本文關(guān)鍵詞： 糖尿病腎病 microRNAs miRNA芯片　出處：《蘭州大學》2014年碩士論文　論文類型：學位論文

【摘要】：研究背景和目的 糖尿病微血管病變包括腎臟病變、神經(jīng)病變、視網(wǎng)膜病變。并發(fā)癥發(fā)展的風險因素受許多因素影響,包括糖尿病持續(xù)時間和基因因素。[1]糖尿病(DM)最常見的微血管并發(fā)癥之一糖尿病腎病(DN),是導(dǎo)致西方國家終末期腎病(ESRD)和DM患者死亡的重要原因,也是成為終末期腎病(ESRD)的首要原因。在我國,DN在導(dǎo)致ESRD的數(shù)量也逐年增加。[2]當前DN的致病機制尚不完全清楚,有效的治療手段尚不足,因此,有效地早期干預(yù)和預(yù)防、治療DN已是醫(yī)學領(lǐng)域中重要的問題,尤其是隨著人口老齡化,糖尿病基礎(chǔ)人群數(shù)量的增加,糖尿病腎病的發(fā)病率也將增加,這將極為重要。不管是1型糖尿病還是2型糖尿病均可導(dǎo)致血管并發(fā)癥,除了高血糖、高低密度脂蛋白、晚期糖代謝產(chǎn)物、生長因子及炎癥因子外,需要更好的理解糖尿病的分子機制來確定新的治療目標。[3]理解糖尿病并發(fā)癥潛在的發(fā)病機制,為這種慢性病狀態(tài)發(fā)展新的和改進的治療策略仍然非常重要。 新的方法來自線蟲研究確定了一種新的內(nèi)源性、小片段、單鏈、非編碼RNA分子,被命名為微小RNA,這種微小RNA作為生長的調(diào)控因子。[4] MicroRNA (miRNAs)是微小RNA,普遍存在真核生物中。由前體(pre-miRNA)進一步成熟為miRNA,具體為內(nèi)源性發(fā)夾狀結(jié)構(gòu)長度為70~80nt的miRNA具體(pre-miRNA)剪切后生成,成熟miRNA是一類長約21~24nt的通過轉(zhuǎn)錄后負調(diào)控靶基因表達的非蛋白編碼RNA分子,可結(jié)合靶基因mRNA3'端非翻譯區(qū)(3'-untranslatedregion,3'-UTR)互補結(jié)合而調(diào)控靶mRNA表達。[4]體液循環(huán)microRNA是存在于細胞外的microRNA特殊存在方式。研究發(fā)現(xiàn)在人體的血清、血漿、尿液、唾液、乳汁等體液中都能檢測到多種microRNAs。體液中的microRNA分子多數(shù)不是以游離形式存在的,它被脂質(zhì)、蛋白復(fù)合體、微囊泡(microvesicle, MV)、外夾體(exosome)等包裹和結(jié)合,因此具有良好的抗RNase降解能力,有較高的穩(wěn)定性。[5]在來源細胞受到生物學因素刺激后,可以選擇性地把特定的microRNA包裹進MV,同時細胞表面標記也攜帶在MV表面。用不同組織、細胞的表面標記抗體磁珠可以容易的檢測到不同組織來源的MV及microRNA。正是由于血清循環(huán)microRNA的特點,它非常適合作為生物標記物用于疾病的預(yù)警和早期診斷。因其變化早于靶基因的變化、具有體液中的穩(wěn)定性、細胞間的穿梭性以及組織來源特異性的特點,它非常適合作為生物標志物用于疾病的預(yù)警和早期診斷。[24] 上世紀50年代自從messager RNA的發(fā)現(xiàn),蛋白質(zhì)和mRNA這個生物聚合物之間的關(guān)系已經(jīng)被主要研究在遺傳信息的傳遞和蛋白質(zhì)的合成。而近年研究熱點在mRNA的調(diào)控,尤其是miRNA在mRNA調(diào)控中的作用。MiRNA是短的RNA序列,源自發(fā)夾結(jié)構(gòu)前體Pre-miRNA,其在轉(zhuǎn)錄后調(diào)控中發(fā)揮關(guān)鍵的作用,通過形成雙重作用,與靶mRNA完全互補則清除靶基因,不完全互補則抑制靶基因。[15]microRNA作為一種潛在的載體,在蛋白質(zhì)表達穩(wěn)定傳遞和動態(tài)競爭的內(nèi)源性RNA網(wǎng)絡(luò)中。