Effects of angiogenin-1 on adhesion, activity and anti-apoptosis of rat bone marrow mesenchymal stem cellsviaintegrin*,⋆

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Abstract

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, β1 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.

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