血清miRNA在糖尿病肾病中预警作用研究

发布时间：2018-01-28 04:34

本文关键词： 糖尿病肾病 microRNAs miRNA芯片　出处：《兰州大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景和目的糖尿病微血管病变包括肾脏病变、神经病变、视网膜病变。并发症发展的风险因素受许多因素影响,包括糖尿病持续时间和基因因素。[1]糖尿病(DM)最常见的微血管并发症之一糖尿病肾病(DN),是导致西方国家终末期肾病(ESRD)和DM患者死亡的重要原因,也是成为终末期肾病(ESRD)的首要原因。在我国,DN在导致ESRD的数量也逐年增加。[2]当前DN的致病机制尚不完全清楚,有效的治疗手段尚不足,因此,有效地早期干预和预防、治疗DN已是医学领域中重要的问题,尤其是随着人口老龄化,糖尿病基础人群数量的增加,糖尿病肾病的发病率也将增加,这将极为重要。不管是1型糖尿病还是2型糖尿病均可导致血管并发症,除了高血糖、高低密度脂蛋白、晚期糖代谢产物、生长因子及炎症因子外,需要更好的理解糖尿病的分子机制来确定新的治疗目标。[3]理解糖尿病并发症潜在的发病机制,为这种慢性病状态发展新的和改进的治疗策略仍然非常重要。新的方法来自线虫研究确定了一种新的内源性、小片段、单链、非编码RNA分子,被命名为微小RNA,这种微小RNA作为生长的调控因子。[4] MicroRNA (miRNAs)是微小RNA,普遍存在真核生物中。由前体(pre-miRNA)进一步成熟为miRNA,具体为内源性发夹状结构长度为70~80nt的miRNA具体(pre-miRNA)剪切后生成,成熟miRNA是一类长约21~24nt的通过转录后负调控靶基因表达的非蛋白编码RNA分子,可结合靶基因mRNA3'端非翻译区(3'-untranslatedregion,3'-UTR)互补结合而调控靶mRNA表达。[4]体液循环microRNA是存在于细胞外的microRNA特殊存在方式。研究发现在人体的血清、血浆、尿液、唾液、乳汁等体液中都能检测到多种microRNAs。体液中的microRNA分子多数不是以游离形式存在的,它被脂质、蛋白复合体、微囊泡(microvesicle, MV)、外夹体(exosome)等包裹和结合,因此具有良好的抗RNase降解能力,有较高的稳定性。[5]在来源细胞受到生物学因素刺激后,可以选择性地把特定的microRNA包裹进MV,同时细胞表面标记也携带在MV表面。用不同组织、细胞的表面标记抗体磁珠可以容易的检测到不同组织来源的MV及microRNA。正是由于血清循环microRNA的特点,它非常适合作为生物标记物用于疾病的预警和早期诊断。因其变化早于靶基因的变化、具有体液中的稳定性、细胞间的穿梭性以及组织来源特异性的特点,它非常适合作为生物标志物用于疾病的预警和早期诊断。[24] 上世纪50年代自从messager RNA的发现,蛋白质和mRNA这个生物聚合物之间的关系已经被主要研究在遗传信息的传递和蛋白质的合成。而近年研究热点在mRNA的调控,尤其是miRNA在mRNA调控中的作用。MiRNA是短的RNA序列,源自发夹结构前体Pre-miRNA,其在转录后调控中发挥关键的作用,通过形成双重作用,与靶mRNA完全互补则清除靶基因,不完全互补则抑制靶基因。[15]microRNA作为一种潜在的载体,在蛋白质表达稳定传递和动态竞争的内源性RNA网络中。miRNAs能诱导蛋白质合成的阈值,这种蛋白产量的阈值能被聚集反应的转录因子完成。所以miRNA虽然小但发挥调控蛋白合成的作用。[16]DNA编码mRNA再翻译成蛋白质,miRNA对靶基因mRNA起到抑制或清除的作用,所以miRNAs与蛋白质可能呈负相关。可以怀疑某种蛋白的增加可能与抑制它的miRNAs减少有关,因此在与正常对照中检测降低的miRNA可能更有意义。通过miRCURYTMLNAexpression array芯片技术筛选糖尿病肾病组、糖尿病组、健康组差异miRNAs谱,并结合TargetScan、MirBase等数据库寻找可能与糖尿病肾病发病相关的miRNAs,并进一步经RT-PCR验证,对揭示糖尿病患者发生糖尿病肾病的microRNA机制,为后续研究及探讨miRNAs在DN发病中的作用奠定基础,并有望确定出1-2个可作为预警标记物的血清miRNA。研究对象和方法通过选取年龄、性别匹配的糖尿病肾病患者、单纯糖尿病患者、健康对照组各20人经上海康成生物公司完成miRCURYTM LNA expression array芯片筛选三组中microRNAs的差异,进一步结合miRBase、TargetScan Human、miRtarBase等数据库分析可能与糖尿病肾病发病相关的miRNAs15个,进一步通过RT-PCR验证,并使用2-ΔΔcT法分析组间倍数关系。