pLVX-CD19-CD28-CD137-TCRζ慢病毒载体构建与鉴定及在B细胞白血病中的应用
发布时间：2018-11-05 09:07
【摘要】：目的:建立PLVX-CD19-CD28-CD137-TCRl慢病毒载体并进行鉴定,讨论构建的慢病毒载体转染目的T细胞在急性B淋巴细胞白血病中的治疗情况,进一步确定CD19靶向抗原在B淋巴细胞白血病治疗的重要作用,联合CD28、CD137抗原共刺激T细胞,加强T细胞增殖能力,发挥更好的抗肿瘤效果。方法:通过基因工程和分子生物学实验技术构建CD19-28-137-TCRl目标基因序列,通过慢病毒载体与基因序列相结合。通过PCR扩增技术、琼脂糖凝胶电泳检测CD19-28-137-TCRl的表达并对目标基因序列进行验证。将构建好的基因载体用CaCL2法体外转染到293FT细胞中,运用免疫荧光的方法48 h时观察目标载体转染效率和进行慢病毒滴度的计算。通过Weston blot方法检测酶切pLVX-CD19-28-137-TCRl在293FT细胞蛋白中的表达。在云南省第一人民医院血液科寻找1名适合并愿意做CAR-T细胞治疗的B-ALL患者,分离病人T淋巴细胞,用pLVX-CD19-28-137-TCRl慢病毒载体进行体外转染T细胞。将CD19-28-137-TCRl抗体基因导入T细胞。扩大培养建立好的CD19-CAR-T细胞。同时,通过此法由北京免疫治疗研究院构建临床治疗所用CD19-CAR-T细胞,对患者进行细胞回输。治疗后,流式细胞术对病人骨髓细胞流式免疫分型,骨髓活检切片免疫组化分析,电镜观察骨髓细胞来判断临床效应。结果:通过PCR扩增的方法检测到CD19-28-137-TCRl得到稳定的表达。通过CaCl2法成功将慢病毒载体转染到293FT细胞中并得到成功包装。荧光显微镜观察慢病毒对T细胞的转染效率近90%,慢病毒滴度计算结果为5.5X107TU/ml。Weston blot方法检测经过酶切质粒包装后的293FT细胞蛋白以及没有进行酶切质粒的293FT细胞蛋白CD19-CAR基因的表达。结果为β-actin蛋白条带很清晰,经过酶切后的质粒蛋白得到在293FT细胞中得到相对较高的表达,而没有经过酶切的CD19单链抗体基因表达不明显。应用CD19-CAR-T在1例急性B淋巴细胞白血病临床治疗结果中,电子显微镜观察骨髓增生活跃,病人经CAR-T治疗后伴有骨髓纤维化产生。CAR-T治疗后比治疗前淋巴细胞亚群淋巴细胞、粒细胞、单核细胞、原始-髓系前体细胞下降,有核红区域细胞、CD34+占有核细胞上升。免疫组化结果为,CD2、CD3、CD5、CD7、CD19、CD20抗体均为阴性不表达,病变细胞表达TdT,部分表达Pax-5抗体以及Ki-67抗体,结果显示为早期病变的B细胞。结论:成功构建pLVX-CD19-28-137-TCRl慢病毒载体,并经过琼脂糖凝胶电泳进行鉴定PLVX-CD19-28-137-TCRl慢病毒载体,WB成功检测慢病毒用293FT细胞得到成功包装,病毒滴度相对较高达到5.5X107TU/ml。pLVX-CD19-28-137-TCRl成功转染目的T细胞,且对B淋巴细胞白血病的治疗起到一定的作用,但副作用伴随有骨髓纤维化。
[Abstract]:Objective: to establish and identify the PLVX-CD19-CD28-CD137-TCRl lentivirus vector and to discuss the therapeutic effect of the constructed lentivirus vector transfection target T cells in acute B lymphocytic leukemia. To further determine the important role of CD19 target antigen in the treatment of B lymphocyte leukemia, combined with CD28,CD137 antigen to stimulate T cells, enhance the ability of T cell proliferation, play a better anti-tumor effect. Methods: the target gene sequence of CD19-28-137-TCRl was constructed by gene engineering and molecular biology experiment, and the gene sequence was combined with lentivirus vector. The expression of CD19-28-137-TCRl was detected by agarose gel electrophoresis and the target gene sequence was verified by PCR amplification. The constructed gene vector was transfected into 293FT cells by CaCL2 method in vitro. The transfection efficiency of the target vector and the calculation of the lentivirus titer were observed by immunofluorescence method at 48 h. The expression of pLVX-CD19-28-137-TCRl in 293FT cells was detected by Weston blot method. A suitable B-ALL patient who was willing to be treated with CAR-T cells was selected in hematology department of the first people's Hospital of Yunnan Province. The T lymphocytes were isolated and transfected with pLVX-CD19-28-137-TCRl lentivirus vector in vitro. CD19-28-137-TCRl antibody gene was introduced into T cells. CD19-CAR-T cells were established by expanded culture. At the same time, the CD19-CAR-T cells for clinical therapy were constructed by this method. After treatment, flow cytometry was used to determine the clinical effects of bone marrow cells by flow cytometry, immunohistochemical analysis of bone marrow biopsy sections and observation of bone marrow cells by electron microscope. Results: stable expression of CD19-28-137-TCRl was detected by PCR amplification. The lentivirus vector was successfully transfected into 293FT cells by CaCl2 and packaged successfully. Fluorescence microscope was used to observe the transfection efficiency of lentivirus on T cells. The results of lentivirus titer calculation were 5.5X107TU/ml.Weston blot method to detect the expression of 293FT cell protein and 293FT cell protein CD19-CAR gene which were packaged by enzyme digestion plasmid and 293FT cell protein without plasmids digested by lentivirus. The results showed that the band of 尾-actin protein was clear, the plasmid protein digested by enzyme was relatively high expression in 293FT cells, but the expression of CD19 single chain antibody gene without enzyme digestion was not obvious. Bone marrow hyperplasia was observed by electron microscopy in a case of acute B lymphocyte leukemia treated with CD19-CAR-T. After CAR-T treatment, the lymphocyte subsets, granulocytes, monocytes, primordial progenitor cells, nucleated red region cells decreased after CAR-T treatment. CD34 has increased in nuclear cells. The results of immunohistochemistry showed that the CD2,CD3,CD5,CD7,CD19,CD20 antibody was negative, and the TdT, partial expression of Pax-5 antibody and the Ki-67 antibody were expressed in the pathological cells. The results showed that the cells were B cells with early pathological changes. Conclusion: pLVX-CD19-28-137-TCRl lentivirus vector was successfully constructed and PLVX-CD19-28-137-TCRl lentivirus vector was identified by agarose gel electrophoresis. WB was successfully packaged with 293FT cells. The titer of the virus was relatively high and the target T cells were successfully transfected with 5.5X107TU/ml.pLVX-CD19-28-137-TCRl, which played a certain role in the treatment of B lymphocytic leukemia, but the side effects were accompanied by bone marrow fibrosis.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7