miRNAs能誘導(dǎo)蛋白質(zhì)合成的閾值,這種蛋白產(chǎn)量的閾值能被聚集反應(yīng)的轉(zhuǎn)錄因子完成。所以miRNA雖然小但發(fā)揮調(diào)控蛋白合成的作用。[16]DNA編碼mRNA再翻譯成蛋白質(zhì),miRNA對靶基因mRNA起到抑制或清除的作用,所以miRNAs與蛋白質(zhì)可能呈負相關(guān)�？梢詰岩赡撤N蛋白的增加可能與抑制它的miRNAs減少有關(guān),因此在與正常對照中檢測降低的miRNA可能更有意義。通過miRCURYTMLNAexpression array芯片技術(shù)篩選糖尿病腎病組、糖尿病組、健康組差異miRNAs譜,并結(jié)合TargetScan、MirBase等數(shù)據(jù)庫尋找可能與糖尿病腎病發(fā)病相關(guān)的miRNAs,并進一步經(jīng)RT-PCR驗證,對揭示糖尿病患者發(fā)生糖尿病腎病的microRNA機制,為后續(xù)研究及探討miRNAs在DN發(fā)病中的作用奠定基礎(chǔ),并有望確定出1-2個可作為預(yù)警標記物的血清miRNA。 研究對象和方法 通過選取年齡、性別匹配的糖尿病腎病患者、單純糖尿病患者、健康對照組各20人經(jīng)上�？党缮锕就瓿蒻iRCURYTM LNA expression array芯片篩選三組中microRNAs的差異,進一步結(jié)合miRBase、TargetScan Human、miRtarBase等數(shù)據(jù)庫分析可能與糖尿病腎病發(fā)病相關(guān)的miRNAs15個,進一步通過RT-PCR驗證,并使用2-ΔΔcT法分析組間倍數(shù)關(guān)系。結(jié)果 1、miRCURYTM LNA expression array芯片結(jié)果：根據(jù)microRAN array信號強度2倍以上三組之間的差異有：糖尿病腎病組比健康組上調(diào)的microRNAs158個,下調(diào)的microRNAs150個;糖尿病腎病組比糖尿病組上調(diào)的microRNAs14個,下調(diào)的microRNAs6個；糖尿病組比健康組上調(diào)106個,下調(diào)125個。 2、應(yīng)用生物信息學在miRBase、miRtarBase、TargetScan數(shù)據(jù)庫中獲得可能與糖尿病腎病發(fā)病機制相關(guān)的microRNAs15個,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驗證：分別設(shè)計15個microRNAs的引物,根據(jù)QIAGEN公司的miRNAs內(nèi)參miR-39為內(nèi)參照,經(jīng)RT-PCR驗證,糖尿病腎病組miR-150比健康組明顯減少,P0.05,而糖尿病組和健康組無意義。 結(jié)論： 糖尿病腎病患者循環(huán)miR-150較健康對照組減少,與miRCURYTM LNA expression array芯片結(jié)果一致,miRNA-150可能作為糖尿病腎病的早期預(yù)警標記物。
[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.

【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R587.2;R692

【參考文獻】

相關(guān)期刊論文 前2條

1 張昌明;張婉芬;劉春蓓;曾科;劉志紅;;循環(huán)microRNAs與局灶節(jié)段性腎小球硬化患者疾病活動的關(guān)系[J];腎臟病與透析腎移植雜志;2013年04期

2 張婉芬;張昌明;劉春蓓;秦衛(wèi)松;鄭春霞;曾科;劉志紅;;尿液microRNAs作為局灶節(jié)段性腎小球硬化疾病活動生物標志物的研究[J];腎臟病與透析腎移植雜志;2013年04期

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