结果 1、miRCURYTM LNA expression array芯片结果：根据microRAN array信号强度2倍以上三组之间的差异有：糖尿病肾病组比健康组上调的microRNAs158个,下调的microRNAs150个;糖尿病肾病组比糖尿病组上调的microRNAs14个,下调的microRNAs6个；糖尿病组比健康组上调106个,下调125个。 2、应用生物信息学在miRBase、miRtarBase、TargetScan数据库中获得可能与糖尿病肾病发病机制相关的microRNAs15个,hsa-miR-150-5p、 hsa-miR-485-3p、hsa-miR-205-3p、hsa-miR-302e、hsa-miR-495-5p、 hsa-miR-361-3p、hsa-miR-377-3p、hsa-miR-484、hsa-let-7a-2-3p、 hsa-miR-532-3p、hsa-miR-302a-3p、hsa-miR-519e-3p、hsa-miR-301a-5p、hsa-miR-216a-3p、hsa-miR-328-3p。 3. RT-PCR验证：分别设计15个microRNAs的引物,根据QIAGEN公司的miRNAs内参miR-39为内参照,经RT-PCR验证,糖尿病肾病组miR-150比健康组明显减少,P0.05,而糖尿病组和健康组无意义。结论：糖尿病肾病患者循环miR-150较健康对照组减少,与miRCURYTM LNA expression array芯片结果一致,miRNA-150可能作为糖尿病肾病的早期预警标记物。
[Abstract]:Background and purpose of research
Diabetic microangiopathy including nephropathy, neuropathy and retinopathy. The risk factors of complications of development are influenced by many factors, including the duration of diabetes and genetic factors.[1] (DM) diabetic microvascular complications of diabetic nephropathy (DN), the most common cause of end-stage renal disease in western countries (ESRD) and DM were important reasons death, but also become the end-stage renal disease (ESRD). The primary reason in our country, resulting in the number of ESRD DN also increased year by year the current DN.[2] pathogenesis is not completely understood, effective treatment is still inadequate, therefore, effective prevention and early intervention and treatment of DN is an important problem in medical field in particular, with the aging of the population, increasing the number of the diabetic population, the incidence of diabetic nephropathy will also increase, it will be extremely important. Whether type 1 diabetes or type 2 diabetes can guide In addition to vascular complications, high blood glucose, high density lipoprotein, late sugar metabolites, growth factor and inflammatory factor, the molecular mechanism of the need for better understanding of diabetes to identify novel therapeutic targets for.[3] understanding of the pathogenesis of diabetic complications potential, is very important to the development of new and improved treatment strategies for chronic diseases in this state.
