本文選題：IgA腎病　切入點(diǎn)：miRNAs　出處：《鄭州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：背景 IgA腎病是全世界最常見的原發(fā)性腎小球疾病，容易進(jìn)展至終末期腎臟病(end stage renal disease, ESRD)。但是，IgA腎病的診斷和動(dòng)態(tài)檢測(cè)依賴腎組織活檢，而腎活檢為有創(chuàng)性的檢查，且重復(fù)腎活檢患者多難以接受。因而，探索IgA腎病非侵入性的診斷方法任重道遠(yuǎn)。miRNAs是一種參與轉(zhuǎn)錄后水平調(diào)控的非編碼RNA，其表達(dá)異�？赡軐�(dǎo)致人類多種疾病，包括IgA腎病，異常表達(dá)的miRNAs可以導(dǎo)致IgA腎病患者腎組織細(xì)胞結(jié)構(gòu)的改變及類型的轉(zhuǎn)化、免疫分子結(jié)構(gòu)的改變、細(xì)胞信號(hào)傳導(dǎo)的異常等，從而引起腎組織損傷。目前，眾多的研究發(fā)現(xiàn)血漿及尿液中存在穩(wěn)定表達(dá)的miRNAs，可抵制RNase的降解。因此，血漿或尿液中與IgA腎病病理形態(tài)學(xué)改變相關(guān)的miRNAs或許可以成為其新的診斷標(biāo)志物。然而，miRNAs的表達(dá)水平與IgA腎病病情的關(guān)系尚無(wú)確切結(jié)論，血漿或尿液中miRNAs的表達(dá)水平是否能夠真正取代腎活檢仍值得探討。本研究選取與IgA腎病發(fā)病和腎臟纖維化相關(guān)的幾種miRNAs，通過(guò)對(duì)IgA腎病患者的腎組織、血漿及尿液miRNAs同時(shí)進(jìn)行檢測(cè)，初步研究腎組織、血漿及尿液miRNAs的表達(dá)與IgA腎病病情的相關(guān)性。 目的 通過(guò)檢測(cè)miR-148b、 miR-194、 miR-200a及miR-382在原發(fā)性IgA腎病腎組織、血漿及尿液中的表達(dá)水平，初步探討miRNAs的表達(dá)與該疾病病情的相關(guān)性，為血漿及尿液中的miRNAs在IgA腎病診斷中的應(yīng)用提供參考，也為尋找IgA腎病非侵入性診斷標(biāo)志物提供新思路。 方法 1.選取鄭州大學(xué)第一附屬醫(yī)院腎臟內(nèi)科2012年12月至2013年6月首次經(jīng)腎組織活檢及臨床、實(shí)驗(yàn)室檢查綜合確診為原發(fā)性IgA腎病患者34例，平均年齡(32.74±10.57)歲，男女比例1:1.1。該34例入組患者在腎活檢當(dāng)日清晨同時(shí)留取晨尿及空腹靜脈血標(biāo)本，其中7例患者留取腎穿刺組織標(biāo)本。收集所有入組患者的臨床及病理資料，并對(duì)其病理結(jié)果進(jìn)行Hass分級(jí)，其中符合Hass分級(jí)I-II級(jí)患者10例，III級(jí)17例，IV-V級(jí)7例。對(duì)照組選自我院同期門診年齡及性別匹配的健康體檢者13例，每個(gè)入組對(duì)象同時(shí)留取晨尿及空腹靜脈血標(biāo)本，對(duì)照組腎組織標(biāo)本選自同期在我院行腎腫瘤切除術(shù)的患者7例，取遠(yuǎn)離腫瘤的正常腎組織。 2.采用TRIzol LS Reagent（Invitrogen life technologies）抽提腎組織、血漿及尿液中的RNA，使用NanoDrop ND-1000(Nanodrop, Wilmington, Delaware,USA)測(cè)定RNA的濃度和純度，應(yīng)用實(shí)時(shí)熒光定量PCR(real-time PCR)技術(shù)定量檢測(cè)腎組織、血漿及尿液中miR-148b、miR-194、miR-200a及miR-382的表達(dá)水平。選擇RNU6B作為內(nèi)參，數(shù)據(jù)采用2-△△Ct法進(jìn)行分析。 3.比較（1）IgA腎病組和對(duì)照組間腎組織、血漿及尿液4種miRNAs的表達(dá)水平是否有差異；（2） IgA腎病患者血漿及尿液4種miRNAs的表達(dá)水平與Hass分級(jí)的關(guān)系；（3）IgA腎病患者血漿及尿液4種miRNAs的表達(dá)水平與臨床指標(biāo)的關(guān)系；（4）IgA腎病患者血漿及尿液4種miRNAs的表達(dá)水平與牛津病理分型的關(guān)系。正態(tài)分布數(shù)據(jù)兩組間比較采用t檢驗(yàn)，多組間比較采用One-way ANOVA，多個(gè)變量間的兩兩比較采用Bonferroni檢驗(yàn)，將檢驗(yàn)水準(zhǔn)校正為0.017（0.05/3）。相關(guān)性檢驗(yàn)應(yīng)用Pearson相關(guān)（服從正態(tài)分布的資料）或Spearman秩相關(guān)（不服從正態(tài)分布的資料）。以P＜0.05表示差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果 納入IgA腎病組血漿及尿液標(biāo)本各34例，腎穿刺組織標(biāo)本7例；對(duì)照組血漿及尿液標(biāo)本各13例，正常腎組織標(biāo)本7例。 （1）IgA腎病組和對(duì)照組間腎組織、血漿及尿液miRNAs表達(dá)水平的比較：腎組織miR-148b、miR-194、miR-200a的表達(dá)水平下調(diào)（P＜0.05），miR-382的表達(dá)水平上調(diào)（P＜0.05）；血漿miR-148b、miR-194、miR-382的表達(dá)水平下調(diào)（P＜0.05），miR-200a的表達(dá)水平上調(diào)（P＜0.05）；尿液miR-148b、miR-382的表達(dá)水平上調(diào)（P＜0.05），miR-194、miR-200a的表達(dá)水平下調(diào)（P＜0.05）。 （2）不同Hass分級(jí)IgA腎病患者血漿miRNAs表達(dá)水平分析：miR-148b和miR-194的表達(dá)水平隨IgA腎病Hass分級(jí)的升高而下降（P＜0.017）。Hass分級(jí)III級(jí)和IV-V級(jí)患者miR-382的表達(dá)水平低于I-II級(jí)（P＜0.017）。Hass分級(jí)I-II級(jí)、III級(jí)和IV-V級(jí)患者miR-200a的表達(dá)水平無(wú)顯著性差異（P＞0.05）。 （3）不同Hass分級(jí)IgA腎病患者尿液miRNAs表達(dá)水平的分析：IgA腎病Hass分級(jí)IV-V級(jí)患者miR-148b的表達(dá)水平高于III級(jí)（P＜0.017）。Hass分級(jí)I-II級(jí)和III級(jí)患者miR-194的表達(dá)水平高于IV-V級(jí)（P＜0.017）。Hass分級(jí)I-II級(jí)、III級(jí)和IV-V級(jí)患者miR-200a、miR-382的表達(dá)水平無(wú)顯著性差異（P＞0.05）。 （4）IgA腎病患者血漿及尿液miRNAs的表達(dá)水平與臨床指標(biāo)的相關(guān)性分析：IgA腎病患者尿液miR-148b的表達(dá)水平與血清尿酸、血清補(bǔ)體C4呈負(fù)相關(guān)性（P＜0.05），尿液miR-382的表達(dá)水平與尿紅細(xì)胞計(jì)數(shù)呈正相關(guān)性（P＜0.05）。尿液miR-194、miR-200a及血漿4種miRNAs與血清肌酐、腎小球?yàn)V過(guò)率、血尿素氮、血尿酸、血胱抑素、血總蛋白、血白蛋白、血球蛋白、血清總膽固醇、血清三酰甘油、血清高密度脂蛋白、血清低密度脂蛋白、血紅蛋白、24小時(shí)尿蛋白定量、尿紅細(xì)胞計(jì)數(shù)，，血清補(bǔ)體C3、血清補(bǔ)體C4、血壓均無(wú)顯著相關(guān)性（P＞0.05）。 （5）不同牛津病理分型IgA腎病患者血漿及尿液miRNAs表達(dá)水平的比較：牛津病理分型T0型IgA腎病患者尿液miR-200a的表達(dá)水平高于T1-2型（P＜0.05），尿液miR-148b、miR-194、miR-382及血漿4種miRNAs的表達(dá)水平在E、S、T分型間無(wú)顯著性差異（P＞0.05）。 結(jié)論 IgA腎病組腎組織、血漿及尿液miR-148b、miR-194、miR-200a、miR-382的表達(dá)水平與對(duì)照組相比均存在顯著性差異，且尿液miRNAs的表達(dá)水平與腎組織一致性較好。血漿miR-148b、miR-194、miR-382及尿液miR-148b、miR-194、miR-200a的表達(dá)與IgA腎病病理?yè)p傷程度相關(guān)，尿液miR-148b、miR-382的表達(dá)與部分臨床指標(biāo)相關(guān)。提示miRNAs可能參與了IgA腎病的發(fā)生及進(jìn)展，miRNAs是否能夠成為IgA腎病無(wú)創(chuàng)性診斷標(biāo)志物，有待進(jìn)一步研究。
[Abstract]:background
IgA nephropathy is the world's most common primary glomerular disease, easy progression to end-stage renal disease (end stage renal disease, ESRD). However, the diagnosis and dynamic detection of IgA nephropathy on renal biopsy, and renal biopsy is an invasive examination, and repeated renal biopsy patients more difficult to accept. Therefore, to explore IgA nephropathy by non-invasive diagnostic methods to.MiRNAs is a non RNA encoding involved in post transcriptional regulation, its abnormal expression may lead to many diseases, including IgA nephropathy, abnormal expression of miRNAs can lead to IgA nephropathy patients with renal cell structural changes and type transformation, the structure of immune molecule change of cell signal conduction abnormalities, resulting in renal tissue damage. At present, many studies have found that the existence of stable expression of miRNAs in plasma and urine, can resist degradation of RNase. Therefore, plasma or urine Associated with the pathological change of IgA nephropathy miRNAs may become a new diagnostic