稳定表达报告基因脂肪间充质干细胞的培养与分子影像鉴定

Culture and molecular imaging identification of adipose derived mesenchymal stem cells with stable reporter gene expression in vitro and in vivo

背景:研究显示分子影像技术可以在活体细胞和分子水平对干细胞进行定性和定量研究,对干细胞长程监测具有独特优势。目的:构建稳定表达报告基因的脂肪间充质干细胞,并运用报告基因成像等方法实现其体外和移植后的评价与鉴定。方法:繁殖与筛选携带β-actin-luc报告基因小鼠后,采用改良胶原酶消化法分离培养其脂肪间充质干细胞,取第3代细胞行表面标志鉴定;将脂肪间充质干细胞(1×106)植入BALB/c裸鼠后肢肌肉内,采用萤火虫荧光素酶(fluc)生物发光成像进行体外和体内移植后脂肪间充质干细胞的示踪和定量评价。结果与结论:转基因小鼠稳定携带β-actin-luc报告基因,分离培养出的脂肪间充质干细胞高表达CD90及CD44,而CD45、CD34和CD31呈现低表达,其细胞数与fluc报告基因生物发光信号强度呈显著直线相关(r2=0.96)。细胞活体肌肉移植24h后存活,并显示较强的生物发光信号,在底物荧光素腹腔注射后0～42min内先迅速增强后平缓减弱,21min时最强,达(6.92×106±4.11×105)Photons·s-1·cm-2·sr-1。提示由携带β-actin-luc报告基因小鼠脂肪组织分离的脂肪间充质干细胞高表达间充质干细胞表面标志,可在体外和肌肉组织移植后稳定表达该报告基因并实现细胞的分子影像示踪与定量。

BACKGROUND：Previous studies show that molecular imaging can monitor the stem cell qualitatively and quantitatively at cellular and molecular levels in vivo and hold promise in long-time evaluation of the stem cell.OBJECTIVE：To construct adipose derived mesenchymal stem cells（ADMSCs） with stable reporter genes expression and identify it by reporter gene imaging in vitro and in vivo.METHODS：β-actin-luc transgenic mice were selected firstly.Then ADMSCs were cultured from β-actin-luc mice by modified collagenase method.ADMSCs at passage 3 were identified by flow cytometry.ADMSCs（1×106） were implanted into the muscles of hindlimb of BALB/c nude mice,then tracked and quantified by firefly luciferase（fluc） bioluminescence imaging（BLI） in vitro and in vivo.RESULTS AND CONCLUSION：The transgenic mice stably carried β-actin-luc reporter gene and the ADMSCs were positive for CD90 and CD44,while negative for CD45,CD34 and CD31.ADMSCs consistently expressed the fluc and a robust correlation existed between different cell numbers and fluc average radiance（r2=0.96）.After 24-hours engrafted ADMSCs survived and displayed strong BLI signal,which increased rapidly and then decreased smoothly during 0-42 minutes after peritoneal injection of D-luciferin,the peak being（6.92×106±4.11×105） Photons·s-1·cm-2·sr-1 at 21 minutes.Results indicated that ADMSCs of β-actin-luc transgenic mice could highly express the markers of MSCs,stably carry the reporter gene and facilitate the tracking and quantifying by BLI in vitro and in vivo.