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CTLA4-Ig基因修饰骨髓间充质干细胞抑制大鼠肝移植排斥反应

尹东亮 孙翀 朱焕斌 李坤 李浩辉 张剑

尹东亮, 孙翀, 朱焕斌, 等. CTLA4-Ig基因修饰骨髓间充质干细胞抑制大鼠肝移植排斥反应[J]. 器官移植, 2014, 5(4): 231-236. doi: 10.3969/j.issn.1674-7445.2014.04.008
引用本文: 尹东亮, 孙翀, 朱焕斌, 等. CTLA4-Ig基因修饰骨髓间充质干细胞抑制大鼠肝移植排斥反应[J]. 器官移植, 2014, 5(4): 231-236. doi: 10.3969/j.issn.1674-7445.2014.04.008
Yin Dongliang, Sun Chong, Zhu Huanbin, et al. Bone marrow mesenchymal stem cell modified by CTLA4-Ig gene can inhibit the rejection of liver transplantation in rats[J]. ORGAN TRANSPLANTATION, 2014, 5(4): 231-236. doi: 10.3969/j.issn.1674-7445.2014.04.008
Citation: Yin Dongliang, Sun Chong, Zhu Huanbin, et al. Bone marrow mesenchymal stem cell modified by CTLA4-Ig gene can inhibit the rejection of liver transplantation in rats[J]. ORGAN TRANSPLANTATION, 2014, 5(4): 231-236. doi: 10.3969/j.issn.1674-7445.2014.04.008

CTLA4-Ig基因修饰骨髓间充质干细胞抑制大鼠肝移植排斥反应

doi: 10.3969/j.issn.1674-7445.2014.04.008
基金项目: 

国家自然科学基金 81070382

广东省科技计划项目 2011B061300028

详细信息
    通讯作者:

    张剑,Email:zhangjian629@sina.com

  • 中图分类号: R617

Bone marrow mesenchymal stem cell modified by CTLA4-Ig gene can inhibit the rejection of liver transplantation in rats

  • 摘要:   目的  探讨细胞毒性T淋巴细胞相关抗原4免疫球蛋白(CTLA4-Ig)基因转染骨髓间充质干细胞(MSC)在抑制大鼠原位肝移植排斥反应的作用及机制。   方法  采用重组腺病毒(Ad)5-CTLA4-Ig转染MSC。转染72 h后,提取细胞总蛋白,采用蛋白质印迹法检测转染后MSC中CTLA4-Ig的蛋白表达。采用细胞计数试剂盒(CCK)-8方法检测未转染和转染后的MSC对外周血淋巴细胞增殖的抑制作用。以雄性Lewis大鼠为供体(40只);以雄性Brown Norway(BN)大鼠为受体(40只)。采用改良的Kamada两袖套法进行原位肝移植,建立大鼠原位肝移植急性排斥反应模型。40只受体大鼠随机分为4组,每组10只。其中对照组(A组),于肝移植时门静脉输注生理盐水;MSC治疗组(B组),于肝移植时门静脉输注MSC;转基因MSC治疗组(C组),于肝移植时门静脉输注转基因MSC;免疫抑制剂治疗组(D组),于肝移植时门静脉输注生理盐水,术后即给予环孢素(CsA)1.5 mg/(kg·d)肌内注射,连续8 d。每组大鼠取5只观察生存情况。每组其余5只于术后第9日处死,检测外周血细胞因子白细胞介素(IL)-2、IL-4、干扰素(IFN)-γ水平,光学显微镜下观察肝组织病理学变化和排斥反应程度。   结果  重组Ad5-CTLA4-Ig转染MSC 72 h后, 蛋白质印迹法可检测到转染后的MSC中有CTLA4-Ig的蛋白表达。当未转染的MSC:外周血单核细胞比例为1:10、1:20时,MSC抑制淋巴细胞增殖的作用分别为85.60%、76.69%。重组Ad5-CTLA4-Ig转染MSC 72 h后,在相同的数量比下,其抑制淋巴细胞增殖的作用分别为90.50%、84.20%;与未转染的MSC比较,转染后抑制淋巴细胞增殖的作用增强(P<0.05)。A、B、C、D组大鼠肝移植术后存活时间分别为(13±3),(41±6),(90±15),(102±18)d。A、B、C组的大鼠术后存活时间比较差异有统计学意义(P<0.05),C组和D组的大鼠术后存活时间比较差异无统计学意义(P>0.05)。与A组比较,B组和C组的IL-4水平明显升高;与B组比较,C组的IL-4水平明显升高,差异均有统计学意义(均为P<0.05);C组和D组的IL-4水平比较,差异无统计学意义(P>0.05)。与A组比较,B组和C组的IL-2、IFN-γ水平明显降低,C组的IL-2、IFN-γ水平亦低于B组,差异均有统计学意义(均为P<0.05),C组和D组的IL-2、IFN-γ水平比较,差异无统计学意义(P>0.05)。大鼠肝组织病理检查结果显示,A组移植肝发生重度排斥反应,B组移植肝亦发生排斥反应,但与A组比较程度较轻。C组与D组移植肝有轻度排斥反应。   结论  重组Ad-CTLA4-Ig转染MSC可抑制肝移植排斥反应,其效果优于MSC单独应用。

     

  • 图  1  重组Ad5-CTLA4-Ig转染MSC后CTLA4-Ig蛋白的表达

    注:条带1样本为MSC裂解液;条带2样本为表达增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP)的重组Ad(Ad-eGFP)转染MSC 72 h后的裂解液;条带3为Ad5-CTLA4-Ig转染MSC 72 h后的裂解液

    Figure  1.  The protein expression of CTLA4-Ig in transgenic MSC infected with recombinant Ad5-CTLA4-Ig

    图  2  4组大鼠的Kaplan-Meier生存曲线

    Figure  2.  Kaplan-Meier survival curves of rats in 4 groups

    图  3  4组大鼠术后第9日的肝组织病理学变化(HE,×100)

    注:A图为SD大鼠正常肝组织;B图为A组大鼠肝组织,可见汇管区及其周围大量炎症细胞浸润,伴中重度静脉炎和肝细胞坏死; C图为B组大鼠肝组织,可见汇管区内胆管和血管被较多炎症细胞浸润,但程度明显减轻;D图和E图分别为C组和D组大鼠肝组织,汇管区内胆管和血管仅见少量炎症细胞浸润

    Figure  3.  Pathological changes of the liver tissues of rats in 4 groups on the 9th day after transplantation

    表  1  4组大鼠肝移植术后细胞因子水平的比较

    Table  1.   Comparison of the levels of cytokines of the rats in 4 groups after liver transplantation ( (x±s),ng/L)

    组 别nIL-2 IFN-γ IL- 4
    A组586±10239±1631±6
    B组542±6a140±15a52±7a
    C组529±4a,b52±10a,b89±15a,b
    D组527±456±886±14
    注:与A组比较,aP<0.05;与B组比较,bP<0.05
    下载: 导出CSV
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出版历程
  • 收稿日期:  2014-03-27
  • 网络出版日期:  2021-01-19
  • 刊出日期:  2014-07-15

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