marker. However, the relationship between the expression level of miRNAs in patients with IgA nephropathy is no definite conclusion, whether the expression level of miRNAs in plasma or urine can truly replace renal biopsy is still worthy of discussion. With the selection of several miRNAs related disease and renal fibrosis IgA nephropathy in this study, patients with IgA nephropathy, plasma and urinary miRNAs were detected at the same time, a preliminary study on the correlation between the expression of renal tissue, plasma and urine miRNAs in patients with IgA nephropathy.
objective
Through the detection of miR-148b, miR-194, miR-200a and miR-382 in primary renal tissue of IgA nephropathy, the expression level in plasma and urine, to study the correlation between the expression of miRNAs and severity of the disease, to provide reference for the application of plasma and urine miRNAs in the diagnosis of IgA nephropathy, also for non-invasive diagnosis of IgA nephropathy markers provide a new way of thinking.
Method
1. the First Affiliated Hospital of Zhengzhou University Department of nephrology in December 2012 to June 2013 for the first time by biopsy and clinical laboratory examination of renal tissue, comprehensive diagnosis of primary IgA nephropathy patients 34 cases, average age (32.74 + 10.57) years of age, the proportion of men and women 1:1.1. the 34 patients in the renal biopsy at the same time, day morning urine and fasting blood samples were collected, including 7 cases of patients with renal biopsies were collected. All the patients enrolled in the clinical and pathological data, and the pathological results were Hass grade, with 10 cases of Hass grade I-II patients, 17 cases of grade III, IV-V grade 7 cases. The control group self selected hospital healthy outpatient age and sex matched 13 cases, each group and urine and fasting venous blood samples of normal renal specimens were selected from the same period in our hospital underwent renal tumor resection in 7 cases, from normal renal tumor Organization.
2. using TRIzol LS Reagent (Invitrogen life technologies) in renal tissue from plasma and urine in RNA, using NanoDrop ND-1000 (Nanodrop, Wilmington, Delaware, USA) for determination of the concentration and purity of RNA, the application of real-time fluorescence quantitative PCR (real-time PCR) in renal tissue of quantitative detection technique, miR-148b, plasma and urine miR-194 expression the level of miR-200a and miR-382. RNU6B is selected as the reference data, using 2- Delta Ct method were analyzed.
