摘要
背景：前期实验已证实骨髓间充质于细胞表面存在整合素受体，但血管生成素1是否能通过整合素受体促进骨髓间充质干细胞的生存、抗凋亡目前尚不明确。目的：探讨血管生成素1对大鼠骨髓间充质干细胞黏附、活力、抗凋亡的影响，并初步分析其信号转导机制。设计、时间及地点：细胞学体外对照观察，于2008—01/10在厦门大学附属中山医院开放实验室完成。材料：清洁级SD大鼠40只，由中科院上海实验动物中心提供。人血管生成素1为R＆D公司产品。方法：密度梯度离心结合贴壁分离法体外分离、培养大鼠骨髓间充质干细胞，第346代细胞用于实验。分别以不同的基质包被24孔培养板1h，清洗后再加入单细胞悬液，设立7组：第1组作为对照，以含0．1％牛血清白蛋白的PBS包被；第2-4组分别以玻粘连蛋白、纤维粘连蛋白、血管生成素1包被；第5-7组单细胞悬液预先分别与乙二胺四乙酸、β1抗体、RGD抗体孵育10min后再加入24孔板，行血管生成素1包被。主要观察指标：通过黏附测定、锥虫蓝染色、MTT试验、Hoechst染色和AnnexinV／PI法检测血管生成素1对骨髓间充质干细胞的黏附、活力、抗凋亡等生物学效应，结合整合索抗体和信号转导阻滞剂通过Western blotting法检测磷酸化丝氨酸，苏氨酸蛋白激酶B、总丝氨酸，苏氨酸蛋白激酶B、Bcl．2和B-actin水平。结果：成功分离培养大鼠骨髓间充质干细胞，流式细胞仪鉴定其纯度达98．3％。黏附试验显示与对照组比较，培养1，2，3d血管生成素1均能促进骨髓间充质干细胞的黏附（P〈0．01），该效应能被乙二胺四乙酸、整合索亚单位B1抗体和RGD抗体所抑制（P〈0．01）。血管生成素1能促进骨髓间充质干细胞在血清饥饿下的存活（P〈0．01）、增殖（P〈0．01）和提高磷酸化丝氨酸，苏氨酸蛋白激酶B、Bcl-2的表达来拮抗紫杉醇诱导的凋C（P〈0．01），RGD抗体能抑制上述作用。结论：血管生成素1可与整合素受体结合促进骨髓间充质干细胞的黏附、存活和增殖，并通过上调磷酸化丝氨酸，苏氨酸蛋白激酶B和Bcl-2的表达发挥抗凋亡作用。
BACKGROUND： Experiments have confirmed that there are integrin receptors on bone marrow mesenchymal stem cells （BMSCs）, but it is still uncertain whether angiogenin-1（Ang-1） can promote survival of BMSCs and protest against its apoptosis via integrins.
OBJECTIVE： To investigate the effect of human recombinant angiopoietin-1 on adhesion, vigour, anti-apoptosis of rat BMSCs and its mechanism of signal transduction.
DESIGN, TIME AND SETTING： The cytological in vitro controlled study was performed at the Opening Laboratory, Zhongshan Hospital Affiliated to Xiamen University from January to October 2008. MATERIALS： A total of 40 clean Sprague Dawley rats were supplied by Shanghai Experimental Animal Center, Chinese Academy of Sciences. Human Ang-1 （R＆D） was used in this study.
METHODS： Combining density gradient centrifugation and adherence separation was used to separate, cultivate and identify rat BMSCs. At the third to sixth passages, BMSCs were used for this study. Various matrix was used to coat 24-well culture plates for 1 hour. Following washing, monoplast suspension was added. Seven groups were set up. Group 1 served as control, BMSCs were coated with phosphate buffered saline containing 0.1% bovine serum albumin. BMSCs in the groups 2, 3 and 4 were incubated with vitronectin, fibronectin and Ang-1. BMSCs in the groups 5, 6 and 7 were firstly incubated in ethylenediaminetetraacetic acid, 131 antibody and RGD antibody for 10 minutes, and then placed in a 24-well plate, and coated with Ang-1.
MAIN OUTCOME MEASURES： Adhesion, trypan blue staining method, MTT test, Hoechst staining and Annexin V/PI were used to measure biological effects of Ang-1 on adherence, activity and anti-apoptosis of BMSCs. Combined with integrin antibody and signal transduction blocker, the expression of phosphoserine/threonine protein kinase B （pAkt）, total Akt, Bcl-2 and β-actin was examined by western blotting.
RESULTS： Rat BMSCs were successfully isolated. The purity of BMSCs was 98.3% using flow cytometry. Cell adhesion assay shows Ang-1 promoted the adhesion of BMSCs at 1, 2 and 3 days compared with control group （P 〈 0.01 ）. This effect could be inhibited by ethylenediaminetetraacetic acid, subunit of integrin β1 antibody and RGD antibody （P 〈 0.01 ）. Ang-1 promoted BMSC survival （P 〈 0.01） and proliferation （P 〈 0.01 ） under serum starvation. Moreover, Ang-1 could protest BMSCs from apoptosis induced by taxinol through activating Phosphorylation of Akt and Bcl-2 （P 〈 0.01）, while inhibited by RGD antibody.
CONCLUSION： Combined with integrin receptor, Ang-1 can promote the adhesion, survival and proliferation of MSC, and exert the anti-apoptosis effect by up-regulating phosphorylation of Akt and Bcl-2.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第23期4510-4516,共7页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
厦门市科技计划项目~~
