低温机械灌注在边缘供肾保存中的优势及其机制的研究

发布时间：2018-04-10 12:30

  本文选题：热缺血 + 家兔　； 参考：《南昌大学》2014年硕士论文

【摘要】：目的： 建立家兔肾脏在体机械灌注与冷保存模型;低温机械灌注对比传统冷保存在边缘供肾保存中的优势并探讨其机理。试验分为两部分 第一部分：建立家兔DCD(Donation After Cardiac Death, DCD)肾脏在体机械灌注与冷保存模型。 方法：健康成年家兔10只，雌雄不限，体重（3.0±0.3）Kg，随机分为低温机械灌注组（MP组）与静态冷保存组（CS组），每组5只。MP、CS组均开腹，游离肾动、静脉，夹闭肾动、静脉35分钟，开放血流60分钟后肾动脉穿刺置管行冷灌洗并将肾脏移至体外，MP组灌洗后接Lifeport（美国OrganRecovery Systems公司）,选择20mmHg灌注压，4小时低温机械灌注；CS组灌洗后将肾脏放入器官保存液中并低温保存4小时；每半小时测量一次两组保存液的温度。4小时后移除MP、CS组动静脉所置导管并缝合血管，开放血流，左肾复灌、复温，切除右肾，膀胱造瘘留置尿管。术前、术中2小时（B2）、术毕（B3）检测三次血气并据此调整水、电解质与酸碱平衡。夹闭肾动、静脉期间每5分钟记录一次体温。MP组器械灌注期间每小时记录一次灌注流量。术后记录24小时尿量与生存率。 第二部分：机械灌注与冷保存对免肾保存效果比较研究 方法：健康成年家兔15只，雌雄不限，体重（3.0±0.3）Kg，随机分为假手术组(SO组)、低温机械灌注组（MP组）及冷保存组（CS组），每组5只。SO组开腹游离肾动、静脉； MP组复制机械灌注模型，CS组复制冷保存模型。24小时后摘取肾脏标本，心脏取血液标本。检测血清指标：肌酐(CRE)、尿素氮(BUN)（自动生化仪检测）、L-6（酶联免疫吸附测定法）。肾脏组织检测指标：ICAM-1mRNA转录水平（实时荧光定量PCR）。NF-κB p65蛋白的表达情况（免疫组织化学），肾组织病理学改变（HE染色，，光镜观察）。 结果： 第一部分：1、体温：肾动、静脉夹闭期间各组家兔体温较为恒定，组间、组内无明显差异(P＞0.05)。 2、灌注流量：60min及此后的时间点流量显著高于0点，具有统计学意义（P0.001）；其他各时间点灌注流量也高于0点，具有统计学意义（P0.05）。 3、24小时生存率：MP、CS组24小时生存率均为100%，无统计学差异(P＞0.05)。 第二部分：1、尿量: SO组、MP术后24小时尿量显著高于CS组（P＜0.05）；MP组显著高于SO组; 2、血清CRE、BUN：N、MP组CRE、BUN显著低于CS组（P＜0.05＝；MP组中CRE、BUN水平显著高于SO组（P＜0.05＝；3组组内无显著差异(P0.05)。 3、血清IL-6水平: IL-6表达CS组高于MP组与SO组，存在统计学差异(P0.05)；MP组显著高于SO组(P0.05);3组组内无显著差异(P0.05)。 4、肾脏组织NF-κB p65蛋白表达免疫组化法检测,采用Image-Pro Plus6.0图像定量分析软件，测量平均光密度值并对结果进行定量分析。肾脏组织NF-κBp65蛋白表达CS组高于SO组、MP组，(P0.05)；差异有显著性意义。SO组与MP组之间无显著差异(P0.05)。 5、ICAM-1mRNA：RTFQ-PCR检测，ICAM-1mRNA：转录水平CS组高于MP组与SO组(P0.05)，MP组高于SO组(P0.05)，均有显著性意义。 6、肾脏组织学检查：HE染色光镜观测，SO组肾脏组织结构正常。MP组中肾小管上皮细胞轻度水肿，肾小管管径增大，尤其近曲小管为甚。CS组中肾小管上皮细胞轻度水肿，近曲小管与远曲小管的管径均变小。虽然MP、CS组肾小管上皮细胞均为轻度水肿但是CS组对比MP组则更为严重。 结论： 1、本实验建立的家兔肾脏在体机械灌注与冷保存模型稳定成功率高，实验条件易于控制，制作方法简单巧妙，适合家免在体肾灌注与冷保存系列研究。 2、低温机械灌注可降低移植术后血清中CRE、BUN、IL-6水平，减少NF-κB与ICAM-1蛋白的表达，提示机械灌注相比静态冷保存能够更好保护肾脏功能。 3、机械灌注可能通过其对肾脏的微循环机械疏通作用，而改善移植肾脏的滤过功能。 4、机械灌注可能通过去除肾脏低温保存时所产生的代谢物从而减轻移植术后的非特异性炎性损伤。
[Abstract]:Purpose :

