MicroRNA-494调节TNF-α诱导髓核细胞凋亡的实验研究

发布时间：2018-04-08 14:27

本文选题：微小RNA　切入点：JunD　出处：《天津医科大学》2015年硕士论文

【摘要】：目的:椎间盘退行性改变的分子生物学机制仍不明确,髓核细胞凋亡增加在椎间盘退变发生发展过程中发挥重要作用。TNF-a可以诱导髓核细胞凋亡,与椎间盘退变密切相关,外源性因素作用于细胞表达受体,内源性因素可能发挥调节作用,其中就包括microRNAs(miRNAs)。课题组前期研究发现椎间盘退变患者的髓核组织中存在异常表达的miRNAs,其中microRNA-494(miR-494)异常高表达,然而,miR-494在TNF-a诱导髓核细胞凋亡中的潜在作用机制仍然不明确。使用TNF-a刺激髓核细胞,建立髓核细胞凋亡模型;运用慢病毒包装miR-494转染髓核细胞,探究miR-494对TNF-a诱导髓核细胞凋亡的影响并探究其潜在作用机制,为椎间盘退变发病机制的研究奠定一定基础,开拓椎间盘退变基因诊断和生物治疗新视野。方法:收集脊柱爆裂骨折患者病例资料及术中摘除的髓核组织,根据患者术前影像学资料(X线,CT,MRI)进行TLISS评分系统和载荷分享评分系统评分,根据MRI进行Pfirrmann分级评价椎间盘退变程度。运用酶消化法进行髓核细胞原代培养,并鉴定获取的髓核细胞。分别使用不同浓度TNF-a(0ng/ml,10ng/ml,50ng/ml,100ng/ml)刺激髓核细胞,在0h,8h,16h,24h时间点,使用流式细胞术(Annexin V-PE/7-AAD)和实时荧光定量PCR检测髓核细胞凋亡率及mi R-494表达量。然后,将慢病毒包装的antigomiR-494感染髓核细胞,成功敲除髓核细胞内源性miR-494,转染后再加入TNF-a(100ng/ml,16h),流式细胞术检测细胞凋亡率和荧光定量PCR检测miR-494表达量。运用生物信息学方法、荧光素酶双报告基因分析技术及免疫蛋白印迹技术(Western-blotting)验证JunD是否是miR-494的靶基因之一。最后,Western-blotting检测细胞色素C表达水平。结果:髓核细胞凋亡率随TNF-a浓度和刺激时间的增加而增加(P0.05),miR-494表达量亦是随TNF-a浓度和刺激时间的增加而增加(P0.05),然而100ng/ml,16h组,髓核细胞凋亡率最合适,用于后续实验。细胞转染后,antigomiR-494+TNF-a组与对照病毒+TNF-a组相比,细胞凋亡率和miR-494表达量明显下降(P0.05)。生物信息学方法预测、荧光素酶双报告基因验证证实miR-494直接靶向JunD基因。Western-blot结果显示antigomiR-494+TNF-a组JunD蛋白表达水平较对照病毒+TNF-a组高,且有统计学意义(P0.05),进一步说明miR-494对JunD基因的靶向作用,antigomiR-494+TNF-a组细胞色素C蛋白表达水平较对照病毒+TNF-a组低,且有统计学意义(P0.05)。结论:本研究证实TNF-a可以诱导髓核细胞凋亡,且呈剂量和时间依赖性。髓核细胞miR-494表达量随TNF-a刺激浓度、时间的增加而增加,miR-494可能是TNF-a诱导的髓核细胞凋亡的关键调节器,mi R-494敲除可以保护髓核细胞,其可能的机制是通过上调靶基因JunD并介导细胞色素C凋亡通路。miR-494-JunD-细胞色素C信号通路可能是椎间盘退变的潜在机制之一,其为未来椎间盘退变生物治疗及预防提供了靶点。
[Abstract]:Objective: the molecular biology mechanism of intervertebral disc degeneration is still unclear, increased apoptosis of nucleus pulposus cells play an important role in the process of.TNF-a occurs in the development of intervertebral disc degeneration can induce the apoptosis of nucleus pulposus cells, closely associated with the degeneration of intervertebral disc, the effect of exogenous factors on the expression of receptor, endogenous factors may play a regulatory role, including including microRNAs (miRNAs). The abnormal expression of miRNAs previous study found that patients with intervertebral disc degeneration of the nucleus pulposus tissue, including microRNA-494 (miR-494), however, the abnormal high expression, miR-494 in TNF-a induced potential mechanism of nucleus pulposus cell apoptosis remains unclear. The use of TNF-a stimulation of nucleus pulposus cells, the establishment of the nucleus cell apoptosis model; using lentiviral packaging miR-494 transfection of nucleus pulposus cells, explore the effect of miR-494 on TNF-a induced apoptosis of nucleus pulposus cells and explore its potential mechanism, Lay a foundation for study on the pathogenesis of intervertebral disc degeneration, intervertebral disc degeneration to develop gene diagnosis and biological treatment of new horizons. Methods: collect spinal burst fracture nucleus pulposus extirpation of the clinical data and postoperative patients, according to preoperative imaging data (X-ray, CT, MRI) TLISS score