兔骨髓间充质干细胞体外诱导血管内皮细胞
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摘要
目的:探讨兔骨髓间充质干细胞(BMSCs)体外诱导为血管内皮细胞(VECs)的方法,为心血管疾病的血管重建提供治疗策略。方法:抽取兔骨髓,采用全骨髓贴壁法加内皮细胞培养液诱导培养细胞,记录并观察诱导后细胞的形态及生长过程,并进行荧光标记;采用免疫细胞化学显色法鉴定诱导后细胞表面标志物CD31、CD34,利用支架材料Matrigel基质胶观察VECs三维血管化形态。结果:兔BMSCs经诱导后48 h,呈小圆形,可见贴壁生长;3～4d可见少量细胞贴壁,多为短梭形;6～8d完全贴壁,多为长梭形、锥形、不规则形;10～12d后细胞呈集落样生长;第15天成片生长的细胞集落相互连接,呈现典型的"鹅卵石样"或"铺路石样"外观;CM-DiI细胞荧光标记显示VECs的胞质、胞膜呈现红色荧光,DAPI细胞荧光标记显示VECs胞核呈蓝色荧光,细胞形态为长梭形,且细胞生长状况良好;免疫细胞化学显色显示CD31、CD34均呈阳性;在Matrigel基质胶上VECs呈现典型的环状或管状。结论:以全骨髓贴壁法和经内皮细胞培养液诱导法可获得VECs。
Objective :To provide therapeutic strategies for revascularization of cardiovascular diseases and to investigate the method of inducing bone marrow-derived mesenchymal stem cells(BMSCs) into vessel endothelial cells(VECs) in vitro. Methods :Bone marrow was extracted. The adherent method and the method of induction through endothelial cell culture solution were applied. The morphology and growth process of induced cells were recorded and observed. The induced cells were labeled with fluorescence. The surface markers CD31 and CD34 of induced cells were identified by immunocytochemical staining. Three dimensional vascularization of VECs was observed by Matrigel matrix adhesive. Results :Adherent growth of rabbit BMSCs was observed 48 hours after induction. 3-4 d showed a small number of cells adhering to the wall, most of which were in the shape of short rod spindle. By 6-8 d, they were completely adherent, mostly spindle shaped, tapered and irregular. After 10-12 d, the cells grew in the colony shape. By the 15 th day, the growing cell colonies interacted with each other, showing typical appearance of "cobblestone" or "paving stone". Markers showed the morphology and density distribution of VECs. Fluorescence labeling of CM-DiI cells showed red in the cytoplasm and membrane of VECs, and fluorescence labeling of DAPI cells showed blue in the nucleus, the cells grew into spindles and the cells grew well.Immunocytochemical staining showed that both CD31 and CD34 were positive. Typical rings or tubes were found on Matrigel matrix adhesive of VECs. Conclusion :VECs can be obtained with the adherent method to extract and endothelial cell culture solution to induce.
Objective :To provide therapeutic strategies for revascularization of cardiovascular diseases and to investigate the method of inducing bone marrow-derived mesenchymal stem cells(BMSCs) into vessel endothelial cells(VECs) in vitro. Methods :Bone marrow was extracted. The adherent method and the method of induction through endothelial cell culture solution were applied. The morphology and growth process of induced cells were recorded and observed. The induced cells were labeled with fluorescence. The surface markers CD31 and CD34 of induced cells were identified by immunocytochemical staining. Three dimensional vascularization of VECs was observed by Matrigel matrix adhesive. Results :Adherent growth of rabbit BMSCs was observed 48 hours after induction. 3-4 d showed a small number of cells adhering to the wall, most of which were in the shape of short rod spindle. By 6-8 d, they were completely adherent, mostly spindle shaped, tapered and irregular. After 10-12 d, the cells grew in the colony shape. By the 15 th day, the growing cell colonies interacted with each other, showing typical appearance of "cobblestone" or "paving stone". Markers showed the morphology and density distribution of VECs. Fluorescence labeling of CM-DiI cells showed red in the cytoplasm and membrane of VECs, and fluorescence labeling of DAPI cells showed blue in the nucleus, the cells grew into spindles and the cells grew well.Immunocytochemical staining showed that both CD31 and CD34 were positive. Typical rings or tubes were found on Matrigel matrix adhesive of VECs. Conclusion :VECs can be obtained with the adherent method to extract and endothelial cell culture solution to induce.
引文
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[2] Aird W C. Endothelial cell heterogeneity[J]. Cold Spring Harb Perspect Med, 2012, 2(1):1-13.
[3] Zhang L,Yu Y,Xia X,et al. Transcription factor E2-2 inhibits the proliferation of endothelial progenitor cells by suppressing autophagy[J]. Int J Mol Med, 2016, 5(37):1254-1262.
[4]董献成,殷建,恽波.G蛋白偶联受体激酶相互作用蛋白1对BMSCs向内皮细胞分化的影响机制研究[J].中国修复重建外科杂志,2018, 32(3):257-263.
[5]张培岩,陈丁.内皮祖细胞的迁移及对血管修复作用的研究进展[J].海峡药学,2017, 29(3):10-15.
[6] Roelandt P, Sancho-Bru P, Pauwelyn K, et al. Differentiation of rat multipotent adult progenitor cells to functional hepatocyte-like cells by mimicking embryonic liver development[J]. Nat Protoc,2010,(7):1324-1336.
[7] Ward M R, Abadeh A, Connelly K A, et al. Concise review:Rational use of mesenchymal stem cells in the treatment of ischemic heart disease[J]. Stem Cells Transl Med, 2018, 4(17):1-20.
[8] Bickford P C,Kaneko Y,Grimmig B,et al. Nutraceutical intervention reverses the negative effects of blood from aged rats on stem cells[J]. Age(Dordr), 2015, 37(5):103-109.
[9]杜利,刘亢丁.内皮细胞生物学特征及其临床应用[J].中国组织工程研究与临床康复,2011,15(40):7581-7583.
[10]胡洋,郝兰坡,赵炳飞.骨髓源性血管内皮细胞体外分离、培养及分化为内皮细胞的研究[J].现代中西医结合杂志,2015, 24(22):2421-2424.
[11]葛权虎.间充质干细胞对血管内皮前体细胞分化影响的研究[D].石河子:石河子大学,2017.
[12]夏鑫,吴明华.血管相关内皮祖细胞标志物研究[J].脑与神经疾病杂志,2018, 26(1):65-67.
[13] Zou H X, Jia J, Zhang W F, et al. Propranolol inhibits endothelialprogenitor cell homing:a possible treatment mechanism of infantile hemangioma[J]. Cardiovasc Pathol, 2013,22(3):203-210.
[14] Lin C P,Lin F Y,Huang P H,et al. Endothelial progenitor cell dysfunction in cardiovascular diseases:role of reactive oxygen species and inflammation[J]. Biomed Res Int,2013,2013:845037.