miR-663在腎移植急性排斥反應(yīng)中調(diào)控作用的體外研究
發(fā)布時間：2019-07-06 10:46
【摘要】：研究背景腎移植術(shù)后,如何減輕急性排斥反應(yīng)(acute rejection AR)所帶來的損傷仍是移植臨床面臨的重要問題。盡管免疫抑制藥物使AR的發(fā)生率及預(yù)后大為改善,但由于傳統(tǒng)的用于診斷AR的指標(biāo)(如:腎小球濾過率、血肌酐水平,彩色多普勒超聲等影像學(xué)檢查、移植腎組織活檢等)存在一定的滯后性,增加了治療難度及費用,對患者預(yù)后帶來較大負面影響。AR過程中,分子信號早于病理損害,更早于臨床癥狀,新近探索的無創(chuàng)診斷方法有血、尿標(biāo)記物、蛋白質(zhì)組學(xué)、mRNA等的檢測,但在敏感性、特異性、穩(wěn)定性等方面存在不同程度的缺陷,使之診斷價值有限。miRNrA是一類非編碼小分子單鏈RNA,在不同疾病中差異表達、外周血液中理化性質(zhì)穩(wěn)定、參與調(diào)控移植腎AR等特點使其有望成為早期診斷移植腎AR的外周血標(biāo)志物。miRNA參與細胞增殖、分化、凋亡、周期調(diào)控等生物學(xué)過程,在生物發(fā)育、病理生理過程中發(fā)揮著重要的調(diào)控作用。有研究表明,miR-663參與移植腎AR并可調(diào)控細胞凋亡的過程。因此,本研究擬證實外周血miR-663在移植腎AR患者及非AR患者中差異表達,明確miR-663是否可作為特異性的指標(biāo)用于AR的診斷,并探討miR-663在AR過程中的作用機制,為臨床上早期準(zhǔn)確診治AR提供新思路。研究目的1、比較移植術(shù)后發(fā)生AR的患者及非AR患者血清中miR-663的表達水平,驗證miR-663在移植腎AR過程中起調(diào)控作用;2、從細胞水平上探討miR-663在腎移植AR過程中的調(diào)控機制。研究方法1、實時熒光定量PCR檢測移植腎AR患者及非AR患者血清miR-663的表達水平;2、實驗設(shè)置miR-663 mimic組、miR-663 inhibitor組、陰性對照組及空白對照組;應(yīng)用MTT及AnnexinV-FITC分別檢測增加miR-663表達及抑制miR-663表達對人腎小球內(nèi)皮細胞(Human renal glomerular endothelial cells,HRGEC)的生存率及凋亡率的影響;3、運用ELISA檢測過表達miR-663及抑制miR-663表達對IL-6、IFN-γ、CCL-2及TNF-α的炎性因子表達水平的影響;4、通過Transwell實驗檢測過表達miR-663和抑制miR-663表達對巨噬細胞趨化性的影響。研究結(jié)果AR組患者血清miR-663的表達較非AR組的腎移植患者顯著升高((4.73±0.28 vs 1.06±0.04;P0.01)。MTT 結(jié)果顯示過表達 miR-663 可以顯著降低HRGEC的生存率并增加細胞凋亡率,而抑制miR-663的表達則可以降低HRGEC凋亡。過表達miR-663可以顯著提高相關(guān)炎性因子的表達,同時顯著增加巨噬細胞的趨化性。研究結(jié)論miR-663在移植腎AR過程中發(fā)揮重要作用,有望做為早期準(zhǔn)確診斷移植腎AR的外周血標(biāo)志物及治療腎移植AR的一個潛在分子靶點。
文內(nèi)圖片：
圖片說明：圖１－１：邋ＡＲ的時間順序和治療效果示意圖［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- 緯, CCL-2 and TNF- 偽 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 鹵0.28 vs 1.06 鹵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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R699.2
本文編號：2510956
文內(nèi)圖片：
圖片說明：圖１－１：邋ＡＲ的時間順序和治療效果示意圖［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- 緯, CCL-2 and TNF- 偽 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 鹵0.28 vs 1.06 鹵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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R699.2
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