
目的体外建立健康人外周血的B淋巴母细胞样细胞系(LCLs)并鉴定其生物学活性。方法光学显微镜下观察,并做Giemsa染色,观察LCLs的形态特征;提取LCLs的总RNA,检测EB病毒潜伏膜蛋白基因LMP1的存在;利用流式细胞术分析LCLs膜表面分子CD19、CD40的表达水平。结果PBMC经EBV感染3周后转化成永生化B淋巴母细胞系。转化后的B淋巴母细胞体积增大积聚成团,可进一步分裂增殖并长期传代培养。转化后的LCLs含有LMP1基因,并保持了成熟B淋巴细胞的生物学特征。结论用EBV体外转染方法成功建立B淋巴母细胞样细胞系,转化后的B淋巴母细胞能持续分裂,并保持了成熟B淋巴细胞的生物学特性。
B cells are lymphocytes that play a critical role in the humoral immune response(HIR). In this case, the HIR is a process that B cells recognition of antigen undergo activation, proliferation and differentiation. The study of B cell characters and immunoglobulin secretion has significant clinical value for immune diseases pathogenesis and medical therapy. To detect the cell surface markers in different stages of B cell activation and proliferation, could be helpful for the comprehension of correlations between B cells and diseases and clinical treatments for deseases.
Epstein-Barr virus (EBV), a prominent member ofγ-herpesviruses, causes many human malignant diseases, especially tumors derived from epithelial or lymphocytic origin. It could infect primary resting B lymphocytes and transform B cells into EBV-immortalized LCLs. As a result, EBV is thought to be an effective method for cell immortalization with stable chromosome and original genetic character. B-LCLs are convenient sources of antibody-secreting cells and antigen-presenting cells. In recent studies, scientists used B-LCLs as an important mean to produce humanized antibodies. CpG motifs could activate human immune cells and induce Th1 type immune response. It has been reported that CpG motif-containing oligodeoxynucleotides could activeate human B lymphocytes, lead to cytokine secretion and cell surface molecules expression. B cells can avoid apoptosis and improve its living ability for the important moleculear CD40 could binds with CD40mAb. We use CpG DNA motifs and agonist CD40mAb(5C11) to raise EBV transfection efficiency. Optimization of transfected human B lymphoblastoid cell lines by EBV makes great sense to study B cells and its application in vitro.
The optimization of transfected human B lymphoblastoid cell lines by EBV in vitro
AIM: Investigation of optimized methods and the influencing factors for the establishment of B lymphoblastoid cell lines (LCLs) derived from human PBMC in vitro. METHODS: The peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and incubated with EB virus (EBV) while agonist CD40mAb(5C11), CpG DNA motifs for stimulating B lymphocytes and cyclospofin A (CyA) for inhibiting T lymphocytes were added at the same time. Morphological characteristics of immortalized B-lymphocytes were observed under optical microscope. Flow cytometry analysis was used to identify the expression of surface molecule CD19 on LCLs. RESULTS: PBMC incubated with EBV for 3 weeks were transformed into immortalized B lymphoblastoid cell lines. These immortalized cells accumulated into groups and were able to be cultured in the long term proliferation. The agonist CD40mAb and CpG DNA motifs markedly promoted the EBV transformation process. EBV-immortalized LCLs maintained the biological features of mature B lymphocytes. CONCLUSION: The combination of CD40 signal activation and CpG DNA motifs could effectively promote the EB virus transformation into human B lymphocytes and cell line establishment.
The establishment of human B lymphoblastoid cell lines in vitro and its biological function
AIM: Establish B lymphoblastoid cell lines (LCLs) derived from healthy human PBMC in vitro and study its biological function. METHODS: Optical microscope was used to observe B lymphoblastoid cells stained by Giemsa technique. We extracted total RNA from LCLs and detected the expression of EpsteinBarr virus (EBV) encoded latent membrane protein 1(LMP1) gene. Flow cytometry analysis identified the expression of surface molecule CD19 and CD40 on LCLs. RESULTS: PBMC incubated with EBV for 3 weeks were transformed into immortalized B lymphoblastoid cell lines. These immortalized cells accumulated into groups and were able to be cultured in the long term proliferation. EBV-immortalized LCLs expressing LMP1 maintained the biological features of mature B lymphocytes. CONCLUSION: LCLs was successfully established by EBV transformation method. The transfected B lymphoblastoid cells not only continue to proliferate but also keep the biological features as mature B cells.
引文
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