肾移植受者HLA-G5的基础和临床研究

发布时间：2018-01-13 15:17

本文关键词：肾移植受者HLA-G5的基础和临床研究　出处：《中国人民解放军军医进修学院》2010年博士论文　论文类型：学位论文

更多相关文章： HLA-G5 肾移植 排斥反应 免疫耐受

【摘要】： 研究背景：HLA-G,非经典MHC-Ib类分子,具有多重免疫调节特性。在生理条件下,HLA-G表达于妊娠期的绒毛膜外的细胞滋养层,在诱导和维持母胎耐受机制中发挥重要的作用。在自身免疫性疾病、肿瘤、病毒感染、器官移植等情况下,HLA-G在血清、血浆中均有不同水平表达,其表现出的免疫调节作用也受到越来越多的关注。目的：本课题通过对比肾移植受者术前、术后与健康对照组HLA-G5的表达,了解HLA-G5与移植耐受的相关性以及不同免疫抑制剂对HLA-G5在’肾移植受者体内表达影响；通过分析对比HLA-G5表达高低两组的不同临床表现,研究HLA-G5在临床监测排斥反应、肾功能恢复、移植肾功能延迟恢复中的意义,对临床免疫抑制剂的应用以及减少免疫抑制剂用量及毒副作用有何指导作用；通过HLA-G5与肾移植受者体内的CD4+CD25+Treg细胞表达的相关性等,探讨HLA-G5与移植物排斥反应和诱导免疫耐受作用机制。方法：应用酶联免疫方法(ELISA)检测HLA-G5的在肾移植受者及健康对照组内表达情况；应用RT-PCR技术检测HLA-G5mRNA在肾移植受者体内表达情况；应用流式细胞术检测CD4+CD25+Treg细胞表达及HLA-G5+T细胞检测;应用体外单向混合淋巴细胞培养技术进一步探讨HLA-G5的免疫抑制性；应用SPSS13.0统计软件统计临床及实验数据。结果：ELISA检测健康对照组、肾移植术前组、肾移植后受者组HLA-G5值,分别为,健康对照组4.922±7.305 ng/ml,肾移植术前组152.575±64.462 ng/ml,肾移植后84天组210.232±107.285 ng/ml,三组间两两比较P值＜0.01,差异有统计学意义,HLA-G5的表达随病程阶段呈现先增高后降低趋势；RT-PCR结果显示HLA-G5mRNA条带密度随病程变化而变化,其密度呈现增高后降低至术前水平趋势；无论是Tac组还是CsA组,HLA-G5表达水平随病程变化而变化,HLA-G5术后1天水平较术前下降,在术后14-28天HLA-G5表达水平较高,之后水平呈下降至术前趋势,两组间HLA-G5表达水平差异不显著,P值＞0.05；移植术后早期肾功能恢复与HLA-G5表达水平相关性研究结果显示：HLA-G5高表达组( 300ng/ml)血肌酐下降斜率为193.779±80.845 umol/天,而HLA-G5低表达组(100ng/ml)血肌酐下降斜率为29.863±25.465 umol/天,P＜0.01,差异有统计学意义；肾移植术后血红蛋白恢复至正常(或术前水平)所需时间在两组间比较,HLA-G5高表达组为21.556±6.464天,而低表达组为80.143±38.555天,P＜0.05,差异有统计学意义；在Tac+MMF+Pred用药组中HLA-G5高表达组免疫抑制剂减药斜率为8.58 0±4.690,低表达组为1.433±0.752,P值＜0.01,差异有统计学意义,在CsA+MMF+Pred用药组中HLA-G5高表达组免疫抑制剂减药斜率为3.633±1.041,低表达组为1.810±0.183,P值＜0.05,差异有统计学意义,；HLA-G5高表达组无一例发生AR,而HLA-G5低表达组发生率43%,P值＜0.05,差异具有统计学意义；HLA-G5高表达组术后1天、4天、7天CD4+CD25+Treg细胞出现频率与术前比较呈下降趋势,P值＜0.05,差异具有统计学意义,术后14天、28天、56天、84天CD4+CD25+Treg细胞出现频率呈逐渐回升至术前水平趋势,而HLA-G5低表达组中,术后1天、4天、7天、14天、28天、56天、84天CD4+CD25+Treg细胞出现频率与术前比较维持在低位水平,差异有统计学意义；肾移植供、受者单向混合淋巴细胞培养增殖实验结果显示HLA-G+组淋巴细胞增殖明显低于空白对照组和HLA-G-组,P值＜0.05,差异具有统计学意义；HLA-G5高表达组无一例发生DGF,而低表达组发生率为57%,P值＜0.05,差异具有统计学意义。结论：HLA-G5在健康人群中可有表达,肾移植术前组、肾移植术后组浓度明显高于健康对照组,差异显著；基因水平HLA-G5的表达,随病程阶段不同表达也不同,不同时间点HLA-G5mRNA条带的相对丰度值呈先升高后降低趋势；无论是Tac组还是CsA组,HLA-G5表达水平随病程呈先增高后降低趋势,免疫抑制剂可能参与了诱导HLA-G5表达；肾移植术后早期HLA-G5表达水平与移植肾功能早期恢复密切相关,HLA-G5高表达者血肌酐下降速率较HLA-G5低表达者为快,且血红蛋白恢复至正常水平或术前水平所需时间短,急性排斥反应发生率低；肾移植术后早期HLA-G5高表达者免疫抑制剂减药速率较HLA-G5低表达者为快,在指导临床用药方面有一定的指导意义；HLA-G5可能通过诱导CD4+CD25+Treg细胞诱导移植物功能耐受,而具体机制有待进一步实验研究；DGF的发生与HLA-G5表达水平密切相关,HLA-G5可能通过减少急性排斥反应这一因素减少DGF的发生。
[Abstract]:Background: HLA-G, a non classical class MHC-Ib molecules with multiple immunomodulatory properties. Under physiological conditions, the expression of HLA-G in chorionic pregnancy outside the cytotrophoblast cells, play an important role in the induction and maintenance of maternal fetal tolerance mechanism. In autoimmune diseases, cancer, viral infection, organ transplantation cases the expression of HLA-G in serum, plasma, have different levels of immune function which showed that has attracted more and more attention.
