Construction Of Ex Vivo Normothermic Machine Perfusion System Of Rat Liver Based On Polymerized Porcine Hemoglobin

Study objective:Hemoglobin based oxygen carriers（HBOCs）belong to a kind of red blood cell substitutes.Its preparation method is to cross-link hemoglobin within or between molecules to form polymeric hemoglobin oxygen carriers.At present,the common hemoglobin oxygen carriers on the market include human,porcine or bovine sources.These products have the function of carrying oxygen and releasing oxygen.They can be used for emergency blood transfusion in clinical practice,and play the role of maintaining blood osmotic pressure balance and expanding blood volume.Compared with human red blood cells,hemoglobin oxygen carrier is polymerized by hemoglobin and inactivated by virus.It can be used directly without considering blood matching,and can avoid pathogenic microbial infection.Its half-life is long,and its storage cycle at room temperature is more than one year,which can be produced on a large scale.Porcine hemoglobin and human hemoglobin have similar structures,similar biological regulation mechanisms,and a wide range of raw material sources.Therefore,in this study,porcine polymerized hemoglobin（p Poly Hb）was used as one of the main components to construct a liver preservation solution.Most preservation solutions usually add red blood cells from the organ donor or artificial blood in order to carry oxygen.Artificial blood is mostly fluorocarbons,and its formula is an emulsion,which can reduce the content of O₂,and requires a higher p O₂to maximize the O₂content.Hemoglobin oxygen carrier is easy to release oxygen,sterile and pyrogen free.It has a wide temperature range（4℃～37℃）,and is less viscous than red blood cells.Moreover,it has a long shelf life,and is compatible with all blood groups.Adding hemoglobin oxygen carrier to the perfusate can better exert the ability to carry oxygen and release oxygen,and it is the most economical and applicable.In order to verify the safety and effectiveness of this perfusate and to explore the effect of adding hemoglobin oxygen carrier on the perfusate,we also designed and constructed a set of matching perfusion equipment to study the protective effect of the liver perfusate.Study methods:In this experiment,SD rats were selected as the experimental subjects and divided into four groups for different treatments:Control（without the addition of polymerized hemoglobin）,12%p Poly Hb,24%p Poly Hb,36%p Poly Hb,with 8 rats in each group.After ligation of the inferior vena cava,the blood flow of the rat liver was blocked and the liver entered the stage of ischemic injury.Then the diaphragm was opened to ligation of the connection between the superior vena cava and the heart.After blocking the cardiac blood flow for 30min,remove the liver and connect it to the pipeline of normal temperature mechanical perfusion system for 6h.The first 10min was the debugging stage,and the perfusion was serious after the end of this stage.At 10min,1h,3h and 6h after the beginning of the perfusion,appropriate amount of the perfusion solution was taken for blood gas analysis and blood biochemical detection,and the levels of p H,p CO₂,p O₂,K⁺,Cl-,Ca²⁺,Glu and Lac were detected,and the levels of AST,ALT and LDH were detected.After the perfusion,bile was collected and some liver tissue samples were collected for HE staining and TUNEL staining to observe the morphology of liver tissue and detect the apoptosis rate of liver cells.Part of the liver tissue samples were collected for Western Blot test to detect the expression levels of Bcl-2 and Bax proteins in the tissues.Study results:1.The p H value of each group increased with time during perfusion.For p CO₂and p O₂,the addition of p Poly Hb can effectively increase p O₂and appropriately reduce p CO₂,which makes the perfusate can effectively carry oxygen and balance the p H of the perfusate.2.The change trend of electrolyte ion analysis in each group was similar,the content of lactic acid and glucose continued to rise with time,and the level of lactic acid in control group was the highest.This indicates that the liver gradually begins aerobic metabolism after the addition of p Poly Hb.3.The enzymatic level analysis showed that the value of the Control group was significantly higher than that of the three groups added with p Poly Hb,and the 24%p Poly Hb group had the lowest level,but the changes of AST and LDH in the 12%p Poly Hb group were consistent with the 24%p Poly Hb group.The degree of liver damage was significantly reduced after the addition of p Poly Hb,but 24%could not be completely determined as the optimal addition amount.4.The bile output of the Control group was lower than that of the three groups added with p Poly Hb,and there was no significant difference among the three groups added with p Poly Hb.The pathological test results of liver tissue were consistent with the results of TUNEL staining.The control group had the most severe morphological damage and the highest apoptosis rate,and the 24%p Poly Hb group had the best liver morphology.5.After analyzing the results of WB experiment,it can be seen that among the four groups,the expression level of hepatocyte apoptotic protein in the Control group is the highest,and the 24%p Poly Hb group is the lowest,but the difference between the 36%p Poly Hb group and the 24%p Poly Hb group is small.Preliminary exploration shows that p Poly Hb may inhibit the apoptosis of liver cells by down-regulating the expression of Bax and up-regulating the expression of Bcl-2,thereby protecting the liver.Study conclusion:The above experimental results prove that the addition of glutaraldehyde-polymerized porcine hemoglobin in normal temperature mechanical perfusion has a protective effect on the liver.Although the 24%p Poly Hb group performed well in all tests,it was not optimal in all tests.Therefore,the optimal addition amount still needs to be further explored,but the addition amount of 24%can be used as a reference to narrow the scope of inquiry.