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中华移植杂志(电子版)

中华移植杂志(电子版) ›› 2022, Vol. 16 ›› Issue (06) : 353 -358. doi: 10.3877/cma.j.issn.1674-3903.2022.06.005

论著

胰肾联合移植供者来源细胞游离DNA水平变化与术后急性排斥反应的相关研究
刘路浩1, 李国君2, 熊韫祎1, 陈荣鑫1, 尹威1, 张磊1, 李光辉1, 方佳丽1, 马俊杰1, 陈正1,†()   
  1. 1. 510260　广州医科大学附属第二医院器官移植科
    2. 528403　中山市人民医院器官移植科
  • 收稿日期:2022-08-13 出版日期:2022-12-25
  • 通信作者: 陈正
  • 基金资助:
    广州医科大学临床重点专科(010004001); 广州市基础与应用基础研究项目(202102020417); 广东省医学科研基金项目(A2021384); 广州市泌尿疾病学重点学科(2021-2023)

Predictive value of donor-derived cell-free DNA in simultaneous pancreas-kidney transplantation recipients with acute rejection

Luhao Liu1, Guojun Li2, Yunyi Xiong1, Rongxin Chen1, Wei Yin1, Lei Zhang1, Guanghui Li1, Jiali Fang1, Junjie Ma1, Zheng Chen1,†()   

  1. 1. Department of Organ Transplantation, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China
    2. Department of Organ Transplantation, Zhongshan City People′s Hospital, Zhongshan 528403, China
  • Received:2022-08-13 Published:2022-12-25
  • Corresponding author: Zheng Chen
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引用本文：

刘路浩, 李国君, 熊韫祎, 陈荣鑫, 尹威, 张磊, 李光辉, 方佳丽, 马俊杰, 陈正. 胰肾联合移植供者来源细胞游离DNA水平变化与术后急性排斥反应的相关研究[J]. 中华移植杂志(电子版), 2022, 16(06): 353-358.

Luhao Liu, Guojun Li, Yunyi Xiong, Rongxin Chen, Wei Yin, Lei Zhang, Guanghui Li, Jiali Fang, Junjie Ma, Zheng Chen. Predictive value of donor-derived cell-free DNA in simultaneous pancreas-kidney transplantation recipients with acute rejection[J]. Chinese Journal of Transplantation(Electronic Edition), 2022, 16(06): 353-358.

目的

应用高通量二代测序技术检测胰肾联合移植受者血清细胞游离DNA(cf-DNA)及供者来源细胞游离DNA(ddcf-DNA)含量，探讨胰肾联合移植术后ddcf-DNA含量与急性排斥反应(AR)的关系。

方法

回顾性分析2019年6月至2021年6月在广州医科大学附属第二医院器官移植科行胰肾联合移植20例受者临床资料，根据术后是否并发AR将其分为对照组(10例)和AR组(10例)。采用随机数字表法选取对照组3例受者作为观察组，检测其移植前、术中移植胰腺血流开放后2 h及术后1、3、7、14、30和60 d血清ddcf-DNA水平；对照组其余7例受者分别检测其移植前及移植后14、30 d血清ddcf-DNA水平。AR组受者分别检测其术前、AR发生时和AR逆转后血清ddcf-DNA水平。组间正态分布计量资料比较采用成组t检验，计数资料比较采用Fisher确切概率法。P<0.05为差异有统计学意义。

结果

ddcf-DNA片段长度为110~350 bp，受者来源cf-DNA片段长度为100~420 bp。观察组3例胰肾联合移植受者分别标记为病例1、2和3，术前ddcf-DNA含量分别为0.49%、0.55%和0.32%，移植胰腺血流开放后2 h ddcf-DNA含量急剧增至12.05%、10.64%和11.05%，并达到峰值；术后第1天开始，ddcf-DNA水平呈明显下降趋势，术后30 d左右达到稳定状态。对照组和AR组受者术前ddcf-DNA含量分别为(0.71±0.09)%和(0.66±0.10)%，差异无统计学意义(t＝1.126，P>0.05)；AR组受者确诊排斥反应的时间为术后(17±4)d，确诊AR发生时血清ddcf-DNA含量为(5.4±2.0)%，对照组受者术后2周血清ddcf-DNA含量为(1.70±0.48)%，ddcf-DNA含量差异有统计学意义(t＝－5.59，P<0.05)。AR组受者排斥反应被逆转且稳定的时间为术后(27±4)d，此时ddcf-DNA含量为(0.93±0.32)%，对照组术后第30天血清ddcf-DNA含量稳定在(0.87±0.25)%，ddcf-DNA含量差异无统计学意义(t＝－0.497，P>0.05)。

结论

胰肾联合移植术后并发AR时受者血清ddcf-DNA含量呈明显升高趋势，当排斥反应被逆转后呈明显下降趋势。

关键词: 胰肾联合移植, 急性排斥反应, 供者来源细胞游离DNA, 生物标志物
Objective

To measure serum cell-free DNA (cf-DNA) and donor-derived cell-free DNA (ddcf-DNA) in simultaneous pancreas-kidney transplantation recipients by the next generation sequencing technology and evaluate the relationship between ddcf-DNA and early acute rejection (AR).

