Effect of hypothermic machine perfusion on expression levels of inflammatory cytokines in rat kidney
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摘要:目的 探讨低温机械灌注(HMP)对大鼠肾脏炎症因子表达水平的影响。方法 雄性大鼠30只,随机分为对照组(Control组),静态冷保存组(SCS组)和HMP组,每组10只。记录HMP过程中流速、肾内阻力和灌注流出液pH值。收集每组肾脏组织,采用逆转录聚合酶链反应(RT-PCR)检测CXC趋化因子配体(CXCL)1、CXCL2、干扰素(IFN)-β1、IFN-α4、CC趋化因子配体(CCL)2、CCL20、白细胞介素(IL)-17α、IL-17C和肿瘤坏死因子(TNF)-α的信使RNA(mRNA)表达水平,苏木素-伊红(HE)染色观察肾脏组织病理学改变。结果 HMP过程中的流速、肾内阻力保持稳定,灌注流出液pH值缓慢降低。RT-PCR结果显示,与Control组比较,SCS组和HMP组CXCL1、CXCL2、CCL2、CCL20、IL-17α、IL-17C和TNF-α的mRNA相对表达量升高;与SCS组比较,HMP组CXCL1、CXCL2、CCL2、CCL20、IL-17α和TNF-α的mRNA相对表达量升高(均为P<0.05)。HE染色结果显示,Control组肾脏细胞形态正常,SCS组出现明显的上皮坏死、胞质空泡化、刷状缘丢失、上皮脱落,与SCS组比较,HMP组病理改变程度减轻。结论 HMP能激活肾脏炎症反应,抑制HMP过程中的炎症反应激活有望进一步提高移植物保存效果。Abstract:Objective To evaluate the effect of hypothermic machine perfusion (HMP) on the expression levels of inflammatory cytokines in rat kidney.Methods Thirty male rats were randomly divided into the control (Control group), static cold storage group (SCS group) and HMP group, with 10 rats in each group. The velocity, intrarenal resistance and pH value of perfusion effluent were recorded during HMP. The expression levels of CXC chemokine ligand (CXCL)1, CXCL2, interferon (IFN)-β1, IFN-α4, CC chemokine ligand (CCL)2, CCL20, interleukin (IL)-17α, IL-17C and tumor necrosis factor (TNF)-α messenger RNA (mRNA) in renal tissues were evaluated by reverse transcription polymerase chain reaction (RT-PCR). Pathological changes of the kidney were observed by hematoxylin-eosin (HE) staining.Results During HMP, the velocity and intrarenal resistance remained stable, and the pH value of perfusion effluent was decreased slowly. RT-PCR showed that the relative expression levels of CXCL1, CXCL2, CCL2, CCL20, IL-17α, IL-17C and TNF-α mRNA in the SCS and HMP groups were higher compared with those in the Control group. Compared with the SCS group, the relative expression levels of CXCL1, CXCL2, CCL2, CCL20, IL-17α and TNF-α mRNA were up-regulated in the HMP group (all P<0.05). HE staining revealed that the morphology of renal cells was normal in the Control group, whereas evident epithelial necrosis, cytoplasmic vacuolation, brush border loss and epithelial shedding were observed in the SCS group. Compared with the SCS group, pathological changes in the HMP group were alleviated.Conclusions HMP may activate renal inflammation, and inhibiting the activation of inflammation during HMP is expected to further improve the effect of allograft preservation.