The new method from nematode study identifies a novel endogenous, small fragments, single stranded, non encoding RNA molecule, was named as the tiny tiny RNA, RNA transcription factor.[4] and the growth of MicroRNA (miRNAs) RNA is small, ubiquitous in eukaryotes. The precursor (pre-miRNA) further mature miRNA specifically, the endogenous hairpin structure length 70~80nt miRNA concrete (pre-miRNA) shear generated after the mature, miRNA is a length of about 21~24nt by non protein encoding RNA molecules after transcription negatively regulate the expression of target genes, can be combined with the target gene mRNA3'untranslated region (3'-untranslatedregion, 3'-UTR) and the combination of complementary target the expression of mRNA.[4] microRNA circulation is present in the extracellular microRNA special existence way. The study found that in human serum, plasma, urine, saliva, body fluids such as milk can be detected by a variety of microRNAs. in body fluids The majority of microRNA molecule does not exist in free form, it is lipid, protein complex, microvesicles (microvesicle, MV), the outer clamping body (exosome) and combined with other packages, so it has good ability to anti RNase degradation, stability of.[5] is higher in the cells by biological factors stimulation, can be selectively the specific microRNA encapsulated into MV, and cell surface markers also carry on the surface of MV. In different tissues, the detection of surface labeled antibody magnetic bead cell can be easily to MV and microRNA. from different tissues is due to the characteristics of blood serum microRNA, it is very suitable to be used as a biomarker for early warning and early diagnosis of disease. Because of its changes as early as target gene changes with stability in body fluids, and the characteristics of shuttle tissue specificity between cells, it is very suitable as a biomarker for disease. Early warning and early diagnosis of disease.[24]
In 50s the last century since messager RNA found that the relationship between mRNA protein and the biological polymers have been mainly studied in the synthesis of protein and the transfer of genetic information. The research focus in recent years in the regulation of mRNA, especially the role of.MiRNA miRNA in mRNA regulation is a short RNA sequence, from hairpin precursor Pre-miRNA. The in the post transcriptional regulation plays a key role, through the formation of the dual role, and fully complementary target mRNA clear target gene, incomplete complementary inhibition of target gene.[15]microRNA as a potential carrier,.MiRNAs network transmission and stable expression of endogenous RNA in dynamic competition protein could induce a threshold of protein synthesis. Complete the transcription factor protein yield threshold can be clustered reaction. So miRNA is small but play a regulatory role of.[16]DNA encoding mRNA protein synthesis and then translated into MiRNA protein, to inhibit or remove the effect of the target gene mRNA, so miRNAs and protein may be negatively correlated. Can be suspected of increasing the protein may be related to the inhibition of its miRNAs reduction, therefore may reduce miRNA makes more sense in detection and normal control. Through the miRCURYTMLNAexpression array microarray in screening of diabetic nephropathy group. Diabetic group and healthy group differences in miRNAs spectra, and TargetScan may be related to the pathogenesis of diabetic nephropathy miRNAs for MirBase database, and further verified by RT-PCR microRNA, to reveal the mechanism of diabetic patients with diabetic nephropathy, which lays a foundation for further study and explore the role of miRNAs in the pathogenesis of DN, and is expected to identify 1-2 can be used as early warning marker serum miRNA.
Research objects and methods
Through the selection of age, sex matched patients with diabetic nephropathy, simple diabetes, differences between healthy control group of 20 by the Shanghai Kang biotechnology company LNA expression miRCURYTM array chip microRNAs screening in the three group, TargetScan Human, further combined with miRBase miRtarBase database analysis may be related to the pathogenesis of diabetic nephropathy miRNAs15, further through the verification of RT-PCR, and use the 2- Delta Delta cT method were analyzed. The multiple relationship
1, miRCURYTM LNA expression array microRAN array chip according to the signal strength of more than 2 times the differences between the three groups: diabetic nephropathy group than healthy group microRNAs158 increased, microRNAs150 decreased; diabetic nephropathy diabetic group than in the microRNAs14 group increased, microRNAs6 decreased; diabetic group than the healthy group by 106, down 125.
2, the application of bioinformatics in miRBase, miRtarBase, microRNAs15 may obtain, associated with the pathogenesis of diabetic nephropathy in TargetScan database hsa-miR-150-5p, hsa-miR-485-3p, hsa-miR-205-3p, hsa-miR-302e, hsa-miR-495-5p, hsa-miR-361-3p, hsa-miR-377-3p, hsa-miR-484, hsa-let-7a-2-3p, hsa-miR-532-3p, hsa-miR-302a-3p, hsa-miR-519e-3p, hsa-miR-301a-5p, hsa-miR-216a-3p, hsa-miR-328-3p.
3. RT-PCR test: 15 microRNAs primers were designed according to the QIAGEN, according to company miRNAs miR-39 as the internal reference, verified by RT-PCR, diabetic nephropathy group miR-150 significantly reduced than the healthy group P0.05 and diabetic group and healthy group had no significance.
Conclusion:
The circulating miR-150 in patients with diabetic nephropathy is lower than that in healthy controls, which is consistent with the results of miRCURYTM LNA expression array chip. MiRNA-150 may be used as an early warning marker for diabetic nephropathy.

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2;R692

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