Effect Of Hemoglobin On The Neutrophil Responses In Rat Blood To The Bacterial Stimulation

Hemoglobin based oxygen carrier (HBOCs) has became a research key point of the red blood substitutes because of its unique oxygen affinity and natural metabolism pathway. Porcine hemoglobin has the high comparability and stability which seems to be a very attractive choice of the source of HBOCs. With the continuously developing study of HBOCs, the security and validity study become more and more essential. Recent years, there are many researches focus on the validity shows that HBOCs has a good ability of carrying and releasing oxygen, however, during the clinical trails of HBOCs study, many serious side effects were discovered and which kind of confine the development of HBOCs. Therefore, the study of HBOCs security is a very important research field.Neutrophil is the most significant component of nonspecific immune system in organism, in the very acceptable theory, neutrophil work through phagocytosis, inactivation and digestion to fulfill its killing and destroying function. Recently, we found that after incubation of bacteria with fresh rat blood in vitro, two distinct peaks of production of reactive oxygen species were observed, which is, Peakâ… and Peakâ…¡. The Peak I period started shortly after addition of the bacteria to the blood sample, characterized as a short duration (less than 30 min) and small peak value with significant oxygen consumption. The Peakâ…¡period started at about 60 min after bacterial addition, with a prolonged duration (up to about 150 min), a peak value is much higher than that of Peakâ… and no observable oxygen consumption. However, the study of killing ability of neutropil demonstrated that, the rate of phagocytosing neutrophils was enhanced with the incubation time. Thus, according to neutrophils' two apparent different behavior, neutrophil in peripheral blood can be divided into two different major functional classes, called early-responsive neutrophils (ERN) and delayed responsive neutrophils (DRN). ERN reacts quickly to stimulus with significant oxygen consumption. DRN exhibits a delayed response to stimulus without oxygen consumption.Based on the previous research achievements, this thesis was focused on the effect of hemoglobin on the neutrophil responses in rat blood to the bacteria stimulation. Chemiluminescence detection approaches were used to study the change of production of ROS. Viable bacteria in testing samples were quantitatively measured by bacterial culture method. Optical microscopic method was used to determine the phagocytosis neutrophil of bacteria. First of all, we established a stable Luminol-H₂O₂-HRP chemiluminescence detection system, in which it could reach to the peak value of about 400,000 within 6 min. Then we tested the entire factors that would appear to the system of rat blood response after bacteria stimulation, the results pointed out that, heparin, MEM and hemoglobin can all enhance the reaction of Luminol-H₂O₂-HRP chemiluminescence detection system by different level. But in the reaction system of rat blood response after bacteria stimulation, hemoglobin played a completely different role, comparing with Luminol-H₂O₂-HRP chemiluminescence system, no matter pFreeHb or pPolyHb, with increasing of the concentration of hemoglobin, the Luminol-H₂O₂-HRP chemiluminescence system was further enhanced while the cellular chemiluminescence system was further inhibited especially in Peakâ…¡. After eliminated the entire possible influence factor, we put the functional point on the neutrophil, more precisely was the delayed responsive neutrophils, which cause the inhibition of Peakâ…¡at the cellular chemiluminescence system. Also, this inhibition was found to work out through some extracellular pathway to affect the production of ROS.At the same time, we found that the addition of hemoglobin could boost up the killing activity of bacteria of rat blood. This enhancement was not achieved by increasing phagocytosis ability. Therefore, we believed that, the addition of hemoglobin could increase the sensibility to bacteria of neutrophil, and it worked through an extracellular pathway.Based on the results of this study, we have discussed the effect of hemoglobin on ROS productive reaction and the killing ability of bacteria under the physiological condition. This study had offered an intuitive, liable and brand new detection method and research angle towards the character of hemoglobin as well as the security of HBOCs products.