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肾移植被视为终末期肾病的最佳治疗选择。然而,器官保存过程中的缺血-再灌注损伤(ischemia-reperfusion injury,IRI)严重影响移植物功能,是移植成功的主要障碍[1]。因此,减轻器官保存中的IRI并改善器官功能成为当前移植领域亟须解决的问题。静态冷保存(static cold storage,SCS)是一种通过降低保存温度和减缓细胞代谢来实现器官保存的技术[2-3]。然而,大量研究证实,在SCS过程中,能量耗竭、氧化应激和免疫炎症激活等因素可能导致细胞结构和功能的受损,从而引发冷缺血损伤[4-6]。这种损伤在边缘供者中尤为显著,并且随着保存时间的延长,损伤程度会进一步加重。低温机械灌注(hypothermic machine perfusion,HMP)通过持续供应氧气和能量物质,并及时清除代谢废物,能够减轻IRI并改善器官保存质量和效果,提高移植成功率并降低移植后并发症的风险[7-8]。在IRI中,炎症反应激活起着关键的作用,适度的炎症反应有助于组织修复和再生,但过度激活则会加剧组织损伤[9-10]。因此,控制炎症反应激活对于减轻IRI和保护器官功能至关重要。然而,目前关于肾脏HMP过程中炎症反应激活的研究报道非常有限。因此,本研究通过构建大鼠肾脏离体SCS和HMP模型,比较这两种器官保存方式对肾脏炎症因子水平的影响,为进一步研究器官保存提供参考。
1. 材料与方法
1.1 实验动物及主要试剂和仪器
雄性Fisher大鼠30只,体质量260~300 g,饲养于西南医科大学动物实验中心。组氨酸-色氨酸-酮戊二酸盐液(histidine-tryptophan-ketoglutarate solution,HTK液)购于德国Custodiol公司,威斯康星大学保存液(University of Wisconsin solution,UW液)购于美国Bridge to Life公司,QuantiTect逆转录试剂盒购于德国Qiagen公司,TaqMan fast universal PCR master mix购于美国赛默飞公司,CXC趋化因子配体(CXC chemokine ligand,CXCL)1、CXCL2、干扰素(interferon,IFN)-β1、IFN-α4、CC趋化因子配体(CC chemokine ligand,CCL)2、CCL20、白细胞介素(interleukin,IL)-17α、IL-17C、肿瘤坏死因子(tumor necrosis factor,TNF)-α、β-actin引物购于美国赛默飞公司,StepOne逆转录聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)系统购于美国赛默飞公司,机械灌注系统购于德国Hugo Sachs Elektronik公司。本实验通过西南医科大学附属医院伦理委员会审批(批号:20211119-067)。
1.2 实验分组及处理方式
将30只大鼠随机分为对照组(Control组)、SCS组和HMP组,每组10只,术前自由饮水。4.5%异氟醚深度麻醉后,打开胸腹腔并剪破心脏,经腹主动脉使用4 ℃ HTK液冲洗肾脏,直至肾脏颜色变为均匀黄白色,剪破心脏到开始冲洗肾脏的时间大约为5 min。Control组冲洗结束后获取肾脏置于10%多聚甲醛中固定,用于后续的病理组织学检查;SCS组冲洗结束后获取肾脏置于4 ℃ HTK液中保存12 h;HMP组冲洗结束后获取肾脏置于4 ℃ HTK液中保存12 h,再使用4 ℃ UW液以80 mmHg(1 mmHg=0.133 kPa)的恒定压力进行2 h低温机械灌注,采用95% O2、5% CO2通过纤维膜氧合器对灌注液进行氧合。
1.3 研究内容及方法
1.3.1 灌注参数
机械灌注系统能实时记录流速及肾内阻力,灌注结束后收集灌注过程中的流速和阻力数据,并用大鼠肾脏质量进行标准化后进行后续分析。灌注过程中每15 min收集静脉流出液,并使用酸碱度测定计测量其pH值。
