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目的探讨一种人高活性血管基质层细胞(stromal vascular fraction cells,SVFs)快速分离及荧光标记的有效方法,为SVFs用于临床奠定理论基础。方法取植皮手术患者自愿捐赠的腹部皮下脂肪组织,分别用0.065%、0.125%、0.185%3种浓度的Ⅰ型胶原酶消化,提取SVFs。收集细胞悬液进行细胞计数,锥虫蓝染色计算细胞成活率。用1’双十八烷-3,3,3’,3’-四甲基吲哚羧化青-高氯酸盐(1,1’-dioctadecyl-3,3,3’,3’-2-tetramethy-lindocyanine perchlorate,DiI)荧光标记0.125%浓度组SVFs,倒置荧光显微镜下观察标记情况;将DiI标记的SVFs接种培养,倒置显微镜观察细胞形态学变化,MTT法检测DiI标记前后细胞增殖能力。结果 0.065%、0.125%及0.185%浓度组SVFs细胞总数分别为(138.68±11.64)×104、(183.80±10.16)×104、(293.07±8.31)×104个,组间比较差异均有统计学意义(P<0.01);细胞成活率分别为91%±2%、90%±2%、81%±2%,0.065%浓度组和0.125%浓度组均显著高于0.185%浓度组(P<0.01),0.065%浓度组和0.125%浓度组比较差异无统计学意义(P=0.881)。倒置荧光显微镜观察示,0.125%浓度组细胞均可被DiI荧光标记,标记后的细胞膜完整,细胞质丰富,细胞形态正常,细胞核未染荧光;DiI标记的SVFs细胞悬液接种培养后,细胞能顺利贴壁及传代。MTT法检测示DiI标记前后SVFs细胞生长曲线相似,生长趋势吻合。结论 0.125%浓度的Ⅰ型胶原酶可有效消化脂肪中的胶原组织,获取大量SVFs,同时对细胞损伤较小,是理想的工作浓度;DiI可有效标记SVFs。
Objective To explore an efficient method for rapid isolation and fluorescent labeling of human highly active stromal vascular fraction cells (SVFs) and to lay a theoretical foundation for the clinical application of SVFs. Methods Abdominal subcutaneous adipose tissue donated voluntarily by skin graft patients was used to digest SVFs with collagenase type Ⅰ of 0.065%, 0.125% and 0.185%, respectively. The cell suspension was collected for cell counting and trypan blue staining to calculate cell viability. Dihydrochloride (1,1’-dioctadecyl-3,3,3 ’, 3’-2) with 1’-dioctadeca-3,3,3’ Di-labeled SVFs were transfected into SVFs with 0.125% concentration of fluorescein. Fluorescence labeling of SVFs was performed under inverted fluorescent microscope. DiV-labeled SVFs were seeded in inverted microscope to observe the morphological changes of cells. MTT assay was used to detect the proliferation of SVFs before and after DiI labeling. Results The total number of SVFs in the groups of 0.065%, 0.125% and 0.185% were (138.68 ± 11.64) × 104, (183.80 ± 10.16) × 104 and (293.07 ± 8.31) × 104, respectively. There was significant difference between the two groups (P <0.01). The cell survival rates were 91% ± 2%, 90% ± 2%, 81% ± 2%, 0.065% and 0.125% ), There was no significant difference between 0.065% concentration group and 0.125% concentration group (P = 0.881). Inverted fluorescence microscopy showed that the cells in 0.125% concentration group could be labeled with DiI fluorescence, the labeled cell membrane was intact, the cytoplasm was abundant, the cell morphology was normal, and the nuclei were not stained with fluorescent light. After inoculation of DiI-labeled SVFs cell suspension, Adherence and passage. The MTT assay showed that the growth curves of SVFs before and after DiI labeling were similar and the growth trend was consistent. Conclusion Type Ⅰ collagenase with 0.125% concentration can effectively digest collagen in adipose tissue and obtain a large number of SVFs with less damage to cells. It is an ideal working concentration. DiI can effectively label SVFs.