本文選題：胞嘧啶脫氨酶 + 基因轉染　； 參考：《大連理工大學》2007年碩士論文

【摘要】： 胞嘧啶脫氨酶(Cytosine deaminase，CD)基因是來源于真菌及大腸桿菌的一種自殺基因，它表達的CD酶可以使抗真菌藥5-氟胞嘧啶(5-FC)脫氨基轉化成具有細胞毒性的化療藥物5-氟胞嘧啶(5-FU)。將外源性CD基因轉移到腫瘤細胞，給予前藥5-FC，CD基因可將對真核細胞相對無毒的5-氟胞嘧啶(5-FC)脫氨基轉化成腫瘤化療藥5-氟胞嘧啶(5-FU)，從而在腫瘤局部產生高濃度毒性作用以殺傷腫瘤細胞而全身反應較輕。 骨髓間充質干細胞是目前研究較多的干細胞之一。其具有強大的自我復制和多向分化潛能；取材容易；能迅速進行體外培養(yǎng)和增殖；不存在免疫反應和倫理問題；且能夠轉染和長期表達外源性基因，是多種疾病細胞移植治療和基因治療的理想載體細胞。 本文選用兔骨髓間充質干細胞做為載體細胞介導CD熒光真核表達載體的表達，旨在構建可在真核細胞中表達CD的真核熒光表達質粒，并且轉染骨髓間充質干細胞，為進一步開展CD基因治療中樞神經系統疾病奠定實驗基礎。 根據CD基因開放閱讀框的5’端和3’端設計CD特異性引物，分別引入了XhoI和BamHI酶切位點，以大腸桿菌JM109基因組DNA為模板，用PCR方法獲得目的基因，將其回收、純化后，經TA克隆法與克隆載體pMD19-T連接，轉化DH5α感受態(tài)細胞。在含氨卞青霉素的LB平板上篩選出陽性質�？寺�，限制性內切酶消化鑒定、基因測序后，將其定向亞克隆至表達載體，構建pIRES2-AcGFP1-CD真核表達質粒。酶切鑒定后，采用Lipofectamine 2000脂質體介導法轉染兔骨髓間充質干細胞。經轉染后24h的骨髓間充質干細胞在倒置熒光顯微鏡下可見綠色熒光，，說明轉染成功。脂質體介導pIRES2-AcGFP1-CD真核表達質粒轉染骨髓間充質干細胞的方法簡單、效率高、易成功，是一較為理想的基因轉染方法，同時骨髓間充質干細胞也有望成為CD基因治療中的理想載體。
[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 偽 competent cells were transformed into DH5 偽 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.
【學位授予單位】：大連理工大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R346

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