CM-DiI标记大鼠脂肪干细胞的效力

Fluorescent dye CM-DiI labeled rat adipose derived stem cells

背景:CM-DiI是DiI的衍生物,因具有一定的水溶性,且含有氯甲基化活性巯基部分,使其更易嵌入、弥散并稳固地结合到整个细胞膜上,使染色更快捷、均匀、持久。目的:进一步验证荧光染料CM-DiI标记大鼠脂肪干细胞的可行性。方法:切取大鼠腹股沟区皮下脂肪组织,采用胶原酶消化法体外分离培养脂肪干细胞,取第3代细胞分为实验组和对照组,用质量浓度4mg/L的CM-DiI对实验组细胞进行标记,于6,12,24,48h观察CM-DiI标记脂肪干细胞体内示踪效果。结果与结论:荧光显微镜下CM-DiI标记细胞的胞浆、胞膜均显红色荧光,标记的脂肪干细胞呈梭形,保持了良好的正常形态,CM-DiI标记阳性率为100%,标记早期细胞形态呈荧光环状,48h后细胞中荧光颗粒增多,荧光增强,细胞核未染荧光。与对照组比较,CM-DiI标记的细胞增殖后形态、上清液乳酸脱氢酶含量及MTT值均无明显变化(P>0.05)。细胞移植后4h后,在心、肺组织可见到发出红色荧光的标记细胞。提示CM-DiI能有效标记体外培养的脂肪干细胞,并在细胞内稳定表达,且标记细胞形态良好,对活体细胞无毒性。体内移植后,有良好的示踪效果。

BACKGROUND： Dil derivative CM-Dil easily insets, disperses and stably binds to the whole cell membrane because of water-solubility and chloromethylated active sulfydryl, which leads to rapid, even and long-lasting staining. OBJECTIVE： To further validate the feasibility of fluorescent dye CM-Dil in labeling rat adipose derived stem cells. METHODS： Rat inguinal fat tissue was resected and adipose derived stem cells were isolated in vitro by collagenase digestion. Passage 3 cells were divided into a control group and an experimental group. The experimental group cells were labeled with 4 mg/L CM-Dil. At 6, 12, 24 and 48 hours, CM-Dil-labeled adipose derived stem cells were traced in vivo RESULTS AND CONCLUSION： Under fluorescent microscope, CM-Dil labeled cells showed cytoplasm and cell membrane with red fluorescence, CM-Dil labeled adipose derived stem cells exhibited a shuttle-shaped normal appearance. CM-Dil positive rate was 100%. In the early period, the cells were fluorescent ring-shaped, and 48 hours later fluorescent granules increased and fluorescence intensity enhanced. No fluorescence was found in cell nucleus. There was no significant difference in cell morphology, content of lactic dehydrogenase in supernatant and MTT value between experimental group and control group （P 〉 0.05）. At 4 hours after cell transplantation, cells with red fluorescence could be observed in the heart and lung tissue. CM-Dil can effectively label adipose derived stem cells cultured in vitro and express stably in cells. CM-Dil-labeled cells have good morphology and no toxicity to living cells.