摘要
背景:骨髓间充质干细胞在体外易分离和扩增,还易于外源基因的转入及表达,是一种理想的治疗性细胞和基因治疗的靶细胞。目的:观察脂质体介导胞嘧啶脱氨酶基因转染兔骨髓间充质干细胞及其表达。设计、时间及地点:单一样本观察的细胞基因工程实验,于2006-03/2007-04在大连理工大学干细胞与组织工程研发中心完成。材料:5月龄新西兰大白耳兔用于骨髓间充质干细胞的分离培养。方法:采用密度梯度离心法分离骨髓间充质干细胞。以大肠杆菌JM109基因组DNA为模板,用PCR方法获得目的基因片段,定向克隆至载体pMD19-T,限制性内切酶消化鉴定、基因测序后,构建pIRES2-AcGFP1-CD真核表达质粒。酶切鉴定后,采用Lipofectamine2000介导胞嘧啶脱氨酶基因转染骨髓间充质干细胞。主要观察指标:倒置荧光显微镜下观察绿色荧光蛋白表达情况。结果:成功克隆了胞嘧啶脱氨酶基因,基因测序结果同Genbank中公布的序列一致。将胞嘧啶脱氨酶基因亚克隆到pIRES2-AcGFP1质粒上,构建了pIRES2-AcGFP1-CD真核表达载体。利用脂质体介导法将胞嘧啶脱氨酶基因转染骨髓间充质干细胞,24h后在倒置荧光显微镜下观察到绿色荧光蛋白的表达。结论:pIRES2-AcGFP1-CD真核表达载体已成功转入骨髓间充质干细胞中,说明骨髓间充质干细胞有望成为胞嘧啶脱氨酶基因治疗中的理想载体。
BACKGROUND： Bone marrow mesenchymal stem cells （BMSCs） are easy to isolate and amplify in vitro, and easy for transferring and expressing of exogenous gene. BMSCs are ideal therapeutic cells and target cells for gene therapy. OBJECTIVE： To investigate transfection and expression of liposome-mediated cytosine deaminase gene in rabbit BMSCs. DESIGN, TIME AND SETTING： The single sample cell gene engineering experiment was performed at the Dalian Research ＆ Development Center for Stem Cells and Tissue Engineering, Dalian University of Technology from March 2006 to April 2007. MATERIALS： New Zealand big-eared rabbits aged 5 months were used for isolation and culture of BMSCs. METHODS： BMSCs were harvested by density gradient centrifugation. The cytosine deaminase gene was obtained from E.coli JM 109 genome DNA by polymerase chain reaction. The fragment was cloned into pMD 19-T vector. Restriction enzyme BamHI/Xhol digestion analysis and DNA sequence analysis showed that CD gene was identical with the published sequence. Constructing the plRES2-AcGFP1-CD plasmid and identified by Restriction enzyme BamHl/Xhol digestion analysis. Lipofectamine 2000-mediated cytosine deaminase gene was used to transfect BMSCs. MAIN OUTCOME MEASURE： Expression of green fluorescent protein was identified by inverted fluorescent microscope. RESULTS： We cloned cytosine deaminase gene and the results from gene sequencing were the same as Genbank. Eukaryotic expression plasmid was constructed after cytosine deaminase gene was cloned on plRES2-AcGFP1 plasmid. Cytosine deaminase gene was transfected on BMSCs by liposome-mediated method. Twenty-four hours later, expression of green fluorescent protein was measured with an inverted fluorescent microscope. CONCLUSION： plRES2-AcGFPI-CD eukaryotic expression vector has been successfully transfected into BMSCs, which indicates that BMSCs can be an ideal vector for cytosine deaminase gene therapy.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第38期7525-7530,共6页
Journal of Clinical Rehabilitative Tissue Engineering Research