3. (1) IgA nephropathy group and control group in renal tissues, the expression level of plasma and urine of 4 kinds of miRNAs whether there are differences; (2) the relationship between the expression level and Hass grade in the plasma and urine of IgA nephropathy in 4 miRNAs; (3) the relationship between the plasma and urine of IgA nephropathy 4 miRNAs. Level and clinical index; (4) the relationship between the expression level of the plasma and urine of IgA nephropathy in 4 miRNAs Oxford and pathologic type. The normal distribution data of the two groups were compared using t test and multiple comparisons with One-way ANOVA, a variable between 22 compared with Bonferroni test, the test level the correction was 0.017 (0.05/3). The correlation test using Pearson (normal distribution data) or Spearman rank correlation (not subject to normal distribution data). P < 0.05 said the difference was statistically significant.
Result
The plasma and urine specimens of IgA nephrotic group were included in 34 cases, renal biopsy specimens in 7 cases, and in the control group, 13 cases of plasma and urine specimens, and 7 normal renal tissue specimens.
(1) IgA nephropathy group and the control group of renal tissue, plasma and urine miRNAs levels: miR-194, miR-148b in renal tissue, the expression level of miR-200a decreased (P < 0.05), the expression level of miR-382 (P < 0.05); plasma miR-148b, miR-194, the expression level of miR-382 decreased (P < 0.05), the expression level of miR-200a (P < 0.05); urine miR-148b, the expression level of miR-382 (P < 0.05), miR-194, the expression level of miR-200a decreased (P < 0.05).
(2) the level of analysis of classification of IgA nephropathy patients plasma miRNAs with different Hass expression: the expression of miR-148b and miR-194 decreased with the increase of IgA nephropathy Hass grading (P < 0.017) the expression level of.Hass III grade and IV-V grade in patients with miR-382 less than I-II (P < 0.017).Hass grade I-II, no significant difference the expression level of III and IV-V in patients with miR-200a (P > 0.05).
(3) the level of classification analysis of urine miRNAs in patients with IgA nephropathy with different Hass expression: the expression level of IgA nephropathy Hass IV-V grade miR-148b patients was higher than that of grade III (P < 0.017) expression of.Hass class I-II and III level in miR-194 patients was higher than that of grade IV-V (P < 0.017).Hass grade I-II, grade III and IV-V patients with grade miR-200a, no significant difference in the expression level of miR-382 (P > 0.05).
(4) analysis of the correlation between the expression level of IgA in plasma and urine of miRNAs nephropathy patients with clinical indicators: the expression level of serum uric acid and urine miR-148b in patients with IgA nephropathy, there was a negative correlation between serum C4 (P < 0.05), urine miR-382 expression level and urine red blood cell count showed a positive correlation (P < 0.05) urine. MiR-194, miR-200a and miRNAs 4 plasma and serum creatinine, glomerular filtration rate, blood urea nitrogen, uric acid, blood cystatin, serum total protein, albumin, globulin, serum total cholesterol, serum three triglycerides, serum high density lipoprotein, serum low density lipoprotein cholesterol, hemoglobin, urine protein in 24 hours, urine red blood cell count, serum C3, serum C4, blood pressure had no significant correlation (P > 0.05).
Oxford (5) of different pathological type of IgA nephropathy patients plasma and urine miRNAs levels: the expression level of Oxford pathological urine miR-200a in patients with type T0 type of IgA nephropathy is higher than that of the T1-2 (P < 0.05), urine miR-148b, miR-194, expression level of miR-382 and plasma water of 4 miRNAs in E, S, T no significant difference (P > 0.05).
conclusion
IgA nephropathy group renal tissue, plasma and urine miR-148b, miR-194, miR-200a, miR-382 expression level compared with the control group there were significant differences, and the expression level of miRNAs in urine and renal tissue consistency. Plasma miR-148b, miR-194, miR-382 and miR-148b in urine, miR-194, expression of miR-200a, IgA and renal pathological injury the degree of urine miR-148b, miR-382 expression correlated with some clinical parameters. It suggests that miRNAs may participate in the occurrence and progression of IgA nephropathy, miRNAs nephropathy can be IgA noninvasive diagnostic markers, require further research.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692.3

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