miR-663在肾移植急性排斥反应中调控作用的体外研究
发布时间：2019-07-06 10:46
【摘要】：研究背景肾移植术后,如何减轻急性排斥反应(acute rejection AR)所带来的损伤仍是移植临床面临的重要问题。尽管免疫抑制药物使AR的发生率及预后大为改善,但由于传统的用于诊断AR的指标(如:肾小球滤过率、血肌酐水平,彩色多普勒超声等影像学检查、移植肾组织活检等)存在一定的滞后性,增加了治疗难度及费用,对患者预后带来较大负面影响。AR过程中,分子信号早于病理损害,更早于临床症状,新近探索的无创诊断方法有血、尿标记物、蛋白质组学、mRNA等的检测,但在敏感性、特异性、稳定性等方面存在不同程度的缺陷,使之诊断价值有限。miRNrA是一类非编码小分子单链RNA,在不同疾病中差异表达、外周血液中理化性质稳定、参与调控移植肾AR等特点使其有望成为早期诊断移植肾AR的外周血标志物。miRNA参与细胞增殖、分化、凋亡、周期调控等生物学过程,在生物发育、病理生理过程中发挥着重要的调控作用。有研究表明,miR-663参与移植肾AR并可调控细胞凋亡的过程。因此,本研究拟证实外周血miR-663在移植肾AR患者及非AR患者中差异表达,明确miR-663是否可作为特异性的指标用于AR的诊断,并探讨miR-663在AR过程中的作用机制,为临床上早期准确诊治AR提供新思路。研究目的1、比较移植术后发生AR的患者及非AR患者血清中miR-663的表达水平,验证miR-663在移植肾AR过程中起调控作用;2、从细胞水平上探讨miR-663在肾移植AR过程中的调控机制。研究方法1、实时荧光定量PCR检测移植肾AR患者及非AR患者血清miR-663的表达水平;2、实验设置miR-663 mimic组、miR-663 inhibitor组、阴性对照组及空白对照组;应用MTT及AnnexinV-FITC分别检测增加miR-663表达及抑制miR-663表达对人肾小球内皮细胞(Human renal glomerular endothelial cells,HRGEC)的生存率及凋亡率的影响;3、运用ELISA检测过表达miR-663及抑制miR-663表达对IL-6、IFN-γ、CCL-2及TNF-α的炎性因子表达水平的影响;4、通过Transwell实验检测过表达miR-663和抑制miR-663表达对巨噬细胞趋化性的影响。研究结果AR组患者血清miR-663的表达较非AR组的肾移植患者显著升高((4.73±0.28 vs 1.06±0.04;P0.01)。MTT 结果显示过表达 miR-663 可以显著降低HRGEC的生存率并增加细胞凋亡率,而抑制miR-663的表达则可以降低HRGEC凋亡。过表达miR-663可以显著提高相关炎性因子的表达,同时显著增加巨噬细胞的趋化性。研究结论miR-663在移植肾AR过程中发挥重要作用,有望做为早期准确诊断移植肾AR的外周血标志物及治疗肾移植AR的一个潜在分子靶点。
文内图片：
图片说明：图１－１：邋ＡＲ的时间顺序和治疗效果示意图［２］逡逑
[Abstract]:Background after renal transplantation, how to reduce the injury caused by acute rejection of (acute rejection AR) is still an important problem in transplantation. Although immunosuppressive drugs greatly improve the incidence and prognosis of AR, the traditional indexes used in the diagnosis of AR (such as glomerular filtration rate, serum creatinine level, color Doppler ultrasound, renal tissue biopsies, etc.) have a certain lag, which increases the difficulty and cost of treatment and has a great negative impact on the prognosis of patients. In AR process, molecular signal is earlier than pathological damage. Earlier than clinical symptoms, the newly explored noninvasive diagnostic methods include blood, urinary markers, proteome, mRNA, etc., but there are different degrees of defects in sensitivity, specificity, stability and so on, which makes the diagnostic value limited. MiRNrA is a kind of non-coding small molecule single chain RNA, differentially expressed in different diseases, and its physical and chemical properties are stable in peripheral blood. It is expected to become a peripheral blood marker for early diagnosis of renal transplantation AR. MiRNA is involved in biological processes such as cell proliferation, differentiation, apoptosis, cycle regulation and so on, and plays an important regulatory role in biological development and pathophysiology. Some studies have shown that miR-663 is involved in renal AR and can regulate apoptosis. Therefore, this study aims to confirm the differential expression of miR-663 in peripheral blood AR patients and non-AR patients, to determine whether miR-663 can be used as a specific index in the diagnosis of AR, and to explore the mechanism of miR-663 in the process of AR, so as to provide a new idea for early and accurate diagnosis and treatment of AR. Objective 1. To compare the expression of miR-663 in serum of patients with AR and non-AR patients after transplantation, and to verify that miR-663 plays a regulatory role in the process of renal AR transplantation. 2. To explore the regulatory mechanism of miR-663 in the process of renal transplantation AR at the cell level. Methods 1. Real-time fluorescence quantitative PCR was used to detect the expression of serum miR-663 in AR patients and non-AR patients. 2, miR-663 mimic group, miR-663 inhibitor group, negative control group and blank control group were set up. MTT and AnnexinV-FITC were used to detect the effect of increasing miR-663 expression and inhibiting miR-663 expression on the survival rate and apoptosis rate of human glomerular endothelial cells (Human renal glomerular endothelial cells,HRGEC). 3. The effects of overexpression of miR-663 and inhibition of miR-663 expression on the expression of IL-6,IFN- 纬, CCL-2 and TNF- 伪 were detected by ELISA, and the effects of overexpression of miR-663 and inhibition of miR-663 expression on the chemotaxis of macrophages were detected by Transwell assay. Results the expression of serum miR-663 in AR group was significantly higher than that in non-AR group (4.73 卤0.28 vs 1.06 卤0.04). MTT). The results showed that overexpression of miR-663 could significantly decrease the survival rate of HRGEC and increase the apoptosis rate, while inhibiting the expression of miR-663 could decrease the apoptosis of HRGEC. Overexpression of miR-663 could significantly increase the expression of related inflammatory factors and the chemotaxis of macrophages. Conclusion miR-663 plays an important role in the process of renal AR transplantation, and is expected to be used as a potential molecular target for early and accurate diagnosis of renal AR and a potential molecular target for the treatment of renal transplantation AR.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R699.2
