The effect of murine adipose-derived stem cells exosomes on hypoxia induced osteocytes apoptosis

doi:10.3877/cma.j.issn.1674-1366.2017.03.005

Abstract

Abstract:

Objective

To investigate the effect of exosomes from murine adipose-derived stem cells (ASCs) on hypoxia and ischemia induced osteocytes apoptosis.

Methods

Primary ASCs were isolated and cultured. Flow cytometry was used to evaluate cell surface markers. Differentiation of ASCs to osteoblasts and adipocytes was used to evaluate multilineage differentiation potential. ASC-derived exosomes were isolated and identified by transmission electron microscope. Western blotting was used to analyze exosomes surface markers CD9 and HSP70. MLO-Y4 cells were cultured under hypoxia with/without ischemia and ASC-derived exosomes. TUNEL assay was used to evaluate cell apoptosis. The mean values between groups were analyzed by Bonferroni Test in SPSS 20.0.

Results

The ASCs we isolated and cultured were confirmed their capacity as mesenchymal stem cells. Flow cytometry results showed that the positive rate of CD31 and CD45 was <2%, while that of CD44 was >95%. Positive staining of Alizarin Red or Oil Red was observed after osteogenic or adipogenic induction of ASC for 21 days respectively. Transmission electron microscope analysis showed ASC-derived exosomes with characteristic morphology and size. Western blot detected the expression of CD9 and HSP70 in ASC-derived exosomes. MLO-Y4 cells were cultured under hypoxia and ischemia for 24 h, and cell apoptosis was increased significantly (cell apoptosis rate_nomorxia= 0.041 ± 0.018, cell apoptosis rate_{normoixa and ischemia}= 0.236 ± 0.539, cell apoptosis rate_hypoxia= 0.058 ± 0.015, cell apoptosis rate_{hypoxia and ischemia}= 0.326 ± 0.108) . Cell apoptosis rate was significantly reduced when 0.5 μg/ml ASC-dervied exosomes were added in (cell apoptosis rate_{hypoxia and exosomes}= 0.111 ± 0.049, P<0.001) .

Conclusion

ASC-derived exosomes can inhibit hypoxia and ischemia induced osteocytes apoptosis, and furthermore promote bone repair and regeneration.

Key words: Adipose-derived stem cells, Exosomes, Osteocytes, Apoptosis, Cell hypoxia

Zijun Song, Chunhuang Huang, Lin Ren, Qingwei Cai, Qiang Fu. The effect of murine adipose-derived stem cells exosomes on hypoxia induced osteocytes apoptosis[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2017, 11(03): 157-163.

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Figures/Tables 5

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