Objective: this topic through the comparison of renal transplantation patients, postoperative and healthy control group HLA-G5 expression, to understand the relationship between HLA-G5 and transplantation tolerance and different immunosuppressive agents on HLA-G5 recipients' influence in the expression of kidney transplantation; through the comparative analysis of HLA-G5 expression in different clinical manifestations of the two groups of HLA-G5 in rejection clinical monitoring and recovery of renal function, delay in the recovery of renal transplantation function, clinical application of immunosuppressive agents and reduce the dosage of immunosuppressant and side effects have a guiding role; through HLA-G5 and renal transplantation in vivo correlation between the expression of CD4+CD25+Treg cells, and explore the HLA-G5 graft rejection and induce immune tolerance. The mechanism.
Methods: using enzyme-linked immunosorbent assay (ELISA) detection of HLA-G5 in renal transplantation expression and healthy control group; the application of RT-PCR technology to detect HLA-G5mRNA expression in recipients of renal transplantation; the application of flow cytometry to detect the expression of CD4+CD25+Treg and HLA-G5+T cells; the application of mixed lymphocyte immune to further explore HLA-G5 inhibition of cultivation technology; application of SPSS13.0 statistical software for clinical and experimental data.
Results: ELISA was detected in healthy control group, before renal transplantation recipients after renal transplantation group, HLA-G5 group, respectively, healthy control group 4.922 + 7.305 ng/ml before renal transplantation group 152.575 + 64.462 ng/ml, 84 days after renal transplantation group 210.232 + 107.285 ng/ml, three groups of comparison between the 22 P < 0.01, the difference was statistically significant, HLA-G5 expression decreased after the first increase with the duration of stages; RT-PCR results showed that the HLA-G5mRNA band density with the course of change, the density is increased and then decreased trend to the preoperative level of either Tac or CsA; group group, the expression level of HLA-G5 varies with the changes of course, after HLA-G5 the 1 day the level decreased, the high expression level of 14-28 after HLA-G5 days, then decreased to the preoperative trend, the expression level of HLA-G5 between the two groups had no significant difference, P value is greater than 0.05; the expression of HLA-G5 and water recovery of early renal function after transplantation Flat association study results show that: HLA-G5 high expression group (300ng/ml) serum creatinine decreased slope of 193.779 + 80.845 umol/ days, and HLA-G5 low expression group (100ng/ml) serum creatinine decreased slope of 29.863 + 25.465 umol/ days, P < 0.01, the difference was statistically significant; after renal transplantation (or hemoglobin recovery to normal before operation the level of required) compares the time between the two groups in the HLA-G5 high expression group was 21.556 + 6.464 days, while the low expression group was 80.143 + 38.555 days, P < 0.05, the difference was statistically significant; high HLA-G5 in Tac+MMF+Pred group expressed group immunosuppressant drug reduction slope is 8.580 + 4.690, low expression group 1.433 + 0.752, P < 0.01, the