pIRES2-AcGFP1-CD真核表达载体的构建及其在MSCs中的表达

发布时间：2018-05-29 20:40

本文选题：胞嘧啶脱氨酶 + 基因转染　；参考：《大连理工大学》2007年硕士论文

【摘要】： 胞嘧啶脱氨酶(Cytosine deaminase，CD)基因是来源于真菌及大肠杆菌的一种自杀基因，它表达的CD酶可以使抗真菌药5-氟胞嘧啶(5-FC)脱氨基转化成具有细胞毒性的化疗药物5-氟胞嘧啶(5-FU)。将外源性CD基因转移到肿瘤细胞，给予前药5-FC，CD基因可将对真核细胞相对无毒的5-氟胞嘧啶(5-FC)脱氨基转化成肿瘤化疗药5-氟胞嘧啶(5-FU)，从而在肿瘤局部产生高浓度毒性作用以杀伤肿瘤细胞而全身反应较轻。骨髓间充质干细胞是目前研究较多的干细胞之一。其具有强大的自我复制和多向分化潜能；取材容易；能迅速进行体外培养和增殖；不存在免疫反应和伦理问题；且能够转染和长期表达外源性基因，是多种疾病细胞移植治疗和基因治疗的理想载体细胞。本文选用兔骨髓间充质干细胞做为载体细胞介导CD荧光真核表达载体的表达，旨在构建可在真核细胞中表达CD的真核荧光表达质粒，并且转染骨髓间充质干细胞，为进一步开展CD基因治疗中枢神经系统疾病奠定实验基础。根据CD基因开放阅读框的5’端和3’端设计CD特异性引物，分别引入了XhoI和BamHI酶切位点，以大肠杆菌JM109基因组DNA为模板，用PCR方法获得目的基因，将其回收、纯化后，经TA克隆法与克隆载体pMD19-T连接，转化DH5α感受态细胞。在含氨卞青霉素的LB平板上筛选出阳性质粒克隆，限制性内切酶消化鉴定、基因测序后，将其定向亚克隆至表达载体，构建pIRES2-AcGFP1-CD真核表达质粒。酶切鉴定后，采用Lipofectamine 2000脂质体介导法转染兔骨髓间充质干细胞。经转染后24h的骨髓间充质干细胞在倒置荧光显微镜下可见绿色荧光，，说明转染成功。脂质体介导pIRES2-AcGFP1-CD真核表达质粒转染骨髓间充质干细胞的方法简单、效率高、易成功，是一较为理想的基因转染方法，同时骨髓间充质干细胞也有望成为CD基因治疗中的理想载体。
[Abstract]:Cytosine deaminase (Cytosine deaminase CDCD) gene is a suicide gene derived from fungi and Escherichia coli. The CD gene expressed by cytosine deaminase can transform 5-fluorocytosine deaminase into 5-fluorocytosine 5-FUU, a cytotoxic chemotherapeutic drug. The exogenous CD gene was transferred to tumor cells. The prodrug 5-FCnCD gene can convert 5-fluorocytosine 5-FC5 deamino, which is relatively non-toxic to eukaryotic cells, into 5-fluorocytosine 5-FUU, a tumor chemotherapeutic drug, thus producing a high concentration of toxic effect in the local tumor to kill tumor cells and less systemic reaction. Bone marrow mesenchymal stem cells are one of the most studied stem cells. It has a strong potential for self-replication and multi-differentiation; easy access to materials; rapid in vitro culture and proliferation; the absence of immune responses and ethical problems; and the ability to transfect and express exogenous genes for a long time. It is an ideal carrier cell for transplantation and gene therapy of many diseases. In this paper, rabbit bone marrow mesenchymal stem cells (BMSCs) were used as vector cells to mediate the expression of CD fluorescence eukaryotic expression vector. The purpose of this study was to construct a eukaryotic fluorescent expression plasmid which could express CD in eukaryotic cells and transfect it into bone marrow mesenchymal stem cells (BMSCs). To lay an experimental foundation for the further development of CD gene therapy for central nervous system diseases. CD-specific primers were designed according to the 5'and 3'end of open reading frame of CD gene. XhoI and BamHI restriction sites were introduced respectively. The target gene was obtained by PCR method using JM109 genomic DNA of E. coli as template, and the target gene was recovered and purified. DH5 伪 competent cells were transformed into DH5 伪 cells by TA cloning and ligation with clone vector pMD19-T. The positive plasmid clones were screened on LB plate containing ampicillin, digested by restriction endonuclease, sequenced and subcloned into the expression vector to construct pIRES2-AcGFP1-CD eukaryotic expression plasmid. Lipofectamine 2000 liposome mediated transfection of rabbit bone marrow mesenchymal stem cells was carried out after restriction endonuclease digestion. The green fluorescence of bone marrow mesenchymal stem cells was observed under inverted fluorescence microscope 24 hours after transfection, indicating that the transfection was successful. Liposome-mediated pIRES2-AcGFP1-CD eukaryotic expression plasmid transfection of bone marrow mesenchymal stem cells (BMSCs) is a simple, efficient and successful method, which is an ideal gene transfection method. At the same time, bone marrow mesenchymal stem cells (BMSCs) are expected to be an ideal vector for CD gene therapy.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

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