Effects of hypoxia on chemokine receptor CXCR4 and CX_3CR1 expression in human bone marrow mesenchymal stem cells

Jie ZHU; Zhujuan ZHOU; Zili GONG; Jian ZHENG

ABSTRACT

BACKGROUND:

Transplanted bone marrow mesenchymal stem cells (BMSCs) migrate to the injured regions and exert their therapeutic effects. The specific mechanisms involved in their directional migration to lesions remain unclear.

OBJECTIVE:

To investigate the chemokine receptor CXCR4 and CX3CR1 expression of human BMSCs in hypoxia culture. DESIGN, TIME AND

SETTING:

The cytology in vitro study was performed at the Central Laboratory, Xingqiao Hospital, Third Military Medical University of Chinese PLA from February 2008 to February 2009. MATERIALS Cells harvested from the iliac heparinized bone marrow were obtained by iliac crest aspiration from healthy adult volunteers, aged 15 to 40 years old, at the Department of Hematology, Xinqiao Hospital, Third Military Medical University of Chinese PLA.

METHODS:

Bone marrow was obtained by puncture. Human BMSCs were harvested by combination of density and gradient centrifugation and different adherent method. Cells at passage 3 were incubated in a 25 cm2 flask. When 70%-80% confluence was found, cells were incubated at 37 ℃ and saturated humidity in an incubator containing 3% O2, 5% CO2, 92% N2 for 48 hours, and those incubated under normal oxygen as controls. MAIN OUTCOME

MEASURES:

Morphology was examined by phase contrast microscopy. Cell surface markers were tested by flow cytometer. The CXCR4 and CX3CR1 mRNA expression were detected by real-time PCR. The CXCR4 and CX3CR1 protein expression were determined by Western blot assay.

RESULTS:

All of the cells had a fibroblast-like morphology cultured in vitro and reached 90% confluence at 12-14 days, with the presence of polarity arrangement and whirlpool-shape. Cells were uniformly positive for CD105 (99.38%) and CD29 (99.13%), but negative for CD14 and CD45. Exposure of BMSCs to 3% O2 increased expression of the CXCR4 mRNA and CX3CR1 mRNA, which were respectively 2.130 times and 2.361 times of normal culture; expression of the CXCR4 protein and CX3CR1 protein was respectively 1.69 times and 1.93 times of normal culture. CXCR4 and CX3CR1 mainly expressed in membrane and cytoplasm of human BMSCs.

CONCLUSION:

Hypoxia (3% O2) can upregulate the expression of CXCR4 and CX3CR1 in human BMSCs, which might be one of the machenisms underlying the migration of BMSCs.