[Abstract]:Objective: to establish and identify the PLVX-CD19-CD28-CD137-TCRl lentivirus vector and to discuss the therapeutic effect of the constructed lentivirus vector transfection target T cells in acute B lymphocytic leukemia. To further determine the important role of CD19 target antigen in the treatment of B lymphocyte leukemia, combined with CD28,CD137 antigen to stimulate T cells, enhance the ability of T cell proliferation, play a better anti-tumor effect. Methods: the target gene sequence of CD19-28-137-TCRl was constructed by gene engineering and molecular biology experiment, and the gene sequence was combined with lentivirus vector. The expression of CD19-28-137-TCRl was detected by agarose gel electrophoresis and the target gene sequence was verified by PCR amplification. The constructed gene vector was transfected into 293FT cells by CaCL2 method in vitro. The transfection efficiency of the target vector and the calculation of the lentivirus titer were observed by immunofluorescence method at 48 h. The expression of pLVX-CD19-28-137-TCRl in 293FT cells was detected by Weston blot method. A suitable B-ALL patient who was willing to be treated with CAR-T cells was selected in hematology department of the first people's Hospital of Yunnan Province. The T lymphocytes were isolated and transfected with pLVX-CD19-28-137-TCRl lentivirus vector in vitro. CD19-28-137-TCRl antibody gene was introduced into T cells. CD19-CAR-T cells were established by expanded culture. At the same time, the CD19-CAR-T cells for clinical therapy were constructed by this method. After treatment, flow cytometry was used to determine the clinical effects of bone marrow cells by flow cytometry, immunohistochemical analysis of bone marrow biopsy sections and observation of bone marrow cells by electron microscope. Results: stable expression of CD19-28-137-TCRl was detected by PCR amplification. The lentivirus vector was successfully transfected into 293FT cells by CaCl2 and packaged successfully. Fluorescence microscope was used to observe the transfection efficiency of lentivirus on T cells. The results of lentivirus titer calculation were 5.5X107TU/ml.Weston blot method to detect the expression of 293FT cell protein and 293FT cell protein CD19-CAR gene which were packaged by enzyme digestion plasmid and 293FT cell protein without plasmids digested by lentivirus. The results showed that the band of 尾-actin protein was clear, the plasmid protein digested by enzyme was relatively high expression in 293FT cells, but the expression of CD19 single chain antibody gene without enzyme digestion was not obvious. Bone marrow hyperplasia was observed by electron microscopy in a case of acute B lymphocyte leukemia treated with CD19-CAR-T. After CAR-T treatment, the lymphocyte subsets, granulocytes, monocytes, primordial progenitor cells, nucleated red region cells decreased after CAR-T treatment. CD34 has increased in nuclear cells. The results of immunohistochemistry showed that the CD2,CD3,CD5,CD7,CD19,CD20 antibody was negative, and the TdT, partial expression of Pax-5 antibody and the Ki-67 antibody were expressed in the pathological cells. The results showed that the cells were B cells with early pathological changes. Conclusion: pLVX-CD19-28-137-TCRl lentivirus vector was successfully constructed and PLVX-CD19-28-137-TCRl lentivirus vector was identified by agarose gel electrophoresis. WB was successfully packaged with 293FT cells. The titer of the virus was relatively high and the target T cells were successfully transfected with 5.5X107TU/ml.pLVX-CD19-28-137-TCRl, which played a certain role in the treatment of B lymphocytic leukemia, but the side effects were accompanied by bone marrow fibrosis.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7
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