【摘要】 【目的】探讨荧光染料CM-DiI直接标记和慢病毒感染法EGFP标记人骨髓间充质干细胞(hMSC)用于体内示踪的优缺点。【方法】利用FUGW质粒构建携带EGFP基因的慢病毒载体,并感染P3代hMSC,荧光倒置显微镜下观察细胞的感染效果及传代前后细胞EGFP表达情况的变化;CM-DiI直接标记P3代hMSC,观察CM-DiI标记对细胞生物学特性的影响及传代前后细胞标记效果的变化。分别将CM-DiI和EGFP标记的P3代hMSC移植入新生大鼠左侧脑室,2周后取大鼠脑组织行冰冻切片,比较两种标记方法用于体内示踪的效果。【结果】慢病毒感染hMSC 48h后,约40%细胞表达绿色荧光,体外培养1个月,EGFP阳性细胞数增至50%左右,荧光强度基本保持不变,细胞生长不受病毒转染的影响。CM-DiI标记hMSC 12h后,95%以上细胞呈现很强的红色荧光,1个月后,阳性细胞减少至50%左右,荧光强度也有一定程度降低,未见染料对细胞形态和生长产生明显影响。CM-DiI标记的hMSC移植后2周脑冰冻切片可以找到红色荧光的标记细胞,EGFP标记的hMSC移植后2周脑冰冻切片未找到绿色荧光细胞。【结论】CM-DiI和EGFP均可在体外高效标记hMSC,但CM-DiI标记的hMSC体内示踪效果优于EGFP标记。更多还原

【Abstract】 【Objective】To investigate the advantages and disadvantages of two cell labeling techniques for tracking human mesenchymal stem cells (hMSC) in vivo: Direct labeling of fluorescent dye CM-DiI and enhanced green fluorescence protein (EGFP) labeling with lentivirus infection. 【Methods】 Lentivirus vector carrying EGFP gene was constructed with plasmid FUGW to infect hMSC P3. Cultured in vitro for a month, the infection effect of cells and the expression changes of EGFP in passage cells were observed under inverted fluorescent microscope. hMSC P3 was directly labeled with CM-DiI, then the influence of CM-DiI label on cell biological characteristics and the changes of labeling effect in passage cells were observed. hMSC P3 labeled with CM-DiI and EGFP were transplanted into the left cerebral ventricle of neonate rats respectively. The frozen sections of the rat brain tissues were obtained after two weeks and the effects of two cell labeling techniques for tracking transplanted hMSC were compared. 【Results】 Forty-eight hours after hMSC being infected with lentivirus, 40% of the cells expressed green fluorescence under fluorescent microscopy. After culture in vitro for one month, the number of EGFP positive cells increased to 50%, while the fluorescence intensity did not change and no effects of virus transfection were found on the cell growth. Twelve hours after labeled with CM-DiI, over 95% of hMSC presented strong red fluorescence under fluorescent microscopy. One month later, the fluorescence intensity decreased in some degree, and the positive cells were reduced to 50%. No significant effect of the dye was found on the cell morphology and growth. Cells labeled with red fluorescence could be observed in the brain frozen sections 2 weeks after transplantation with CM-DiI. Cells labeled with green fluorescence could not be found in the brain frozen sections two weeks after transplantation with EGFP. 【Conclusion】 Both CM-DiI and EGFP are useful markers for labeling hMSC in vitro, while CM-DiI is better than EGFP for tracking the migration of hMSC.更多还原