Angiopoietin-1通过整合素对间充质干细胞粘附存活和凋亡的影响

发布时间：2018-04-30 03:30

本文选题：血管生成素-1 + 间充质干细胞　；参考：《福建医科大学》2009年硕士论文

【摘要】： 目的:探讨重组人血管生成素-1(Ang-1)能否通过整合素促进大鼠骨髓间充质干细胞(MSC)粘附和存活、抑制其凋亡,并初步讨论其抗凋亡的信号转导机制。方法:密度梯度离心法结合贴壁分离法体外分离、培养大鼠骨髓MSC并通过流式细胞仪检查表面抗原做鉴定;在24孔培养板上测定不同时间下Ang-1对MSC的粘附作用;台盼蓝染色法和MTT法测定Ang-1对血清饥饿下MSC的活力影响;紫杉醇诱导MSC凋亡后,Hoechst染色法和Annexin V/PI法检测Ang-1对MSC凋亡的影响,通过Western blot方法检测磷酸化Akt、总Akt、Bcl-2、β-actin表达水平的变化,结合整合素抗体和信号转导阻滞剂来探讨其抗凋亡的可能分子机制。结果:体外成功分离和培养和扩增了MSC,结合细胞形态、生长特点和流式细胞仪表面抗原检测均符合MSC特点,其纯度达98.3%。粘附试验显示在1d、2d、3d三个不同时间点Ang-1均能显著促进MSC的粘附(均有P＜0.01),该效应能被EDTA、整合素亚单位β1抗体和RGD多肽所抑制(P＜0.01)。Ang-1能促进血清饥饿下MSC的存活(1d:P＜0.01;2d:P＜0.005)、增殖(P＜0.01)和拮抗紫杉醇诱导的细胞凋亡(P＜0.01),RGD多肽能抑制Ang-1的上述效应。Ang-1组pAkt和Bcl-2表达上调,RGD多肽能抑制二者的表达(P＜0.01),PI-3K/Akt通路传导阻滞剂Wortmannin仅能抑制pAkt的表达(P＜0.01),对Ang-1作用下的Bcl-2表达上调无抑制作用(P=0.436)。结论:密度梯度离心与贴壁分离法相结合可简单有效地获得高纯度的MSC,Ang-1可与MSC表面的整合素受体结合促进细胞的粘附、存活和增殖,并通过上调Bcl-2的表达而发挥抗凋亡作用。RGD多肽能够抑制Ang-1的抗细胞凋亡保护作用。
[Abstract]:Aim: to investigate whether recombinant human angiopoietin-1 Ang-1) can promote the adhesion and survival of rat bone marrow mesenchymal stem cells (MSCs) and inhibit its apoptosis through integrin, and discuss its signal transduction mechanism of anti-apoptosis. Methods: rat bone marrow MSC was isolated by density gradient centrifugation combined with adherent separation in vitro and identified by flow cytometry. The adhesion of Ang-1 to MSC was measured on 24 well culture plate. Trypan blue staining and MTT assay were used to detect the effect of Ang-1 on the activity of MSC under serum starvation, the effect of Ang-1 on MSC apoptosis was detected by Hoechst staining and Annexin V/PI method after paclitaxel induced MSC apoptosis, and the changes of phosphorylated Akt2, total Aktbcl-2, 尾 -actin were detected by Western blot method. To explore the possible molecular mechanism of integrin antibody and signal transduction blocker. Results: MSCs were successfully isolated, cultured and amplified in vitro. The cell morphology, growth characteristics and flow cytometry surface antigen detection were all in line with the characteristics of MSC, and the purity was 98.33%. The adhesion test showed that Ang-1 could significantly promote the adhesion of MSC at three different time points (all P < 0.01). This effect could be inhibited by EDTA, integrin subunit 尾 _ 1 antibody and RGD polypeptide. P < 0.01).Ang-1 could promote the survival of MSC under serum starvation on 1 day, P < 0.012 days, P < 0.005, proliferation P < 0.01), and could promote the survival of MSC at different time points (P < 0.012, P < 0.005, P < 0.01), and the effect could be inhibited by EDTA, integrin subunit 尾 _ 1 antibody and RGD polypeptide (P < 0.01). Antagonism to apoptosis induced by paclitaxel (P < 0.01). Ang-1 can inhibit the above effects of Ang-1. In Ang-1 group, the up-regulation of pAkt and Bcl-2 expression can inhibit the expression of PI-3K / Akt pathway conduction blocker Wortmannin can only inhibit the expression of pAkt (P < 0.01). There was no inhibitory effect on the upregulation. Conclusion: the combination of density gradient centrifugation and adherent separation can be used to obtain the high purity of MSCG Ang-1, which can bind to the integrin receptor on the surface of MSC to promote cell adhesion, survival and proliferation. By up-regulating the expression of Bcl-2, the anti-apoptosis effect. RGD peptide can inhibit the anti-apoptotic protective effect of Ang-1.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

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