中国组织工程研究与临床康复第13卷第1期 2009–01–01出版
Journal of Clinical Rehabilitative Tissue Engineering Rearch January 1, 2009 Vol.13, No.1 ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH
133 1Department of Orthopedics,
2Department of Neurosurgery,
Second Affiliated Hospital of Dalian Medical University, Dalian 116023, Liaoning Province, China
Wu Chen-huan★, Studying for master’s degree, Department
of Orthopedics,
Second Affiliated Hospital of Dalian Medical University, Dalian 116023, Liaoning Province, China
oldhappy88@sohu.
com
Correspondence to: Wang Hong-fei, Doctor, Professor, Department of Orthopedics, Second Affiliated Hospital of Dalian Medical University, Dalian 116023, Liaoning Province, China
Received: 2008-05-07 Accepted: 2008-11-12
脂质体介导胞嘧啶脱氨酶基因转染鼠骨髓间充质干细胞★
吴陈欢1，王鸿飞1，宋飞2
Transfection of mou bone marrow menchymal stem cells with liposome mediated cytosine deamina genes
Wu Chen-huan1, Wang Hong-fei1, Song Fei2
Abstract
BACKGROUND: The particular bystander effect of suicide gene can remarkably kill tumor cells. It can be ud together with
radiotherapy as well as immune gene therapy, and overcome the defect of low gene transduction efficiency. Cytosine deamina
(CD) can generate a powerful bystander effect.
OBJECTIVE: To obrve the effect of a eukaryotic expression plasmid pIRES2-AcGFP1-CD mediated by liposome transfected
into bone marrow menchymal stem cells (BMSCs) and its gene expression.
DESIGN, TIME AND SETTING: A cytologic experiment of genetic level was performed at Rearch and Development Center of
Stem Cell and Tissue Engineer, Dalian University of Technology from May to December 2007.
MATERIALS: A total of six C57BL mice of SPF degree were provided by Experimental Animal Center of Dalian Medical
University. E. DH5α was provided by Rearch and Development Center of Stem Cell and Tissue Engineer, Dalian University of
Technology. Lipofectamine TM 2000 was the product of Invitrogen, China.
METHODS: The DNA plasmids were extracted from transformed into the competent E. DH5α. pIRES2-AcGFP1-CD plasmid was
identified by BamHI/XhoI double digestion. The BMSCs from mou femur and tibia were cultured and purified by adhesion
method in vitro. Signal cell suspension prepared by BMSCs of the third generation was cultured by adding fluorescence-labeled
CD44, CD45, CD90 and CD105 antibodies. BMSCs of the third generation were transfected by lipofectamine 2000 mediation.
MAIN OUTCOME MEASURES: Identification of recombinant plasmids. The expressions of surface markers on BMSCs were
detected by fluoroscopy. The expressions of CD gene were obrved after transfection.
RESULTS: After agaro gel electrophoresis, a band appeared at 1.0-1.5 kb of the digested products of pIRES2-AcGFP1-CD
plasmids and accorded with the length of CD gene in the length. The cells were negative for CD45, and positive for CD44, CD90,
and CD105. Under the fluorescent inverted pha contrast microscope, the expressions of green fluorescent protein in the
BMSCs were found at 36 hours after transfection. At 48 hours after transfection, the expressions of green fluorescent protein
remained and the intensity incread obviously.
CONCLUSION: The expression of CD gene mediated by liposome in BMSCs is successful, and it reaches the peak 48 hours
after transfection.
