基因修饰供体猪肾脏获取、灌注、保存和运输方法的建立OA北大核心

Objective To establish a method for acquisition,perfusion,preservation and transportation of the genetically modified pig kidneys.Methods An eight genetically modified pig was utilized as experimental subject.Prior to kidneys procurement,the health status of the pig was assessed through hematology examination,and the vascular structure of the kidneys was examined using imaging techniques.Following kidneys acquisition,the pig kidneys were perfused and subsequently packaged into the cryogenic storage container labeled"For Organ Transportation Only"for interprovincial transport after communicating the transportation process with transportation department.To evaluate pathological damage to the pig kidneys,a serious of methods were employed such as hematoxylin-eosin(HE)staining,real-time fluorescent quantitative polymerase chain reaction(RT-qPCR),terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)fluorescence staining and enzyme-linked immune absorbent assay(ELISA).Results The preoperative examination of the eight genetically modified pig showed that the serum creatinine was 73.2 μmol/L,blood urea nitrogen was 2.8 mmol/L and hemoglobin was 116 g/L,all within the normal range,indicating normal renal function.CT angiography revealed no lesions in the pig kidneys,and no dilation,stenosis or premature branching of the blood vessels.The total time of obtaining the left and right kidneys from the eight genetically modified pig was(125±10)min,with a blood loss of(20±2)mL.The warm ischemia times were 3 min and 7 min,respectively.The perfusion and trimming times of the left and right kidneys were 36 min and 41 min,respectively.After perfusion,both kidneys were white and moist.The cold preservation and transportation time was 8 h.HE staining showed that some glomeruli were shrunk,and the lumens of the surrounding renal tubules were slightly depressed and swollen with partial inner membrane shedding and microvacuoles formed when the kidneys were preserved for 8 h.The level of cysteinyl aspartate-specific proteinase-3 messenger RNA in the kidneys tissue gradually increased with the extension of cold preservation time after 2h(P<0.05).TUNEL fluorescence staining showed that only a small number of cells underwent apoptosis after 8 h of cold preservation,which was not significantly different from that at 0 h(P>0.05).ELISA results showed that the contents of lactate dehydrogenase(LDH)and creatinine in the preservation solution remained relatively stable,but the content of kidney injury molecule 1(KIM-1)gradually increased with the extension of preservation time,suggesting that the pig kidneys had mild injury.Conclusions By establishing methods for acquisition,perfusion,preservation and transportation of the kidneys from genetically modified donor pig,it is possible to effectively and reliably use genetically modified pig kidneys for xenotransplantation.