人脂肪组织来源基质干细胞成脂分化及Ad-EGFP体外转染研究

Adipogenic differentiation of human's adipose tissue-derived stem cells and transfection of Ad-EGFP in vitro

目的探讨重组腺相关病毒载体能否有效地转染脂肪组织来源基质干细胞及(ADSC)其影响,同时为组织工程寻找一种理想的病毒载体及细胞示踪标记方法。方法采用抽脂法分离培养ADSC,诱导培养液诱导细胞成脂分化,行细胞形态学检查,细胞表面抗原鉴定,油红O染色,评价细胞成脂情况。在此基础上转染Ad-EGFP,观察细胞形态学改变及荧光表达的时间与强度,计算转染效率,确定转染的理想感染复数(MOI)值,同时通过活力和增殖实验进行Ad-EGFP体外转染对ADSC的影响研究。结果细胞扩增迅速,形态良好,经诱导后呈现明显的成脂改变。实验确定Ad转染细胞的较佳MOI值为50,以此值转染Ad-EGFP后细胞荧光持续高效稳定表达,并可传至子代,可以满足示踪标记的需要。Ad转染效率高,对细胞活性影响小。结论ADSC的培养和分化能满足脂肪组织工程的要求;Ad长期稳定表达目的基因,是一种理想的组织工程病毒载体;基因转染方法高效稳定表达EGFP,可作为种子细胞良好的示踪标记方法。

[Objective] To investigate the transfection efficiency and affect of recombinant adenovirus- enhanced green fluorescent protein in human adipose tissue-derived stem cells by means of a simple method of culturing and adipogenic induction in vitro so as to find an ideal viral vector and cell tracing mark for tissue engineering. [Methods] Human adipose tissue were isolated and cultured the ADSCs of human. Adipogenic differentiations of the 4th generation of ADSC was assessed by Oil Red O. The morphology of the cells was observed continuously, and surface markers were detected by flow cytometry. The ADSC of human was infected with adenovirus expressing the enhanced green fluorscent protein（EGFP） gene by different multiphcity of infection. The expression of EGFP was detected by fluorscent microscope. The cytotoxicity of adenovirus to ADSC was evaluated by cell proliferation cell vitality and cell differentiation. [Results] The morphology ADSCs obtained were good. The adipogenic of the cells were signifi- cant after induction. The best MOI value was found to be 50. The expression intensity of fluorescent light was so strong that the fluorescent light could be observed after cell generations. The transfection e^ciency of Ad was high without significant biologic effects on the cyto-activity. [Conclusions] The ADSC＇s culture and induction can satisfy the requirements for seeding cells in adipose tissue engineering. Ad is an ideal viral vector for tissue engineering. Transfection of Ad-EGFP to cells could be an ideal method for cell tracing mark.