本文選題：胞嘧啶脫氨酶 + 基因轉(zhuǎn)染��； 參考：《大連理工大學(xué)》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(dá)的CD酶可以使抗真菌藥5-氟胞嘧啶(5-FC)脫氨基轉(zhuǎn)化成具有細(xì)胞毒性的化療藥物5-氟胞嘧啶(5-FU)。將外源性CD基因轉(zhuǎn)移到腫瘤細(xì)胞，給予前藥5-FC，CD基因可將對(duì)真核細(xì)胞相對(duì)無毒的5-氟胞嘧啶(5-FC)脫氨基轉(zhuǎn)化成腫瘤化療藥5-氟胞嘧啶(5-FU)，從而在腫瘤局部產(chǎn)生高濃度毒性作用以殺傷腫瘤細(xì)胞而全身反應(yīng)較輕。 骨髓間充質(zhì)干細(xì)胞是目前研究較多的干細(xì)胞之一。其具有強(qiáng)大的自我復(fù)制和多向分化潛能；取材容易；能迅速進(jìn)行體外培養(yǎng)和增殖；不存在免疫反應(yīng)和倫理問題；且能夠轉(zhuǎn)染和長(zhǎng)期表達(dá)外源性基因，是多種疾病細(xì)胞移植治療和基因治療的理想載體細(xì)胞。 本文選用兔骨髓間充質(zhì)干細(xì)胞做為載體細(xì)胞介導(dǎo)CD熒光真核表達(dá)載體的表達(dá)，旨在構(gòu)建可在真核細(xì)胞中表達(dá)CD的真核熒光表達(dá)質(zhì)粒，并且轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞，為進(jìn)一步開展CD基因治療中樞神經(jīng)系統(tǒng)疾病奠定實(shí)驗(yàn)基礎(chǔ)。 根據(jù)CD基因開放閱讀框的5’端和3’端設(shè)計(jì)CD特異性引物，分別引入了XhoI和BamHI酶切位點(diǎn)，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(jīng)TA克隆法與克隆載體pMD19-T連接，轉(zhuǎn)化DH5α感受態(tài)細(xì)胞。在含氨卞青霉素的LB平板上篩選出陽性質(zhì)�？寺�，限制性內(nèi)切酶消化鑒定、基因測(cè)序后，將其定向亞克隆至表達(dá)載體，構(gòu)建pIRES2-AcGFP1-CD真核表達(dá)質(zhì)粒。酶切鑒定后，采用Lipofectamine 2000脂質(zhì)體介導(dǎo)法轉(zhuǎn)染兔骨髓間充質(zhì)干細(xì)胞。經(jīng)轉(zhuǎn)染后24h的骨髓間充質(zhì)干細(xì)胞在倒置熒光顯微鏡下可見綠色熒光，，說明轉(zhuǎn)染成功。脂質(zhì)體介導(dǎo)pIRES2-AcGFP1-CD真核表達(dá)質(zhì)粒轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞的方法簡(jiǎn)單、效率高、易成功，是一較為理想的基因轉(zhuǎn)染方法，同時(shí)骨髓間充質(zhì)干細(xì)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(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 郭惠蘭,謝華,謝東北,劉錫儀,黃紹軒;兔骨髓間充質(zhì)干細(xì)胞分化成骨及VEGF表達(dá)[J];解剖學(xué)研究;2003年04期

2 錢偉峰,趙英宏,黃宗海;大腸癌的自殺基因治療實(shí)驗(yàn)研究進(jìn)展[J];國(guó)外醫(yī)學(xué)(腫瘤學(xué)分冊(cè));2000年02期

3 姚遠(yuǎn),王和,彭芝蘭,劉珊玲,李清麗,姚先瑩;CD基因聯(lián)合5-FC治療人卵巢癌裸鼠移植瘤的實(shí)驗(yàn)研究[J];華西醫(yī)科大學(xué)學(xué)報(bào);2002年04期

4 沈歷宗,吳文溪,華一兵,丁強(qiáng),陳濤;胞嘧啶脫氨酶基因旁觀者效應(yīng)與宿主免疫狀態(tài)關(guān)系[J];臨床腫瘤學(xué)雜志;2004年03期

5 姬舒榮,劉暉,張奕,顧琴龍,劉炳亞,朱正綱,林言箴;CD/5-FC系統(tǒng)對(duì)小鼠胃癌細(xì)胞殺傷作用的研究[J];實(shí)用癌癥雜志;2002年05期

6 王新紅,殷蓮華;胞嘧啶脫氨酶基因/5-氟胞嘧啶聯(lián)合在腫瘤基因治療中的研究[J];中國(guó)病理生理雜志;2000年04期

7 潘雪,李兆申,許國(guó)銘,崔龍,戴觀榮,龔燕芳,屠振興;CD/5-FC系統(tǒng)對(duì)胰腺癌裸鼠移植瘤的抗血管形成作用的初步探討[J];中華肝膽外科雜志;2002年12期

8 殷蓮華,付四清,王新紅,T.Nanakorn,Jongho Won,A.Deisserot;腺病毒載體介導(dǎo)胞嘧啶脫氨酶基因轉(zhuǎn)染的前列腺癌細(xì)胞株對(duì) 5-氟胞嘧啶作用敏感性的研究(英文)[J];Chinese Medical Journal;2001年09期

9 張正望,殷蓮華,張永康,趙鳳娣;原位CD基因轉(zhuǎn)導(dǎo)配合全身應(yīng)用5-FC對(duì)于前列腺癌原位移植瘤模型腫瘤和轉(zhuǎn)移的抑制作用(英文)[J];Chinese Medical Journal;2002年02期

10 曹慧青,孟憲敏,劉冬青,趙秀文,丁金鳳;Killing effect of coexpressing cytosine deaminase and thymidine kinase on rat vascular smooth muscle cells[J];Chinese Medical Journal;2004年10期

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