1.3.2 逆转录聚合酶链反应实验
取20 mg肾脏组织于组织裂解器中使其变成组织匀浆,使用RNAeasy Mini试剂盒提取总RNA,分光光度计测定浓度,使用QuantiTect逆转录试剂盒进行逆转录获得cDNA。随后使用RT-PCR系统检测CXCL1、CXCL2、IFN-β1、IFN-α4、CCL2、CCL20、IL-17α、IL-17C、TNF-α的CT值。每个标本设3个复孔,用β-actin将每个基因平均CT值进行标化,并使用相对定量方法计算信使RNA(messenger RNA,mRNA)的相对表达水平。
1.3.3 肾脏组织病理学检查
多聚甲醛固定肾脏,经过不同浓度的乙醇脱水、透明、浸蜡、包埋、切片、脱蜡、水化、苏木素-伊红(hematoxylin-eosin,HE)染色后封片制成病理切片,进行病理组织学观察并对肾小管损伤进行评分。肾小管损伤采用改良的评分方法:在每张病理切片中,高倍镜下随机选取10个非重叠区域分别对肾小管扩张、空泡形成、刷状缘缺失、上皮坏死和上皮脱落五种组织形态学变化进行半定量评估。评分标准:结构正常0分;轻度改变为1分(< 25%);中度改变为2分(25%~50%);严重改变为3分(> 50%)。计算10个区域的平均分作为每张病理切片的肾小管损伤评分。
1.4 统计学方法
采用SPSS 26.0软件进行统计学分析。对于符合正态分布的计量资料以均数±标准差表示,两组独立样本间比较采用非配对t检验,同一组样本灌注前后比较采用配对t检验,P< 0.05为差异有统计学意义。
2. 结 果
2.1 机械灌注过程中各参数变化
流速在HMP过程中相对稳定,灌注结束时流速并未降低[(2.7±0.4) mL/(min·g)比(2.9±0.4) mL/(min·g),P=0.5,图1A]。肾内阻力在HMP过程中均相对稳定,灌注结束时与灌注开始时比肾内阻力没有明显变化[(22.2±1.6) mmHg/(min·g)比(20.1±2.1) mmHg/(min·g),P=0.3,图1B]。肾静脉灌注流出液pH值在HMP最初阶段下降,随后保持相对稳定,灌注结束时的pH值低于灌注开始时(7.28±0.01比7.35±0.01,P=0.002,图1C)。
2.2 各组肾脏炎症因子mRNA表达水平比较
与Control组比较,SCS组和HMP组CXCL1、CXCL2、CCL2、CCL20、IL-17α、IL-17C、TNF-α的mRNA相对表达量升高;与SCS组比较,HMP组CXCL1、CXCL2、CCL2、CCL20、IL-17α 和 TNF-α的mRNA相对表达量升高(均为P< 0.05,图2)。
2.3 各组肾脏组织病理学结果的比较
肾脏组织切片HE染色结果显示,Control组肾小管刷状缘完整,管腔无细胞碎片,细胞形态正常;SCS组存在大量的细胞质广泛空泡化、刷状缘缺失、上皮坏死、上皮脱落、管腔阻塞,肾小管损伤严重;HMP组仅有少量刷状缘丢失和上皮脱落,肾小管损伤轻微(图3)。
Control组肾小管损伤评分为(0.34±0.11)分,低于SCS组的(6.20±0.59)分和HMP组的(4.10± 0.76)分,而HMP组肾小管损伤评分低于SCS组(均为P< 0.05)。
3. 讨 论
肾移植仍然是终末期肾病的最佳治疗选择,能够改善患者生活质量和延长寿命[11-12]。供者短缺是目前器官移植面临的巨大挑战[13-15]。边缘供者被迫用以补充供者池以改善供者短缺的窘境,这类供者对保存过程中的IRI更加敏感,术后移植物无功能发生率更高[16-17]。随着Collins液、UW液、HTK液等器官保存液相继开发和改进,SCS成为了目前常用的器官保存方式,被多数器官移植中心广泛使用[18-19]。SCS主要通过降低温度来抑制细胞代谢,然而长时间SCS可能会导致细胞活力丧失甚至坏死[20-21]。冷保存时间越长,器官遭受的损伤越严重,特别是对于边缘供器官,这大大降低了SCS的效果[22-24]。近年来,研究人员开始更加关注HMP,它不仅可以保存器官,改善器官功能,还可用于评估器官功能[25-28]。本研究结果表明,HMP虽然激活了肾脏的炎症反应,但减轻了肾脏冷保存损伤。靶向调控HMP过程中的炎症反应为进一步改善器官功能提供了新的思路。