Wu CH, Wang HF, Song F. Transfection of mou bone marrow menchymal stem cells with liposome mediated cytosine
deamina genes.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2009;13(1): 133-136(China)
[ ]
摘要
背景：自杀基因独有的旁观者效应，可显著提供肿瘤细胞杀伤效果，同时还与放射治疗、免疫基因治疗联合应用，并克服
零钱英文了基因转导效率低的缺陷。胞嘧啶脱氨酶（cytosine deamina，CD）即可产生强大的旁观者效应。
目的：观察脂质体介导真核表达载体CD基因转染鼠骨髓间充质干细胞的效果及其基因表达。
设计、时间及地点：细胞学基因水平实验，于2007-05/12在大连理工大学干细胞与组织工程研发中心完成。
材料：SPF级C57BL纯系小鼠6只，由大连医科大学实验动物中心提供。连接产物转化感受态大肠杆菌DH5α由大连理
工大学干细胞与组织工程研发中心提供。Lipofectamine TM 2000脂质体为Invitrogen产品。
方法：取连接产物转化感受态大肠杆菌DH5α，提取质粒DNA，对质粒pIRES2-AcGFP1-CD进行XhoI和BamHI双酶切，
用于转染。取小鼠双侧下肢股骨和胫骨，贴壁法分离纯化骨髓间充质干细胞，传至第3代制成单细胞悬液，加入荧光标记
的CD44，CD45，CD90，CD105抗体后，采用Lipofectamine 2000介导法转染第3代骨髓间充质干细胞。
主要观察指标：重组质粒的鉴定，流式细胞仪检测骨髓间充质干细胞表面标记表达，细胞转染后CD基因的表达。
结果：质粒pIRES2-AcGFP1-CD酶切产物经琼脂糖凝胶电泳后，于1.0~1.5 kb处有一条带出现，符合CD基因长度。细
胞表面标记CD45呈阴性，CD44，CD90，CD105呈阳性。pIRES2-AcGFP1-CD基因转染36 h后，荧光倒置相差显微镜
下可见小鼠骨髓间充质干细胞有绿色荧光蛋白的表达，48 h后细胞仍有荧光表达，且强度明显增强。
结论：脂质体介导的CD基因在鼠骨髓间充质干细胞中成功表达，于转染48 h后达峰值。
关键词：骨髓间充质干细胞；胞嘧啶脱氨酶；基因转染；脂质体
检测不到硬盘吴陈欢，王鸿飞，宋飞.脂质体介导胞嘧啶脱氨酶基因转染鼠骨髓间充质干细胞[J].中国组织工程研究与临床康复，2009，
13(1):133-136 [ ]
基础医学
吴陈欢，等.脂质体介导胞嘧啶脱氨酶基因转染鼠骨髓间充质干细胞
P .O. Box 1200, Shenyang 110004
134
www.CRTER
表白大连医科大学第二附属医院，1 骨
外科，2
神经外科，辽宁省大连市 116023
吴陈欢★，男，1982年生，湖北省荆州市人，汉族，大连医科大学在读硕士，主要从事骨组织工程与脊柱损伤方面的研究。
oldhappy88@
通讯作者：王鸿飞，博士，教授，大连医科大学第二附属医院骨外科，辽宁省大连市 116023
中图分类号:R394.2 文献标识码:B
文章编号:1673-8225 (2008)01-00133-04
收稿日期：2008-05-07
修回日期：2008-11-12 (54200805060021/ ZS ·M)
0 引言
骨髓间充质干细胞是一种中胚层来源具有自我更新和多向分化潜能的多能干细胞，取材方便，体内植入后不良反应弱，易于外源基因的表达和转染，已被认为是一种理想的基因治疗载体
[1-11]
。
基因靶向细胞的成功导入至关重要，使用脂质体是基因转染的一种重要方法。自1987年Felgner 等[8]
首次报道以来，一系列广谱高效的脂质体相继诞生。脂质体与细胞膜之间通过“膜间传递、吸附、细胞吞噬和融合”这4种相互作用，完成脂质体携带遗传物质进入靶细胞的基因转移[12]，具有病毒载体无法比拟的安
全性
[13]
，并且不引起人体免疫反应，转染细胞
类型广泛、高效，操作十分简便易行
[14]
。胞嘧
啶脱氨酶（cytosine deamina ，CD ）是大肠杆菌代谢旁路中的一种酶，哺乳动物细胞中不含该酶。
