Role of endothelial progenitor cell transplantation in rats with sepsis / 中华危重病急救医学

ABSTRACT

Objective To investigate the role of endothelial progenitor cells ( EPCs ) transplantation in rats with sepsis induced by endotoxin ( lipopolysaccharides, LPS ). Methods Sixty clean grade Sprague-Dawley ( SD ) rats with genetic background were divided into three groups according to random number table method:control group, model group, and EPCs transplantation group, with 20 rats in each group. The sepsis model was reproduced by intravenous delivery of LPS 5 mg/kg. Rats in control group were injected with the same amount of normal saline. EPCs were isolated, and cultured and identified were fluorescently labeled with the green fluorescent protein ( GFP ) adenoviral transfection method. The EPC transplantation group was injected with LPS, then a fluorescently labeled EPCs suspension was injected via the tail vein 1 hour later. The expression of fluorescent markers of EPCs was detected with both small animal in vivo imaging instrument and frozen section. Seven days after transplantation, abdominal aorta blood was collected to determine interleukins ( IL-6 and IL-10 ) in peripheral blood with enzyme linked immunosorbent assay ( ELISA ), and the lung, liver, and kidney tissues were harvested, the wet/dry ratio of the lung ( W/D ) was calculated, and hematoxylin and eosin ( HE ) staining was performed to observe, the change in histopathology. Toll-like receptor 4 ( TLR4 ) mRNA expression in lung, liver, and kidney tissues was determined with real-time reverse transcription-polymerase chain reaction ( RT-PCR ). Results The positive rate of EPCs cells with double marking of CD133 and CD34 was 99.0% at the 5th generation of subculture by using flow cytometry. After the transplantation of EPCs labeled with the green fluorescent protein, the appearance of fluorescence indicated that EPCs were mainly localized in the chest, and a stronger fluorescence was observed near the blood vessels. EPCs transplantation could significantly reduce the inflammatory cell infiltration and cell damage in lung, liver, and kidney tissue in septic rats. Compared with control group, the expression of IL-6 and IL-10 in the peripheral blood, W/D ratio, and TLR4 mRNA in lung, liver, and kidney were increased significantly in the model group. Compared with model group, the expressions of IL-6 and IL-10 in the peripheral blood were significantly reduced after EPCs transplantation [ IL-6 (μg/L )2.127±0.118 vs. 2.664±0.438, IL-10 ( ng/L ) 24.5±3.9 vs. 31.5±3.8, both P < 0.01 ]. EPCs transplantation reduced the W/D ratio of lung, liver and kidney tissues ( lung 4.68±0.24 vs. 5.48±0.15, liver 3.33±0.11 vs. 3.94±0.09, kidney 4.08±0.20 vs. 4.84±0.21, all P < 0.05 ], and down-regulated the expression of TLR4 mRNA ( ×103, lung 782±131 vs. 1 136±126, liver 39.1±14.0 vs. 69.2±8.7, kidney 52.2±15.2 vs. 83.5±17.1, all P < 0.01 ). Conclusions EPCs can enter the lung, liver and kidney tissues of the rat successfully after transplantation of EPCs via vein. EPCs transplantation can down-regulate pro-inflammatory process, help to recover the balance of pro-and anti-inflammatory processes, alleviate the damage to the lung, liver, and kidney tissue significantly.