胸腺素β-4对大鼠骨髓源性内皮祖细胞功能的影响

发布时间：2018-11-24 10:47

【摘要】：目的:观察梯度浓度胸腺素β4(Thymosin-β4)对大鼠骨髓源性内皮祖细胞(EPCs)生物学行为的影响,为改善EPCs移植治疗中枢神经系统损伤这一策略的有效性,推动EPCs移植为主体的治疗手段在临床中的应用提供更为广泛的思路。方法:SPF级SD大鼠90~100 g,雌雄不拘,采用密度梯度离心法分离大鼠骨髓源性单个核细胞,接种于鼠纤维连接蛋白(FN)预先包被过夜的24孔培养板中,以含有10%胎牛血清(FBS)的EGM-2完全培养基培养,隔日换液,7d后行细胞形态学及免疫细胞化学双抗体荧光检测(CD133及VEGFR-2),激光共聚焦显微镜观察并记录拍照,同时行Dil标记的乙酰化低密度脂蛋白及异硫氰酸荧光素荆豆凝集素-1(Dil-ac-LDL及FITC-UEA-1)荧光检测吞噬实验,激光共聚焦显微镜观察并记录拍照,进一步鉴定目的细胞为内皮祖细胞。设置梯度浓度的Tβ4(1 000ng/ml、100 ng/ml、10 ng/ml、1 ng/ml)条件培养基分别处理EPCs,分别采用CCK-8细胞增殖实验、粘附能力实验以及Transwell细胞迁移能力实验以观察EPCs的增殖能力、粘附能力及迁移能力的变化。同时在统一浓度(1 000 ng/ml)下干预不同时间(24 h、48 h、72 h)后以上述同样的方法分别进行细胞功能学实验以观察EPCs的增殖能力及迁移能力的变化。结果:Tβ4能够显著增强体外培养EPCs的增殖、迁移和黏附能力,并且呈现一定的剂量效应关系,在1 000 ng/ml条件培养基培养时其增殖、迁移及黏附能力均得到显著增强(与对照组相比,P0.05)。在1 000 ng/ml条件培养基培养时随着干预时间的逐渐推移,各时间点的EPCs增殖能力均显著增强且优于空白对照组(P0.05);黏附能力在48 h达到最大效应(与对照组相比,P0.05)。结论:Tβ4可显著增强体外培养的大鼠骨髓源性EPCs的生物学活性,并且呈现一定的剂量效应关系,1 000 ng/ml Tβ4条件培养基下预处理48 h可获得最优生物学效应。
[Abstract]:Aim: to observe the effect of gradient concentration of thymosin 尾 4 (Thymosin- 尾 4) on the biological behavior of rat bone marrow-derived endothelial progenitor cells (EPCs) in order to improve the efficacy of EPCs transplantation in the treatment of central nervous system injury. Promoting the application of EPCs transplantation as the main therapeutic means in clinical practice provides a more extensive way of thinking. Methods: bone marrow-derived mononuclear cells of SPF grade SD rats were isolated by density gradient centrifugation and inoculated into a 24 well culture plate coated with rat fibronectin (FN) for the night. The cells were cultured on EGM-2 medium containing 10% fetal bovine serum (FBS) and changed into solution every other day. After 7 days, cell morphology and immunocytochemical double antibody fluorescence detection (CD133 and VEGFR-2) were detected. Laser confocal microscopy was used to observe and record the pictures. Dil labeled low density lipoprotein and fluorescein thiocyanate agglutinin 1 (Dil-ac-LDL and FITC-UEA-1) were used to detect phagocytosis, and laser confocal microscopy was used to record and photograph the phagocytosis. The target cells were identified as endothelial progenitor cells. EPCs, was treated with T 尾 _ 4 (1 000 ng 路ml ~ (-1) ng/ml,10 ng/ml,1 ng/ml) condition medium with gradient concentration. CCK-8 cell proliferation test was used. The ability of proliferation, adhesion and migration of EPCs were observed by adhesion assay and migration ability test of Transwell cells. At the same time, the cell function of EPCs was observed by the same method after intervention at different time (24 h, 48 h, 72 h) at the same concentration (1 000 ng/ml) to observe the changes of proliferation and migration ability of EPCs. Results: t 尾 4 significantly enhanced the proliferation, migration and adhesion of EPCs cultured in vitro, and showed a dose-effect relationship. Migration and adhesion were significantly increased (compared with the control group, P0.05). When cultured on 1 000 ng/ml conditioned medium, the proliferative ability of EPCs at each time point was significantly enhanced and better than that of the blank control group (P0.05). The adhesion ability reached the maximum effect at 48 h (compared with the control group, P0.05). Conclusion: t 尾 4 can significantly enhance the biological activity of rat bone marrow derived EPCs cultured in vitro, and has a dose-effect relationship. The optimal biological effect can be obtained by preconditioning on 1 000 ng/ml T 尾 4 medium for 48 h.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R651.2

【相似文献】