吉林大学学报(医学版) ›› 2020, Vol. 46 ›› Issue (05): 955-962.doi: 10.13481/j.1671-587x.20200510

• 基础研究 • 上一篇    

LncRNA-ATB通过调节miR-200c表达对肾移植大鼠急性排斥反应的影响

乔良伟, 曲青山, 李明   

  1. 郑州人民医院器官移植中心肾脏移植科, 河南 郑州 450000
  • 收稿日期:2020-02-26 发布日期:2020-10-23
  • 通讯作者: 曲青山,主任医师,硕士研究生导师(Tel:0371-67077512,E-mail:zzhxqk0371@163.com) E-mail:zzhxqk0371@163.com
  • 作者简介:乔良伟(1975-),男,河南省商丘市人,副主任医师,主要从事肾脏移植方面的研究。
  • 基金资助:
    河南省科技厅医学科技攻关项目资助课题(2018020823)

Effect of LncRNA-ATB on acute rejection of kidney transplanted rats by regulating miR-200c expression

QIAO Liangwei, QU Qingshan, LI Ming   

  1. Department of Kidney Transplantation, Organ Transplant Center, Zhengzhou People's Hospital, Zhengzhou 450000, Henan
  • Received:2020-02-26 Published:2020-10-23

摘要: 目的:探讨被转化生长因子β活化的长链非编码RNA(LncRNA-ATB)调节微小RNA(miRNA)-200c对肾移植大鼠移植后急性排斥反应(AR)和受体组织炎症反应的影响,分析肾移植AR潜在的分子机制,为临床诊断和治疗AR提供理论参考。方法:30只SD大鼠为供体,30只Wistar大鼠为受体,通过手术将供体肾脏移植到受体大鼠体内,建立肾移植AR模型,将建模成功大鼠(27只)随机分为模型组、LncRNA-ATB短发夹RNA(shRNA)组和LncRNA-ATB-NC组,每组9只。选取10只Wistar大鼠为假手术组。术后2 h,LncRNA-ATB-shRNA组和LncRNA-ATB-NC组大鼠按每只200 μL分别尾静脉注射含LncRNA-ATB-shRNA和LncRNA-ATB-NC的psiCHECK2基因重组质粒脂质体复合物,假手术组和模型组大鼠按每只200 μL尾静脉注射Opti-MEM培养基。干预7 d后,RT-PCR法检测各组大鼠外周血中LncRNA-ATB和miR-200c表达水平,全自动生化分析仪检测各组大鼠血肌酐(Scr)和血尿素氮(BUN)水平,酶联免疫吸附法(ELISA)检测各组大鼠血清中白细胞介素6(IL-6)、白细胞介素8(IL-8)和肿瘤坏死因子α(TNF-α)水平,苏木精-伊红(HE)染色法观察各组大鼠肾组织病理形态表现并对AR进行诊断分级和半定量评分,RT-PCR法和Western blotting法检测各组大鼠肾组织中Toll样受体4(TLR4)、髓样分化因子88(MyD88)和核转录因子κB(NF-κB)mRNA及蛋白表达水平。结果:HE染色,与假手术组比较,模型组大鼠肾脏出现AR,表现为炎性细胞浸润、动脉炎症、间质细胞水肿和皮质出血等,LncRNA-ATB-NC组与模型组大鼠肾脏组织病理形态表现相似,LncRNA-ATB-shRNA组大鼠肾组织病理学形态表现较模型组和LncRNA-ATB-NC组均减轻。与假手术组比较,模型组、LncRNA-ATB-NC组和LncRNA-ATB-shRNA组大鼠外周血中LncRNA-ATB表达水平,血清中Scr、BUN、IL-6、IL-8和TNF-α水平,AR半定量评分,肾组织中TLR4、MyD88和NF-κB mRNA及蛋白表达水平均升高(P<0.05),外周血中miR-200c表达水平均降低(P<0.05);与模型组和LncRNA-ATB-NC组比较,LncRNA-ATB-shRNA组大鼠外周血中LncRNA-ATB表达水平,血清中Scr、BUN、IL-6、IL-8和TNF-α水平,AR半定量评分,肾组织中TLR4、MyD88和NF-κB mRNA及蛋白表达水平均降低(P<0.05),外周血中miR-200c表达水平升高(P<0.05);模型组与LncRNA-ATB-NC组大鼠外周血中LncRNA-ATB和miR-200c表达水平,血清中Scr、BUN、IL-6、IL-8和TNF-α水平,AR半定量评分,肾组织中TLR4、MyD88和NF-κB mRNA及蛋白表达水平比较差异均无统计学意义(P>0.05)。结论:沉默LncRNA-ATB可以减轻肾移植大鼠移植后AR,抑制受体组织发生炎症反应,其机制可能是通过上调miR-200c,抑制TLR4、MyD88和NF-κB mRNA及蛋白表达而发挥炎症抑制作用。