胞嘧啶脱氨酶可将无毒性的原药转化为有细胞毒性的代谢产物，导致细胞自杀性死亡。同时，CD 基因还能产生强大的旁观者效应，杀死除转基因细胞之外的大量未转基因细胞。
实验采取脂质体介导真核表达载体pIRES2-AcGFP1-CD 基因转染鼠骨髓间充质干细胞，观察CD 基因的表达。
1 材料和方法
设计：细胞学基因水平实验。
时间及地点：于2007-05/12在大连理工大学干细胞与组织工程研发中心完成。
材料：SPF 级C57BL 纯系小鼠6只，4周龄，雌雄不限，体质量18~20 g ，由大连医科大学实验动物中心提供，动物质量合格证号：SCXK （辽）2002-0002，实验过程中对动物的处置符合2006年科技部《关于善待实验动物的指导性意见》的规定
[15]
。
实验方法：
质粒的扩增、提取与鉴定：取连接产物转化
感受态大肠杆菌DH5α，按1∶100的比例接种于含50 mg/L 卡那霉素的LB 液体培养基中，37 ℃振荡(220 r/min)培养过夜，取菌液 10 mL ，按QIAGEN 质粒纯化试剂盒说明书提取质粒DNA ，分光光度计测定质粒浓度，对质粒pIRES2-AcGFP1-CD 进行XhoI 和BamHI 双酶切，10 g/L 琼脂糖凝胶电泳鉴定。
骨髓间充质干细胞的分离培养与鉴定：小鼠断
颈处死，以体积分数为0.75的乙醇浸泡2.0~ 3.0 min ，无菌条件下取双侧下肢股骨和胫骨，用1 mL 注射器吸取配好的含100 U/mL 青霉素、100 mg/L 链霉素、体积分数为0.15胎牛血清的LG-DMEM 培养基冲洗骨髓腔。收集冲洗骨髓悬液，1 000 r/min 离心5 min ，去上清，培养液重悬细胞，调整细胞浓度至1×1011
L -1
，转入培养瓶，于37 ℃、体积分数为0.05的CO 2培养箱中培养，48 h 后半量换液，4 d 后再次半量换液，6 d 后全量换液，取出非贴壁细胞，待细胞生长至80%~90%融合时，以1.25 g/L 胰酶~0.25 g/L EDTA 消化传代。取第3代细胞制成单细胞悬液，加入荧光标记的CD44，CD45，CD90，CD105抗体后，用流式细胞仪进行表面标识鉴定。
CD 基因的转染：取第3代骨髓间充质细胞，
以5×105
个/孔接种于24孔板，培养24 h 。取 0.8 μL 重组质粒DNA ，加入50 μL 无抗生素无血清培养基中，室温平衡5 min (A 液)。取 2.0 μL Lipofectamine2000，加入50 μL 无抗生素无血清培养基中，室温平衡5 min(B 液)。将A 、B 液混合，室温孵育30 min 。将孵育后的A 、B 混合液加入24孔板中，置于37 ℃、体积分数为0.05的CO 2培养箱中，培养6 h 后更换成含血清的培养基。分别于36，48 h 后在荧光倒置相差显微镜下观察荧光表达。
主要观察指标：①重组质粒的提取与鉴定结果。②骨髓间充质干细胞的形态观察与表面标记表达。③细胞转染后CD 基因的表达。
设计、实施、评估者：设计为一、三作者，干预实施为第一、二、三作者，结果评估为一、二作者，均经过系统培训，未使用盲法评估。
2 结果
2.1 重组质粒的提取与鉴定结果 质粒pIRES2-AcGFP1-CD 经XhoI 和BamHI 双酶切，
菌种与试剂
连接产物转化感受态大肠杆菌
DH5
α Lipofectamine TM
2000脂质体 质粒纯化试剂盒
低糖DMEM 培养基
胎牛血清
来源
大连理工大学干细胞与 组织工程研发中心 Invitrogen QIAGEN Invitrogen GIBCO
吴陈欢，等.脂质体介导胞嘧啶脱氨酶基因转染鼠骨髓间充质干细胞
ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH
135
www.CRTER
其酶切产物在1%琼脂糖凝胶电泳后，于1.0~1.5 kb 有一条带出现，符合CD 基因长度，见图1。
2.2 骨髓间充质干细胞的形态观察与表面标记表达
2.3 细胞转染后CD基因的表达 pIRES2-AcGFP1-CD 基因转染36 h 后，荧光倒置相差显微镜下可见小鼠骨髓间充质干细胞有绿色荧光蛋白的表达，表明pIRES2-AcGFP1-CD 真核表达载体已成功转入骨髓间充质干细胞中，见图5a ；48 h 后细胞仍有荧光表达，且强度明显增强，见图5b 。
