大鼠白色脂肪组织来源血管内皮生长因子受体阳性细胞向内皮细胞的诱导分化

Inducing the differentiation of white adipose tissue-derived vascular endothelial growth factor receptor positive cells into endothelial cells in rats

目的:观察大鼠白色脂肪组织作为内皮祖细胞来源的可能性。方法:实验于2004-12/2005-04在上海组织工程重点实验室完成。取3周龄Wistar大鼠腹股沟白色脂肪组织,消化法得到的细胞接种在培养皿内,用含体积分数为0.1的胎牛血清的DMEM培养基培养,传至第2代,应用免疫磁珠分选仪分选血管内皮生长因子受体阳性细胞。获得的细胞分诱导组(DMEM,体积分数为0.1的胎牛血清,血管内皮生长因子10μg/L,碱性成纤维细胞生长因子2μg/L)和非诱导组(DMEM,体积分数为0.1的胎牛血清)进行培养。流式细胞仪检测分选前后细胞纯度;噻唑蓝比色法检测诱导组与非诱导组细胞的生长曲线;相差倒置显微镜观察细胞形态;免疫细胞荧光检测细胞vonWillebrand因子和血小板-内皮细胞间黏附分子-1的表达;荧光显微镜观察细胞摄取DiI-ac-LDL的功能;诱导组细胞接种于甲基纤维素半固体培养基进行三维培养,观察形成血管样结构的能力。结果:①细胞纯度:流式细胞仪检测显示未分选的第2代细胞血管内皮生长因子受体阳性率为(8.65±1.47)%,刚分选的血管内皮生长因子受体细胞阳性率(94.06±6.86)%,两者比较差异显著。分选前后的第2代细胞血小板-内皮细胞间黏附分子1阳性率分别为(1.26±0.21)%和(0.57±0.06)%,两者比较差异无显著性。②细胞生长曲线测定:诱导组细胞在诱导培养基中生长良好,第3天进入对数生长期,第6天到平台期。非诱导组细胞生长较慢,第5天进入对数生长期,第8天到平台期。第5天后各时间点差异均显著(P<0.05)。③细胞形态观察:培养12d后,非诱导组细胞为梭形,而诱导组细胞呈“铺路石”样排列。④细胞免疫荧光检测:诱导培养12d后诱导组细胞vonWillebrand因子和血小板-内皮细胞间黏附分子1为阳性,非诱导组细胞皆为阴性。⑤内皮细胞功能检测:诱导后12d后的低密度脂蛋白摄取实验,诱导组细胞内出现红色荧光,非诱导组为阴性。⑥三维培养:诱导组细胞接种在甲基纤维素的半固体培养基内3d后形成“树枝样”分叉结构,未诱导组细胞未形成此类结构。结论:大鼠脂肪组织来源的血管内皮生长因子受体阳性细胞能够诱导分化为成熟内皮细胞。

AIM： To observe the feasibility of white adipose tissue as the source of endothelial progenitor cell. METHODS：This experiment was conducted at the Shanghai Key Laboratory of Tissue Engineering from December 2004 to April 2005. White adipose tissue was collected from fold inguen of 3-week-old Wistar rats. The obtained cells after digestion were inoculated in the Petri dish, and cultured with DMEM culture medium containing 0.1 volume fraction of fetal bovine serum. Vascular endothelial growth factor receptor-2^＋ （VEGFR-2^＋） cells of passage 2 were sorrel by MACS. The obtained cells were divided into induction group （ DMEM, 0.1 volume fraction of fetal bovine serum, 10μg/L endothelial growth factor and 2 fl,g/L basic fibroblast growth factor） and non-induction group （DMEM,0.1 volume fraction of fetal bovine serum）for culture. Cellular purity was detected with flow cytometer before and after sorting; The growth curve were measured with MTT colorimetric assay. Cellular morphology was observed under an inverted phase contrast microscope; The expression of cellular yon Willebrand factor and blood platelet-endothelial celullar adhesion molecular with immunofluorescence; Fluorescence microscope was Used to observe the function of taking up DiI-ac-LDL （low-density lipoprotein） by the induced cells. The ability of forming capillary-like structure in threedimensional matrices was observed, and the induced cells were also cultured in methylcellulose. RESULTS： （1） Cellular purity ： Flow cytometer showed that 94,06±6.86% of VEGFR-2-selected cells expressed VEGFR-2^＋, while only 8.65±1.47% of non-selected cells were VEGFR-2^＋. There was no significant difference between the two. （2）Determination of cell growth curve： Cells in the induction group grew well in the inducted culture medium, and they entered the logarithmic phase on the 3^th day and reached the platform on the 6^th day. Cells in the non-induced group grew slowly, entered the logarithmic phase on the 5^th day and reached the platform on the 8^th day. There was significant difference at each time point after 5 days （P 〈 0.05）. （3） Cellular morphology observation： At 12 days after culture, cells in the non-induced group was fusiform, while those in the induced group presented ＂slabstone＇-like arrange. （4） Immunofluorescence ： After 12 days of induction, yon Willebrand factor and endothelial cellular adhesion molecular in the induction group were positive ,and those in the noninduction group were negative. （4） Function detection of endothelial cells： After 12 days of low-density lipoprotion taking up, induction group presented red fluorescence, but non-induction group did not, （6） Threedimensional culture： Cells were inoculated on the methylcellulose in the induction group and ＂branch-like＂ structure formed 3 days later, and there was no such structure in the non-induction group. CONCLUSION： Adipose tissue-derived VEGFR2＋ cells can induce and proliferate into mature endothelial cells.