sCD40L诱导人脐静脉内皮细胞损伤对人脐间充质干细胞趋化性的影响

Chemotaxis of Human Umbilical Cord Mesenchymal Stem Cells to the sCD40L-induced Vascular Endothelial Cells

目的应用人重组CD40L(r-CD40L)诱导人脐静脉内皮细胞(HUVEC)损伤,研究该损伤对人脐间充质干细胞(UC-MSCs)趋化作用的影响。方法培养HUVEC,采用不同浓度r-CD40L刺激HUVEC 24 h,应用Transwel(l滤膜直径6.5 mm,孔径0.4μm)技术建立内皮细胞通透模型并计算通透系数;培养HUVEC,采用不同浓度r-CD40L刺激HUVEC 24 h,将干预后4组细胞经处理后取上清置入EP管,离心后加入Transwel(l滤膜直径6.5 mm,孔径8μm)小室下室,将第5代UC-MSCs用含5%FBS的培养基调整细胞浓度为1×105/mL,接种到预处理后的Transwell小室的上室,5%CO237°C,培养24 h,取出后用1%结晶紫染色,200倍镜下随机计数6个无重叠细胞视野的透膜细胞数,取平均数代表该组细胞的侵袭能力。结果随着r-CD40L刺激浓度的增加,内皮细胞通透系数逐渐升高;不同浓度r-CD40L(0、20、30、40μg/L)刺激HUVEC 24 h后,损伤的内皮细胞诱导UC-MSCs透膜细胞个数分别为10.00±1.76、25.96±2.84、37.88±3.23、49.04±2.81,组间比较差异有统计学意义(P<0.01)。结论 sCD40L能诱导内皮细胞损伤;并且随着内皮细胞损伤程度的增加对人脐间充质干细胞的趋化性增强。

Objective To study the chemotaxis effect of human umbilical cord mesenchymal stem cells to r-CD40L HUVGC.Methods The cultured HUVEC were treated by different concentration of r-CD40L factor.24 hours later,endothelial permeability was measured by fluorescein isothiocyanate labeled bovineserum albumin（FITC.BSA）clearance across the monolayer.The cultured HUVEC were divided into four groups and induced by different concentration of r-CD40L factor for 24hs.The culture solution of the four groups was collected in a sterile EP,centrifuged the solution,then added into the lower chamber of Transwell;then,the fifth passage of UC-MSCs（1×105/mL）was filled to the upper chamber of Transwell.After 24 h of incubation in a 5% CO2 atmosphere at 37 ℃,the invasive cells of the lower surface were fixed and stained by 1% crystal violet solution.Then the cells were redyeing with tetrabromofluorescein.The extent of invasion was determined under a light microscope by counting the number of stained cells on the membranes in six randomly selected,non-overlapping fields at 200× magnification.Results r-CD40L induction lead to a significant increase in the permeability.The number of stained UC-MSCs on the membranes of different groups were：10.00 ±1.76,25.96 ±2.84,37.88 ±3.23,49.04 ±2.81.The differences were statistically significant,P 0.01.Conclusion sCD40L could induce a significant increase in the endothelial permeability,and the r-CD40L-induced HUVEC cell injury could increase the chemotactic characteristic of UC-MSCs.