To establish a model of rabbit kidney perfusion and cold preservation in vivo ; compare the advantages of low temperature mechanical perfusion with the traditional cold storage in the preservation of the marginal donor kidney and discuss its mechanism . The test is divided into two parts .

The first part : To establish the model of rabbit ' s DCD ( DCD ) kidney in vivo mechanical perfusion and cold storage .

Methods : 10 healthy adult rabbits were randomly divided into two groups : low temperature mechanical perfusion group ( MP group ) and static cold storage group ( CS group ) . Each group was divided into low temperature mechanical perfusion group ( MP group ) and static cold storage group ( CS group ) .
the kidney is put into an organ preservation solution after the CS group is filled and washed , and the kidney is stored at low temperature for 4 hours ;
The temperature of the two groups of preservation solution was measured every half an hour . After 4 hours removal of MP and CS group , the catheter was removed and the blood vessel , open blood flow , left renal complex irrigation , rewarming , right kidney and cystostomy indwelling catheter were removed . Before operation , three blood gases were recorded and the water and electrolyte were left in balance with acid and alkali . After operation , the temperature was recorded every 5 minutes during operation . The perfusion flow rate was recorded every hour during the infusion of MP group . The urine volume and survival rate were recorded 24 hours after operation .

The second part : comparative study on the effect of mechanical perfusion and cold preservation on kidney - free preservation

Methods : Fifteen healthy adult rabbits were randomly divided into sham operation group ( SO group ) , low temperature mechanical perfusion group ( MP group ) and cold storage group ( CS group ) .
In MP group , mechanical perfusion model and CS group were used to copy cold storage model . After 24 hours , kidney specimens were taken and blood samples were collected . Serum parameters were measured : creatinine ( CRE ) , urea nitrogen ( BUN ) ( automatic biochemistry analyzer ) , L - 6 ( enzyme linked immunosorbent assay ) . The expression of NF - 魏B P65 protein ( immunohistochemistry ) , pathological changes of renal tissue ( HE staining , light microscope observation ) were detected .

Results :

The first part : 1 , body temperature : the temperature of the rabbits in each group was relatively constant during the clamping and closing of the vein , and there was no significant difference between the groups and the group ( P > 0.05 ) .

2 . The flow rate of perfusion : 60min and later time point flow was significantly higher than 0 point , which had statistical significance ( P0.001 ) ;
The perfusion flow at other time points was also higher than 0 point , which had statistical significance ( P0.05 ) .

The 24 - hour survival rate of MP and CS group was 100 % and no statistical difference ( P > 0.05 ) .

The second part : 1 , urine volume : 24 hours after MP , the urine volume was significantly higher than that in CS group ( P < 0.05 ) ;
MP group was significantly higher than that in SO group .

2 銆

本文编号：1731205