CM-DIL和DAPI标记的骨髓间充质干细胞

Comparison of CM-DIL and DAPI labeled bone marrow-derived mesenchymal stem cells

背景:在有关"细胞移植"的实验研究中,细胞标记技术被广泛应用。CM-DIL与DAPI是细胞标记实验中常用的荧光染料,目前两者的对比研究报道较少。目的:从体外实验与体内实验两方面,探讨两种荧光染料CM-DIL与DAPI在标记大鼠骨髓间充质干细胞方面的差异。方法:用贴壁培养法获取、培养、扩增大鼠骨髓间充质干细胞,分别用CM-DIL与DAPI进行标记,锥虫蓝计数检测骨髓间充质干细胞的活力;MTS法检测骨髓间充质干细胞的增殖能力并绘制增殖曲线;倒置相差荧光显微镜下动态观察标记后1,2,3代骨髓间充质干细胞的荧光衰减情况。结扎SD大鼠冠状动脉前降支致心肌梗死。1周后于心肌梗死边缘处注射CM-DIL与DAPI标记的细胞,细胞移植后3 d取材观察骨髓间充质干细胞的分布情况。结果与结论:体外实验中,CM-DIL与DAPI标记的骨髓间充质干细胞两者的早期增殖能力均低于对照组;两种染料标记后的第1代细胞的荧光阳性率均为100%,但DAPI标记后的第3代细胞的荧光强度明显减弱。体内实验中,CM-DIL组与DAPI组心肌组织冰冻与石蜡切片均检测到集中分布的荧光;CM-DIL组冰冻切片红色荧光比DAPI组的蓝色荧光边界清晰并且背景低;CM-DIL组还可以通过含细胞核染色的封片剂封片排除荧光假阳性。可见,CM-DIL染料比DAPI更适合对骨髓间充质干细胞进行体内示踪。

BACKGROUND： Cell marker technology has been widely applied in many studies concerning cell transplantation Chlormethylbenzamido-l,l-dioctadecyl-3,3,3＇3＇-tetramethylin-docarbocyamine （CM-DIL） and 4＇,6-diamidino-2-phenyUndole （DAPI） are commonly used for labeling cells. To our knowledge, there are few reports on comparing the two fluorescent dyes. OBJECTIVE： To compare the effects of CM-DIL and DAPI on labeling bone marrow mesenchymal stem cells in vitro and in vivo. METHODS： Isolation and expansion of bone marrow-derived mesenchymat stem cells were performed according to attachment culture. The cells were labeled by CM-DIL and DAPI, respectively. Cell viability was assessed via trypan blue exclusion assay. Growth curves of bone marrow-derived mesenchymal stem cells were depicted using MTS assay. The reduction of fluorescent intensity was observed under an inverted fluorescent contrast phase microscope from passage 1 to passage 3 after labeling. Myocardial infarction was induced by left anterior artery ligation in Sprague-Dawley rats. One week later, bone marrow-derived mesenchymal stem cells labeled by CM-DtL or DAPI were injected randomly into the border area of infarct myocardium. After 3 days, transplanted cell distribution was examined under the fluorescent microscope through paraffin sections and frozen sections respectively. RESULTS AND CONCLUSION： In vitro, bone marrow-derived mesenchymal stem cells labeled by both CM-DIL and DAPI showed decreased cell proliferation during the early period; the percentage of fluorescent-positive cellswas approximately 100% in the two groups; however, the fluorescent intensity was significantly reduced from passage 1 to passage 3 in bone marrow-derived mesenchymal stern cells labeled, by DAPI./n vivo, the transplanted cells were detected in a concentrated way both on the paraffin sections and frozen ones; the background color of frozen sections was lower in the CM-DIL group than in the DAPI group; false positive results of fluorescent expression could be eliminated in the CM-DIL group by using fluorescent mounting medium with the fluorescence of DNA staining. These data indicates that CM-DIL is more appropriate to in vivo tracing cells than DAPI.