肾脏机械灌注过程中的血流动力学变化是一个重要的功能参数,可用于评估灌注效果和肾脏损伤程度[6]。由于体外灌注过程中灌注压力恒定,因此流速与肾内阻力成反比。本研究发现,灌注过程中肾内阻力相对稳定,无明显变化。Blum等[29]对猪肾脏进行8 h的HMP,也发现灌注过程中肾内阻力保持基本稳定。IRI过程中存在多种炎症因子的释放和激活,如细胞因子TNF-α、IFN-β1、IFN-α4、IL-17α、IL-17C,趋化因子CXCL1、CXCL2、CCL2、CCL20[30]。这些炎症因子在IRI中发挥着重要的作用,调控这些炎症因子有助于减轻IRI,改善器官保存和移植效果。对机械灌注过程中炎症因子的研究,有助于深入理解机械灌注对器官的影响,并为开发相关的治疗策略提供基础。目前,对于机械灌注过程中移植物炎症细胞因子的变化,大部分研究集中在常温机械灌注(normothermic machine perfusion,NMP)[31],对于HMP过程中炎症因子的变化研究非常有限。一项包括293例肝移植受者的随机临床多中心试验数据显示,相比于SCS,NMP能显著减轻肝小叶炎症,减轻IRI[32]。Beetz等[33]研究发现,相比于SCS,6 h的NMP明显提高了猪肝IL-1α、IL-1β、IL-2、IL-6和IL-18的表达水平。然而,Fontes等[34]研究表明,与SCS相比,机械灌注明显降低猪肝IFN-α、IFN-γ、TNF-α、IL-1β、IL-4和IL-12的水平。Jager等[35]分别对猪和人的肾脏进行4 h和 6 h的NMP,每小时采集灌注液样本以评估促炎因子变化水平,发现IL-6、IL-8和TNF在NMP期间显著增加。Yang等[36]使用夹闭兔子肾脏25 min模拟热缺血损伤,SCS组松开夹子再灌注29 h后在4 ℃高渗柠檬酸腺嘌呤溶液-Ⅱ溶液(hypertonic citrate adenine solution-Ⅱ,HCA-Ⅱ溶液)中进行6 h的SCS,NMP组松开夹子恢复血流1 h,然后再进行HMP保存6 h,结果发现与SCS组相比,HMP组核因子(nuclear factor,NF)-κB和TNF-α的表达明显降低。然而本研究发现与SCS组相比,HMP组肾脏多种促炎因子mRNA表达水平显著升高,且HMP能减轻冷保存损伤,研究结果的差异可能与两个实验方案中热缺血时间、保存液种类以及保存时间不同有关。
赵德芳等[37]比较了SCS和HMP对犬肝脏炎症因子水平的影响,发现移植后HMP组肝脏IL-1β、TNF-α、IL-8、巨噬细胞炎症蛋白(macrophage inflammatory protein,MIP)-1α、MIP-1β、CCL20的mRNA表达水平明显低于SCS组,而在移植前的肝脏中只有TNF-α、MIP-1α、MIP-1β、CCL20的mRNA表达水平差异有统计学意义。本研究采用大鼠肾脏作为实验对象,实验设计也有所不同,获取的肾脏置于4 ℃的HTK液中保存12 h,而赵德芳等[37]对移植前的肝脏采用生理盐水保存约3 h。此外,本研究中的HMP组肾脏在SCS 12 h后,继续进行2 h的HMP,而SCS组仅接受了12 h的SCS,因此HMP组肾脏的冷缺血时间更长。这可能是导致HMP组肾脏炎症因子表达水平明显升高的原因。相较于SCS,虽然HMP升高了肾脏炎症因子的mRNA表达水平,但改善了肾脏病理损伤,这可能与HMP降低移植物代谢率、提供氧气、清除代谢产物、维护细胞膜稳定性和抗氧化等多种机制有关。
综上所述,肾脏移植物在HMP期间的炎症反应被激活,在HMP过程中降低移植肾的免疫炎症活性有望进一步提高机械灌注效果,改善移植物存活率。
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