寻找基因转染人脂肪来源的成体干细胞合适载体:脂质体介导质粒pEGFP-N1、重组腺病毒Ad5-EGFP与重组腺相关病毒rAAV-2/1-EGFP  

Suitable vehicle for gene transfection into human adipose derived adult stem cells: pEGFP-N1, Ad5-EGFP and rAAV-2/1-EGFP

作　　者：靳小兵[1] 孙永生[1] 娄思权[1] 张克[1] 

机构地区：[1]北京大学第三医院骨科,北京市100083

出　　处：《中国组织工程研究与临床康复》2007年第7期1205-1208,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金(30371431)~~

摘　　要：目的:观察脂质体介导质粒pEGFP-N1、重组腺病毒Ad5-EGFP、重组腺相关病毒rAAV-2/1-EGFP基因转染人脂肪来源成体干细胞后增强型绿色荧光蛋白的表达及细胞毒性,探讨适用于脂肪来源的成体干细胞的转基因方法。方法:实验于2006-01/07在国家人类基因组北方中心和北京大学第三医院完成。全髋关节置换术患者的皮下脂肪由北京大学第三医院骨科提供,均征得患者及其家属同意。pEGFP-N1(Clotech公司),Ad5-EGFP,rAAV-2/1-EGFP(本元正阳公司)。②自成人皮下取少量脂肪组织,经机械剪切及Ⅰ型胶原酶消化后获取脂肪来源的成体干细胞,体外培养扩增。③采用脂质体介导质粒pEGFP-N1、重组腺病毒Ad5-EGFP、重组腺相关病毒rAAV-2/1-EGFP3种转染方法,观察增强型绿色荧光蛋白的表达及对细胞毒性的影响。④细胞转染后24h,将5×104细胞悬液接种于24孔板中,每周换液3次,绘制细胞生长曲线。以正常未转染细胞作为正常对照。观察不同转染方法对细胞增殖的影响。结果:①不同转染方法的感染效率比较:pEGFP-N1脂质体转染后细胞毒性较大,且感染效率低,仅为10.5%。重组腺病毒Ad5-EGFP转染率高,当感染复数为5×102时,感染效率达82.5%;当感染复数低于1×103时,对细胞无明显损害。重组腺相关病毒rAAV-2/1-EGFP转染法不能有效感染人脂肪来源的成体干细胞。②不同转染方法对细胞增殖的影响:重组腺病毒Ad5-EGFP转染和重组腺相关病毒AAV-2/1-EGFP转染人脂肪来源的成体干细胞后,对细胞增殖能力无明显影响,基本同正常对照细胞(P>0.05)。而pEGFP-N1脂质体转染后细胞增殖能力较正常对照明显下降,转染后3～10d差异有显著性意义(P<0.05)。结论:重组腺病毒Ad5-EGFP可作为一种高效的脂肪来源成体干细胞的示踪工具,提示5型复制缺陷型腺病毒是其合适的转基因载体。AIM： To observe the enhanced green fluorescent protein （EGFP） gene expression and cytotoxicity in human adipose derived adult stem cells （hADSCs） by pEGFP-N1, Ad5-EGFP and rAAV-2/1-EGFP, and investigate the suitable gene-transferred vehicle. METHODS： The experiment was conducted a.t Chinese National Human Genome Center and the Third Hospital of Peking University from January to July 2006. （1）After the patients and their relative were informed consent, the subcutaneous adipose tissue was obtained from the patients undergoing routine total hip joint replacement in Department of Orthopaedics, Third Hospital of Peking University. pEGFP-N1 was provided by Clotech Company, Ad5-EGFP and rAAV-2/1-EGFP by Vector Gene Technology Company. （2）hADSCs were cultured in vitro after isolated from the adipose tissue after dissected and digested with type I collagenase. （3）hADSCs of passage 3 were infected with pEGFP-N1, Ad5-EGFP and rAAV-2/1-EGFP and the EGFP expression and the cell toxicity were observed. （4） Twenty-four hours after being transfected, 5×10^4 cells were reseeded in a 24-well plate and the solution was changed three times every week. The growth curves of each group were drawn. Normal non-transfected cells served as control. The influence of different transfection ways on the growth of hADSCs was observed. RESULTS： （1）Comparison of transfected efficiency with different ways： pEGFP-N1 transfection showed a higher cytotoxicity and .lower efficiency of 10.5%; Ad5-EGFP could efficiently transfect hADSCs （multiplicity of infection=5×10^2, 82.5%）; when MOI was 〈1×10^3, Ad5-EGFP transfecUon showed no obvious damage to the cells, rAAV-2/1-EGFP almost could not transfect hADSCs. （2）lnfluence of different transfected ways on cell proliferation： The proliferation activity of hADSCs were not obviously influenced by transfection with Ad5-EGFP and AAv2/1-EGFP, and the cell number was similar to that of the control group （P 〉 0.05）; however, the growth capability of hADSCs w

关 键 词：转基因 腺病毒科 重组 遗传 脂质体 转染 

分 类 号：R394.2[医药卫生—医学遗传学]