大鼠脑梗死组织上清液对脂肪干细胞诱导分化的研究

Research on adipose-derived stem cells differentiation induced by supernatant liquid from cerebral infarction tissue in rats

目的探讨大鼠脑梗死组织上清液对脂肪干细胞（ADSCs）向内皮细胞及平滑肌细胞分化的影响。方法取SD大鼠腹膜后脂肪组织进行ADSCs分离培养，流式细胞技术鉴定ADSCs；用正常脑组织上清液、脑梗死组织上清液对ADSCs进行诱导，未干预组作对照，反转录聚合酶链反应技术检测经诱导后的细胞表达血管性血友病因子（vWF）、α-平滑肌肌动蛋白（α—sMA）和平滑肌肌球蛋白重链（SM-MHC）的mRNA水平，免疫荧光法检测内皮细胞及平滑肌细胞标志物的表达，荧光显微镜观察细胞阳性情况。结果ADSCs表面阳性抗原呈高表达CD90（96．7％）、CD29（84．4％）、CD44（98．900），而阴性抗原呈较低表达CD45（6．5％）、CD34（7．4％）、CD31（3．6％）；与未干预组和正常脑组织组比较，脑梗死组诱导后细胞表达vWF、α—sMA、SM—MHC的mRNA相对表达量有显著提高（F=5．962、6．756、6．144，P=0．001、0．004、0．003）；免疫荧光显示脑梗死组诱导后的细胞阳性表达vWF和α-SMA、SM-MHC高于未干预组和正常脑组织组。结论ADSCs在脑梗死组织上清液的诱导下高表达内皮细胞和平滑肌细胞标志物，具有向内皮细胞及平滑肌细胞分化的倾向。

Objective To study adipose-derived stem cells （ADSCs）differentiation into endothelial cells and smooth muscle cells by induction with supernatant liquid from cerebral infarction tissue in rats. Methods The ADSCs were obtained from retroperitoneal adipose tissue of rats and identified by flow cytometry technology. The normal brain tissues and the infarcted cerebral tissue obtained from rats with middle cerebral artery occlusion were used to induce ADSCs differentiation, and no intervention group was as a control. The mRNA expression level of von willebrand factor （vWF） ,α-smooth muscle actin（α-SMA）and smooth muscle myosin heavy chain（SM-MHC）in cells after induction were detected using reverse transcriptase-polymerase chain reaction. Immunofluorescence were used to identify the expression of endothelial cells markers and smooth muscle cell markers, and positive expression cell was detected by fluorescence microscope. Results On the ADSCs surfaces, the high expressed-positive antigens included CD90 （96.7 % ）, CD29 （84.4%）,CD44（98.9%）,and the low expressed negative antigens included CD45（6.5%）,CD34 （7.4%） and CD31 （3.6%）.Compared with no intervention group and normal brain tissue of supernatant liquid group, the cerebral infarction tissue of supernatant liquid-induced group showed the increased mRNA expression level of vWF, a-SMA and SM-MHC（F= 5. 962,6. 756,6. 144, P=0. 001, 0. 004,0. 003）, and showed that the immunofluorescence indicated-cell expression level of vWF, α-SMA and SM-MHC was much more increased （all P 〈 0.05 ）. Conclusions Under the induction by supernatant liquid of cerebral infarction tissue, ADSCs highly expresses the markers of endothelial cells and smooth muscle cells. This suggests that the cerebral infarction tissue of supernatant liquid-induced ADSCs have a tendency to differentiate into endothelial cells and smooth muscle cells.