Methods

A total of 20 recipients who underwent simultaneous pancreas-kidney transplantation in the Department of Organ Transplantation, the Second Affiliated Hospital of Guangzhou Medical University from June 2019 to June 2021 were enrolled. According to whether there was AR after operation, the 20 recipients were divided into two groups: control group (10 cases) and AR group (10 cases). Three recipients in the control group were selected as the observation group by the random number table. The serum levels of ddcf-DNA were measured before transplantation, 2 h after recanalization of blood vessel of transplanted pancreas, and 1, 3, 7, 14, 30, and 60 d after transplantation. The levels of serum ddcf-DNA in the other 7 recipients in the control group were measured before and 14 and 30 d after transplantation. Meanwhile, the testing time points of serum ddcf-DNA in AR group were as follows: before operation, during rejection and after reversal of rejection. The measurement data of normal distribution between the two groups were compared by group t test. Fisher′s exact probability method was used for counting data comparison. P<0.05 means the difference was statistically significant.

Results

The length of ddcf-DNA fragment was 110-350 bp. The length of cf-DNA fragment from the recipients was 100-420 bp. Three recipients in the observation group were marked as cases 1, 2 and 3, respectively. The content of ddcf-DNA of the 3 recipients before operation were 0.49%, 0.55% and 0.32%, respectively. The content of ddcf-DNA increased sharply to 12.05%, 10.64% and 11.05% 2 h after recanalization, and reached its peak after blood flow restored. At the first day after transplantation, the level of ddcf-DNA showed a significant downward trend, and reached a stable state about 30 d after transplantation. The content of ddcf-DNA was (0.71%±0.09%) in the control group and (0.66±0.10)% in the AR group. There was no significant difference between the two groups (t=1.126, P>0.05). The time of diagnosis of rejection in AR group was (17±4) d after transplantation, and the content of serum ddcf-DNA was (5.4±2.0)% when AR was confirmed; while the content of serum ddcf-DNA in the control group was (1.70±0.48)% 2 weeks after transplantation, and the difference was statistically significant (t=-5.59, P<0.05). The rejection was reversed and the recipients condition were stable at (27 ± 4) d after transplantation in the AR group, at which time the content of ddcf-DNA was (0.93 ± 0.32)%. The content of ddcf-DNA in the control group was (0.87±0.25)% on the 30th day after transplantation, and the difference was not statistically significant between the 2 groups (t=-0.497, P>0.05).

Conclusions

The serum ddcf-DNA in simultaneous pancreas-kidney transplantation recipients increased significantly when AR occurred. When AR was reversed, the content of ddcf-DNA showed a significant downward trend.

Key words: Simultaneous pancreas-kidney transplantation, Acute rejection, Donor-derived cell-free DNA, Biomarker
图1 胰肾联合移植供受者来源cf-DNA片段大小的差异注：a．供者来源cf-DNA; b．受者来源cf-DNA; cf-DNA.细胞游离DNA
图2 观察组3例胰肾联合移植受者移植前至术后60 d血清供者来源cf-DNA水平变化注：cf-DNA.细胞游离DNA
表1 对照组和AR组胰肾联合移植受者一般资料比较
组别 例数 年龄(岁，±s) 性别(例，男/女) 体质指数(kg/m2, ±s) 原发病(例，1型/2型糖尿病) 糖尿病病程(月，±s) 透析方式(例，血液/腹膜)
对照组 10 47±5 7/3 24.2±2.9 2/8 166±49 9/1
AR组 10 46±9 9/1 23.7±1.9 3/7 142±42 8/2
t值 － 0.369 － 0.466 － 1.195 －
P值 － >0.05 >0.05 >0.05 >0.05 >0.05 >0.05
组别 例数 透析时间(月，±s) 入院时血糖水平(mmol/L，±s) HLA错配(例) 群体反应性抗体(例)
2个 3个 4个 0 >0~≤10% >10%
对照组 10 18±7 5.94±0.68 4 5 1 6 3 1
AR组 10 16±5 6.36±0.58 3 6 1 7 2 1
t值 － 0.434 －1.482 － －
P值 － >0.05 >0.05 >0.05 >0.05
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