M 1 2
渐渐近义词M: Marker; 1: Identification of pIRES2-AcGFP1-CD by XhoI+BamHI double digestion; 2: Identification of pIRES2-AcGFP1-CD by agaro gel electro-phoresis
Figure 1 Identification of pIRES2-AcGFP1-CD plasmid
图1 pIRES2-AcGFP1-CD 质粒，1为双酶切鉴定结果，2为琼脂糖凝胶电泳结果
拼音占格
Figure 2 Irregular arrangement of cells,spindle cells and little
round cells mainly at the 3rd day after the primary
culture (×200)
图2 原代培养3 d 的骨髓间充质干细胞(×200)
Figure 3 Cell fusion, the vertical arrangement of cells around the center at the7th day after the primary culture(×
200) 图3 原代培养7 d 的骨髓间充质干细胞 (×200) Figure 4 The cells enlargement and cell spreading after the
venth passage culture (×200) 图4 第7代骨髓间充质干细胞(×200)
Figure 5 Bone marrow menchymal stem cells transfected
with pIRES2-AcGFP1 were obrved under
fluorescence microscope （×400） 图5 pIRES2-AcGFP1-CD 转染骨髓间充质干细胞荧光显微镜观察结果（×400）
a: 36 hours after transfection
b: 48 hours after transfection痰湿内阻
吴陈欢，等.脂质体介导胞嘧啶脱氨酶基因转染鼠骨髓间充质干细胞
P .O. Box 1200, Shenyang 110004
136
www.CRTER
3 讨论
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-氟胞嘧啶脱氨基转化成具有细胞毒的化疗药物5-氟尿嘧啶。将外源性CD 基因转移到肿瘤细胞，给予前药5-氟胞嘧啶，CD 基因可将对真核细胞相对无毒的5-氟胞嘧啶脱氨基转化为5-氟尿嘧啶，抑制细胞DNA 合成，杀伤肿瘤细胞
[16-17]
，从而
改善因5-氟尿嘧啶在肿瘤治疗中由于其全身的不良反应较大而应用受限情况。同时CD/5-氟胞嘧啶体系治疗肿瘤时可产生具有临床应用实际价值的旁观者效应
[18]
，
即少量的转基因细胞便可导致更多的肿瘤细胞被抑杀，可能与以下机制有关：细胞间缝隙连接介导的细胞毒性药物或(和)凋亡小体传递[19]
；激活免疫机制
[20]
；瘤体缺
血机制
[21]
。
实验成功地将CD 基因转染骨髓间充质干细胞，36 h 后在荧光显微镜下观察到绿色荧光蛋白的表达，结果表明pIRES2-AcGFP1-CD 真核表达载体已成功转入骨髓间充质干细胞中。
虽然CD 基因作为肿瘤治疗的一种新方法，正日益受到人们的重视，但自杀基因抑癌体系还存在很多
不足，如其可能存在对肿瘤细胞类型的依赖性、转染率有待提高、目的基因如何持续表达与适时终止表达等问题均有待解决。
4 参考文献
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[17] Stefani AL, Barzon L, Castagliuolo I, et al. Systemic efficacy of combined sucide/cytokine gene therapy in a murine model of hepatocellular.J Hepatol.2005;42(5):728-735.
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