异种肝移植手术前后移植肝差异表达基因的筛选及验证  

The screening and proving of genes differentially expressed after liver xenotransplantation

作　　者：李霄 纪洪辰 潘登科[2] 周亮[3] 窦科峰 陶开山 Li Xiao;Ji Hongchen;Pan Dengke;Zhou Liang;Dou Kefeng;Tao Kaishan(Department of hepatobilliary surgery,Xijing hospital,Fourth Military Medical University,Xi＇an 710032,Shaanxi,China;Institute of Animal Science of Chinese Agriculture Sciences Academy,Beijing 100000,China;Institute of Laboratory Animal Sciences,Siehuan Academy of Medical Sciences and Siehuan Provincial People＇S Hospital,Chengdu 610000,Siehuan,China.)

机构地区：[1]空军军医大学西京医院肝胆外科,陕西西安710032 [2]中国农业科学院北京畜牧兽医研究所,北京100193 [3]四川省人民医院.四川省医学科学院实验动物研究所,四川成都610072

出　　处：《实用器官移植电子杂志》2018年第5期349-354,共6页Practical Journal of Organ Transplantation(Electronic Version)

基　　金：陕西省自然科学基础研究计划资助项目(2017JM8014);陕西省社会发展科技攻关项目(2015SF067);国家"973"计划基金资助项目(2015CB554100);国家重点研发计划资助项目(2017YFC1103703);国家"863"计划基金资助项目(2012AA021005);国家自然科学基金资助项目(81300361;81270549;81670593;81470873;81671838);陕西省科技统筹创新工程计划资助项目(2012KTCL03-01;2011KTCL03-15);西京医院学科助推计划基金资助项目(XJZT12M09;XJZT13Z01;XJZT14Z04)

摘　　要：目的探讨异种移植手术前后移植肝内差异表达基因的变化,并且进行筛选及验证。方法以α-1, 3-半乳糖苷转移酶基因敲除(GTKO)小型猪为供体、藏酋猴为受体,完成4例异种脾窝辅助性肝移植手术。于供肝修整时切取适量术前标本,于移植肝开放血流后48小时以肝穿刺活检获得术后标本。对手术前后的移植肝标本进行miRNAs和mRNAs的高通量芯片分析,筛选出关键的差异表达miRNAs和m RNAs,并进行实时定量PCR及Western Blot实验验证。结果与术前相比,有165个miRNAs在术后出现差异表达,其中上调分子112个,下调分子53个。筛选并验证mi-23b等12个出现显著差异表达的miRNAs,除miR-150和miR-126-3p外,其他miRNAs的表达变化情况与miRNAs芯片结果基本相符。与术前相比,有2 035个mRNAs在术后出现显著差异表达,其中922个表达上调、1 113个表达下调,筛选IGFBP6、CDK2AP1、TNFAIP6、FⅦ、NKG2D等5个生物学功能与肝脏移植密切相关的差异表达基因进行m RNA和蛋白水平验证。除NKG2D外,其他4个基因的蛋白水平表达变化与mRNA水平变化基本一致(均P <0.05),但CDK2AP1的蛋白变化幅度较小,差异无统计学差异(P=0.073)。结论异种肝移植术后移植肝的miRNAs和mRNAs表达出现较大变化,这些差异表达基因可能与免疫损伤及凝血功能障碍有关。Objective To investigate the changes of differentially expressed genes before and after in the liver xenotransplantation, and they were screened and verified. Methods Four cases of xenograft splenic fossa heterotopic auxiliary liver transplantation were performed with a - 1,3- galactoside transferase gene knockout （ GTKO ） mini-pig as donor and Tibetan macaque as recipient. Appropriate preoperative liver specimens were harvested from the donor liver at the back table procedure, and postoperative liver specimens were harvested by biopsy at 48 h after opening the blood flow of the graft. High throughput chip analysis of miRNAs and mRNAs was performed on the transplanted liver specimens. The key differential expression of milaNAs and mRNAs were screened, and real-time quantitative PCIR and Western Blot tests were performed. Results There were 165 miRNAs differentially expressed after surgery, and 112 of them were up-regulated and 53 were down-regulated. Twelve differentially expressed miRNAs, such as mi-23b, were screened and verified. In addition to miR-150 and miR-126-3p, the expression changes of other miRNAs were basically consistent with the results of miRNAs chip. Compared with preoperative data,2 035 mRNAs showed significant differential expression after operation, 922 of them were up-regulated and 1 113 were down-regulated,5 differentially expressed genes which were closely related to liver transplantation, such as IGFBP6, CDK2AP1, TNFAIP6, FVII and NKG2D, were screened at mlRNA and protein levels. Except for NKG2D, the changes in the protein level of the other 4 genes were basically consistent with the changes in the mRNA level （P 〈 0.05）, hut the changes in the CDK2AP1 protein were small, and there was no statistical difference （P = 0.073）. Conclusion The expression of miRNAs and inRNAs in transplanted liver after xenotransplantation has greatly changed. These differentially expressed genes may be related to immune injury and coagulation dysfunction.

关 键 词：异种移植 肝脏移植 小型猪 藏酋猴 微小核糖核酸 信使RNA 

分 类 号：R657.3[医药卫生—外科学]