关键词: 被转化生长因子β活化的长链非编码RNA, 微小RNA, 肾移植, 急性排斥反应, 炎症反应, 大鼠,Wistar

Abstract: Objective: To investigate the influence of long non-coding RNA activated by TGF-β (LncRNA-ATB) by regulating microRNA (miRNA)-200c in the acute rejection (AR) and the inflammatory response of recipient tissue after transplantation in the kidney transplantation rats,and to analyze the potential molecular mechanism of AR of kidney transplantation. Methods: Thirty SD rats were used as donors and 30 Wistar rats were used as recipients. The donor kidneys were transplanted into the recipient rats by surgery to establish the models of kidney transplantion AR. The successful modeling rats (n=27) were randomly divided into model group, LncRNA-ATB short hairpin RNA (shRNA) group, and LncRNA-ATB-NC group; there were 9 rats in each group. Ten Wistar rats were selected and used as sham operation group. Two hours after transplantation, the rats in LncRNA-ATB-shRNA group and LncRNA-ATB-NC group were injected with LncRNA-ATB-shRNA and LncRNA-ATB-NC gene recombinant plasmid liposome complex in the tail vein (200 μL/rat), and the rats in sham operation group and model group were injected with Opti-MEM medium (200 μL/rat). After 7 d, the relative expression levels of LncRNA-ATB and miR-200c in peripheral blood of the rats in various groups were detected by RT-PCR method,and the levels of serum creatinine (Scr) and blood urea nitrogen (BUN) of the rats in various groups were detected by automatic biochemical analyzer; the levels of serum interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α(TNF-α) of the rats in various groups were measured by enzyme-linked immunosorbent assay(ELISA); HE staining was used to observe the pathomorphology of kidney tissue of the rats in various groups, and the diagnosis grade of AR and the AR semi-quantitative scores were performed;the expression levels of Toll-like receptor-4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear transcription factor κB (NF-κB) mRNA and proteins in kidney tissue of the rats in various groups were detected by RT-PCR and Western blotting methods. Results: The HE staining results showed that compared with sham operation group, there was AR in kidney tissue of the rats in model group, existing inflammatory cell infiltration, arterial inflammation, interstitial cell edema, and cortical hemorrhage, etc; the phathomorphology of kidney tissue of the rats of the rats in LncRNA-ATB-NC group was similar to model group; the pathomorphology of the kidney tissue in LncRNA-ATB-shRNA group was improved compared with model group and LncRNA-ATB-NC group. Compared with sham operation group, the expression levels of LncRNA-ATB in peripheral blood, the levels of serum Scr, BUN, IL-6, IL-8 and TNF-α,the AR semi-quantitative scores, the expression levels of TLR4, MyD88, NF-κB mRNA and proteins in kidney tissue of the rats in model group, LncRNA-ATB-NC group, and LncRNA-ATB-shRNA group were decreased(P<0.05), the expression levels of miR-200c in peripheral blood were decreased (P<0.05). Compared with model group and LncRNA-ATB-NC group, the expression level of LncRNA-ATB in peripheral blood,the levels of serum Scr, BUN, IL-6, IL-8 and TNF-α,the AR semi-quantitative score, the expression levels of TLR4, MyD88, NF-κB mRNA and proteins of the rats in LncRNA-ATB-shRNA group were reduced (P<0.05), and the expression level of miR-200c in peripheral blood was increased (P<0.05). There were no significant differences in the expression levels of LncRNA-ATB and miR-200c in peripheral blood, the levels of serum Scr, BUN, IL-6, IL-8 and TNF-α,the AR semi-quantitative scores, and the expression levels of TLR4, MyD88, NF-κB mRNA and proteins in kidney tissue of the rats between model group and LncRNA-ATB-NC group (P>0.05). Conclusion: Silencing LncRNA-ATB can reduce AR after kidney transplantation in the rats and inhibit the inflammatory reactions in recipient tissue. Its mechanism may be related to up-regulating the expression of miR-200c kidney transplanted and inhibiting the expressions of TLR4, MyD88, NF-κB mRNA and proteins to exert the inflammatory suppression.

Key words: long non-coding RNA activated by transforming growth factor β, microRNA, kidney transplantion, acute rejection, inflammation response, rats,Wistar

中图分类号: 

  • R692.5