整合素α9在人脂肪源性间充质干细胞向淋巴管内皮细胞分化过程中的表达变化

Changes in the expression of integrin α9 in the differentiation of adipose-derived stem cells to lymphatic endothelial cells

摘要:

目的：从人体脂肪组织中分离培养出脂肪源性间充质干细胞（adipose-derived stem cells，ADSCs），诱导其向淋巴管内皮细胞（lymphatic endothelial cells，LECs）分化，同时探讨分化过程中整合素α9（integrin α9）表达的变化，旨在寻找淋巴水肿分子治疗靶点，为 ADSCs应用于淋巴水肿的分子治疗奠定实验基础。方法：①从吸脂术后废弃的人体脂肪组织中分离培养出ADSCs，倒置相差显微镜观察原代及传代细胞的形态特征。②流式细胞仪（flow cytometry，FCM）检测第3代ADSCs表面特征性抗原CD90、CD29、CD34及CD４５的表达；MTT法绘制生长曲线。③用人血管内皮细胞生长因子（vascular endothelial growth factor，VEGF）C、人VEGF-A、血小板源性生长因子BB（platelet-derived growth factor-BB，PDGF-BB）诱导其向LECs方向分化，同时以人LECs作为阳性对照组。④Western blot检测各组细胞LECs表面特征性抗原淋巴管内皮细胞透明质酸受体-1（lym－phatic endothelial hyaluronan receptor-1，LYVE-1）、血管内皮细胞特征性抗原CD34及integrin α9的表达。⑤FCM检测各组细胞周期，使用SPSS17.0对实验数据进行统计分析。结果：①ADSCs贴壁生长，呈长梭形，第1、3、5代ADSCs的生长曲线均呈S形；细胞表面抗原为CD90+、CD29+、CD34-、CD45-。② 实验组及阳性对照组LYVE-1表达阳性、CD34表达均阴性，integrin α9的表达趋势与LYVE-1一致，且二者在实验组的表达水平低于阳性对照组；三者在阴性对照组中的表达均为阴性。③各组G0/G1期细胞比例有明显统计学差异（P<0.01），实验组与阳性对照组G0/G1期细胞比例显著小于阴性对照组（P<0.01），实验组与阳性对照组之间无明显统计学差异（P=0.083）。结论：ADSCs离体培养，具有向LECs分化的潜能。使用VEGF-C、VEGF-A、PDGF-BB可诱导部分人ADSCs向LECs分化。在人ADSCs向LECs分化过程中integrin α9的表达增强，分化前后细胞的周期发生了改变。该分子在LECs的形成过程中可能发挥着极为重要的作用，将有望在淋巴水肿的再生治疗中成为重要的分子靶点之一。

Abstract:

Objective：To isolate and culture adipose-derived stem cells（ADSCs） from human adipose tissues，to induce the differentiation of ADSCs to lymphatic endothelial cells（LECs），to explore the changes in the expression of integrin α9 in the differentiation pro－cess so as to find a molecular target and to make contribution to the molecular treatment of lymphedema with ADSCs. Methods：①ADSCs were isolated and cultured from human adipose tissue which derived from the liposuction surgery.Morphological characteristics were observed with inverted phase contrast microscope.②Expression of CD90，CD29，CD34 and CD45 in the cell membrane was as－sayed by using flow cytometry（FCM）. Growth curve was drawn with MTT assay. ③Lymphendothelial differentiation of ADSCs was induced with human vascular endothelial growth factor C（VEGF-C），human vascular endothelial growth factor A（VEGF-A） and platelet-derived growth factor BB（PBGF BB） in vitro.Human LECs were taken as positive control group. ④Expression of lymphatic endothelial hyaluronan receptor-1（LYVE-1），integrin α9 and CD34 was detected by Western blot. ⑤Cell cycles of the three groups were determined with FCM. Then the data were ana－lyzed with SPSS 17.0. Results：①ADSCs was in spindle shape and adherent growth.Growth curves of 1th，3th and 5th passage of ADSCs all presented S shape. The expression of CD90+ was positive，and the expression of CD34+ and CD45+ was negative. ②LYVE-1 expressed positively in experiment group and posi－tive control group；CD34 expressed negatively in experiment group and positive control group；expression of integrin α9 was similar to LYVE-1 and expression of integrin α9 and LYVE-1 were lower in experiment group than in positive control group；integrin α9，LYVE-1 and CD34 expressed negatively in negative control group. ③There were differences in proportion of cells at G0/G1 stage，higher in negative control group than in experiment group and positive control group（P<0.01）. There was no difference in proportion of cells at G0/G1 stage between experiment group and positive control group（P=0.083）. Conclusions：Human ADSCs can differentiate into LECs but not vascular endothelial cells when culture in vitro with VEGF-C，VEGF-A and PDGF-BB. Cell cycle will change and the expression of integrin α9 will elevate during this process. Integrin α9 may make an important role in the formation of LECs and may obtain the opportunity to become a molecular target in the treatment of lymphedema.