difference was statistically significant, high HLA-G5 in the CsA+MMF+Pred group in the expression group of immunosuppressive drug reduction slope is 3.633 + 1.041, the low expression group is 1.810 + 0.183, P < 0.05, the difference was statistically significant, HLA-G5 high table; As a group of patients with AR, and the low expression of HLA-G5 group was 43%, P < 0.05, the difference was statistically significant; HLA-G5 high expression in 1 day group after 4 days, 7 days CD4+CD25+Treg cell frequency compared with preoperative decreased, the P value is less than 0.05, the difference was statistically significant, 14 day, after 28 days, 56 days, 84 days CD4+CD25+Treg cell frequency was gradually recovered to the preoperative level trend, and the low expression of HLA-G5 group, the 1 day, after 4 days, 7 days, 14 days, 28 days, 56 days, 84 days CD4+CD25+Treg cell frequency compared with preoperative in maintenance the low water level, the difference was statistically significant; for kidney transplantation recipients, one-way mixed lymphocyte culture proliferation assay showed that the lymphocyte proliferation in group HLA-G+ was significantly lower than the blank control group and HLA-G- group, P < 0.05, the difference was statistically significant; the high expression of HLA-G5 group with no occurrence of DGF, while the low expression group the incidence rate was 57 The value of P was less than 0.05, and the difference was statistically significant.
Conclusion: HLA-G5 in healthy people can be expressed before renal transplantation group after renal transplantation group was significantly higher than that in the healthy control group, the difference was significant; gene expression level of HLA-G5, with the duration of different stages of expression are different, the relative abundance of HLA-G5mRNA at different time point value of bands increased at first and then decreased; no matter the Tac group and CsA group, the expression level of HLA-G5 decreased after the first increase with the duration is, immunosuppression may be involved in the induction of HLA-G5 expression; the early stage of renal transplantation and transplantation of HLA-G5 expression in the early recovery of renal function is closely related to the high expression of HLA-G5 blood creatinine reduction rate than the low expression of HLA-G5 was faster, and the recovery of hemoglobin to normal levels or preoperative levels required for a short time, low rate of acute rejection after renal transplantation; early high expression of HLA-G5 of immunosuppressive drug reduction for low expression rate is HLA-G5 Fast, has a certain significance in guiding the clinical use of drugs; HLA-G5 could induce CD4+CD25+Treg cell tolerance induced graft function, and the specific mechanism needs further study; closely related to the expression level of DGF and HLA-G5, HLA-G5 can reduce the acute rejection of the factors to reduce the incidence of DGF.

【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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