本文编号：2510956
文内图片：
图片说明：图１－１：邋ＡＲ的时间顺序和治疗效果示意图［２］逡逑
[Abstract]:Background after renal transplantation, how to reduce the injury caused by acute rejection of (acute rejection AR) is still an important problem in transplantation. Although immunosuppressive drugs greatly improve the incidence and prognosis of AR, the traditional indexes used in the diagnosis of AR (such as glomerular filtration rate, serum creatinine level, color Doppler ultrasound, renal tissue biopsies, etc.) have a certain lag, which increases the difficulty and cost of treatment and has a great negative impact on the prognosis of patients. In AR process, molecular signal is earlier than pathological damage. Earlier than clinical symptoms, the newly explored noninvasive diagnostic methods include blood, urinary markers, proteome, mRNA, etc., but there are different degrees of defects in sensitivity, specificity, stability and so on, which makes the diagnostic value limited. MiRNrA is a kind of non-coding small molecule single chain RNA, differentially expressed in different diseases, and its physical and chemical properties are stable in peripheral blood. It is expected to become a peripheral blood marker for early diagnosis of renal transplantation AR. MiRNA is involved in biological processes such as cell proliferation, differentiation, apoptosis, cycle regulation and so on, and plays an important regulatory role in biological development and pathophysiology. Some studies have shown that miR-663 is involved in renal AR and can regulate apoptosis. Therefore, this study aims to confirm the differential expression of miR-663 in peripheral blood AR patients and non-AR patients, to determine whether miR-663 can be used as a specific index in the diagnosis of AR, and to explore the mechanism of miR-663 in the process of AR, so as to provide a new idea for early and accurate diagnosis and treatment of AR. Objective 1. To compare the expression of miR-663 in serum of patients with AR and non-AR patients after transplantation, and to verify that miR-663 plays a regulatory role in the process of renal AR transplantation. 2. To explore the regulatory mechanism of miR-663 in the process of renal transplantation AR at the cell level. Methods 1. Real-time fluorescence quantitative PCR was used to detect the expression of serum miR-663 in AR patients and non-AR patients. 2, miR-663 mimic group, miR-663 inhibitor group, negative control group and blank control group were set up. MTT and AnnexinV-FITC were used to detect the effect of increasing miR-663 expression and inhibiting miR-663 expression on the survival rate and apoptosis rate of human glomerular endothelial cells (Human renal glomerular endothelial cells,HRGEC). 3. The effects of overexpression of miR-663 and inhibition of miR-663 expression on the expression of IL-6,IFN- 纬, CCL-2 and TNF- 伪 were detected by ELISA, and the effects of overexpression of miR-663 and inhibition of miR-663 expression on the chemotaxis of macrophages were detected by Transwell assay. Results the expression of serum miR-663 in AR group was significantly higher than that in non-AR group (4.73 卤0.28 vs 1.06 卤0.04). MTT). The results showed that overexpression of miR-663 could significantly decrease the survival rate of HRGEC and increase the apoptosis rate, while inhibiting the expression of miR-663 could decrease the apoptosis of HRGEC. Overexpression of miR-663 could significantly increase the expression of related inflammatory factors and the chemotaxis of macrophages. Conclusion miR-663 plays an important role in the process of renal AR transplantation, and is expected to be used as a potential molecular target for early and accurate diagnosis of renal AR and a potential molecular target for the treatment of renal transplantation AR.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R699.2
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