system and load sharing scores according to the MRI score system, Pfirrmann classification and assessment of the degree of intervertebral disc degeneration. By enzymatic digestion of nucleus pulposus cells were cultured, and identified the nucleus pulposus cells. Using different concentrations of TNF-a (0ng/ml, 10ng/ml, 50ng/ml, 100ng/ml) stimulation of nucleus pulposus cells in 0h, 8h, 16h, 24h time point. The use of flow cytometry (Annexin V-PE/7-AAD) expression and detection of nucleus pulposus cell apoptosis rate and MI R-494 real-time fluorescence quantitative PCR. Then, the lentiviral packaging antigomiR-494 infection of nucleus pulposus cells, knockdown of endogenous nucleus pulposus cells successfully MiR-494 after transfection, then add TNF-a (100ng/ml, 16h), flow cytometry to detect the apoptosis rate and fluorescence quantitative PCR detection of miR-494 expression. By using bioinformatics method, dual luciferase gene analysis technology and protein immune imprinting technology (Western-blotting) verify whether JunD is the target gene of miR-494. Finally, the level of Western-blotting was used to detect the expression of cytochrome C. Results: the apoptosis of nucleus pulposus cells increased with the concentration of TNF-a and the stimulation time increased (P0.05), the expression of miR-494 was increased with the concentration of TNF-a and the stimulation time increased (P0.05), whereas 100ng/ml, 16h group, apoptosis of nucleus pulposus cells was most suitable for subsequent experiments. After transfection, antigomiR-494+TNF-a virus +TNF-a group compared with the control group, the apoptosis rate and the expression of miR-494 was significantly decreased (P0.05). The prediction method of bioinformatics, dual luciferase gene Verification of miR-494 directly targeting.Western-blot JunD gene showed that the expression level of JunD protein in antigomiR-494+TNF-a group than in the control group +TNF-a virus is high, and there was statistical significance (P0.05), suggested that miR-494 of JunD gene targeting, the expression of cytochrome C protein in antigomiR-494+TNF-a group than in the control group +TNF-a virus is low, and there was statistical significance (P0.05). Conclusion: This study showed that TNF-a can induce the apoptosis of nucleus pulposus cells, and showed a dose and time dependent manner. The expression of miR-494 in nucleus pulposus cells with TNF-a concentration and time increased, miR-494 may be the key to the nucleus pulposus cell apoptosis induced by TNF-a regulator, MI R-494 knockout can protect the nucleus cells, its possible mechanism is by up regulating the expression of target gene JunD and cytochrome C mediated apoptosis pathway of cytochrome.MiR-494-JunD- C signaling pathway may be the potential of intervertebral disc degeneration One of the mechanisms is a target for future biotherapy and prevention of intervertebral disc degeneration.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R681.53

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