【摘要】：目的應(yīng)用miRCURY LNATM microRNA Array微陣列芯片技術(shù)檢測非節(jié)段型白癜風(fēng)(Non-Segmental Vitiligo, NSV)患者外周血單個核細(xì)胞(Peripheral blood mononuclear cells, PBMCs)中microRNAs的表達(dá)模式,并篩選其和健康人相比差異性表達(dá)的miRNAs。運用實時熒光定量PCR技術(shù)對差異性表達(dá)的miRNAs進(jìn)行驗證。體外培養(yǎng)白癜風(fēng)患者PBMCs,提取miRNA檢測胸腺肽Tα1對篩選的miRNA表達(dá)變化的直接效應(yīng)。應(yīng)用靶基因預(yù)測數(shù)據(jù)庫選擇白介素2受體作為miR-3940-5p調(diào)控的靶基因,構(gòu)建慢病毒載體抑制miR-3940-5p表達(dá)并感染人皮膚T細(xì)胞株HuT78細(xì)胞,進(jìn)一步研究miR-3940-5p對白癜風(fēng)免疫相關(guān)基因的靶向調(diào)節(jié),探討差異表達(dá)的miRNAs和Tα,在非節(jié)段型白癜風(fēng)中的作用機(jī)制。方法第一部分：收集5例進(jìn)展期非節(jié)段型白癜風(fēng)患者和5例年齡性別相匹配的健康人外周靜脈血,分離單個核細(xì)胞,提取總RNA。應(yīng)用第七代miRCURYTMLNA陣列(v.18.0) (Exiqon)芯片雜交技術(shù)對NSV患者和健康人PBMCs總RNA進(jìn)行miRNAs的表達(dá)譜系差異分析,運用MEV軟件(v4.6, TIGR)進(jìn)行聚類分析；收集32例進(jìn)展期NSV患者和18例健康人外周血并提取單個核細(xì)胞總RNA,莖環(huán)引物法逆轉(zhuǎn)錄合成cDNA,通過實時熒光定量PCR技術(shù)進(jìn)行miRNA差異表達(dá)驗證;體外培養(yǎng)白癜風(fēng)患者外周血單個核細(xì)胞,將不同濃度胸腺肽α.加入細(xì)胞懸液,72h后離心收集細(xì)胞,提取miRNA進(jìn)行RT-qPCR分析,檢測差異性miRNAs的相對表達(dá)量。分析胸腺肽α1對白癜風(fēng)患者PBMCs中差異性miRNAs表達(dá)水平的直接效應(yīng)。第二部分：應(yīng)用生物信息數(shù)據(jù)庫miRDB和Targetscan預(yù)測篩選的miRNA-3940-5p可能調(diào)控的與白癜風(fēng)自身免疫相關(guān)的靶基因IL-2RG。構(gòu)建低表達(dá)miR-3940-5p慢病毒載體,轉(zhuǎn)染至人T淋巴細(xì)胞株HuT78細(xì)胞,設(shè)立慢病毒陰性對照組；miR-3940-5p表達(dá)下調(diào)是通過應(yīng)用預(yù)先設(shè)計的反義RNA序列競爭結(jié)合到miR-3940-5p前體并克隆到GV159載體Hl-MCS-CMV-EGFP來實現(xiàn)的。3-4天后觀察綠色熒光蛋白表達(dá)陽性的細(xì)胞轉(zhuǎn)染率,收獲細(xì)胞沉淀；通過ELISA和Western Blot技術(shù)檢測培養(yǎng)的淋巴細(xì)胞上清液和細(xì)胞中IL-2R Gamma蛋白含量及表達(dá)水平,分析miRNA-3940-5p抑制表達(dá)對靶基因IL-2RG的調(diào)控作用。結(jié)果一、通過高通量miRNA微陣列芯片檢測技術(shù),對NSV患者和健康人外周血免疫細(xì)胞中的miRNA表達(dá)譜進(jìn)行全面地分析,結(jié)果顯示與年齡性別相匹配的健康人相比,NSV患者PBMCs miRNA表達(dá)譜中有4個miRNAs呈顯著差異性表達(dá),其中miR-224-3p, miR-2682-3p和miR-4712-3p表達(dá)明顯上調(diào)(p0.05), miR-3940-5p表達(dá)明顯下調(diào)(p0.05)。二、應(yīng)用莖環(huán)引物逆轉(zhuǎn)錄SYBR Green PCR技術(shù)在較大的患者人群中分析了這四個miRNAs的表達(dá)水平,結(jié)果發(fā)現(xiàn)NSV患者PBMCs中miR-224-3p、 miR-4712-3p表達(dá)均上調(diào),miR-3940-5p表達(dá)下調(diào),差異均有統(tǒng)計學(xué)意義(p0.05),這與miRNA微陣列數(shù)據(jù)分析結(jié)果相一致。微陣列芯片檢測技術(shù)和實時熒光定量PCR技術(shù)這兩種檢測方法呈高度正相關(guān)(相關(guān)系數(shù)r=1.000,p=0.005)。但是和健康人相比,miR-2682-3p的表達(dá)水平升高但差異沒有統(tǒng)計學(xué)意義(p0.05)。三、倒置相差顯微鏡下觀察到體外培養(yǎng)的白癜風(fēng)患者外周血單個核細(xì)胞形態(tài)圓而透亮,懸浮分布。和空白對照組相比,加入不同濃度胸腺肽a1(50、100ug/ml)處理后實驗組細(xì)胞數(shù)量和聚集性明顯增加,與處理前相比差異有統(tǒng)計學(xué)意義(p0.05)。胸腺肽α1處理72h后對4個差異性miRNAs表達(dá)水平進(jìn)行PCR檢測,發(fā)現(xiàn)與未經(jīng)處理的對照組相比,處理組PBMC中miR-224-3p, miR-2682-3p和miR-4712-3p表達(dá)下降,而miR-3940-5p表達(dá)升高,但兩個濃度之間比較差異無統(tǒng)計學(xué)意義(p0.05)。四、體外實驗結(jié)果顯示和慢病毒陰性對照組HuT78細(xì)胞相比,目的基因miRNA-3940-5p低表達(dá)轉(zhuǎn)染實驗組可促進(jìn)T細(xì)胞增殖,淋巴細(xì)胞上清液中的IL-2RG含量明顯升高,實驗組細(xì)胞中的IL-2RG蛋白表達(dá)上調(diào)(p0.05)。結(jié)論1、非節(jié)段型白癜風(fēng)患者外周血單個核細(xì)胞miRNA表達(dá)譜中4個miRNAs發(fā)生了差異性表達(dá),其中3個miRNAs表達(dá)上調(diào),1個miRNA表達(dá)下調(diào)。2、實時熒光定量PCR技術(shù)檢測證實了這四種miRNAs的異常表達(dá)與微陣列芯片分析結(jié)果一致。3、免疫調(diào)節(jié)劑胸腺肽α1干預(yù)對白癜風(fēng)患者外周血單個核細(xì)胞miRNAs的表達(dá)譜產(chǎn)生直接影響。4、miRNA-3940-5p在人皮膚T淋巴細(xì)胞系中存在一定表達(dá)。LV-hsa-miR-3940-5p-inhibit on可轉(zhuǎn)染至人T淋巴細(xì)胞株HuT78細(xì)胞。5、miR-3940-5p通過對細(xì)胞因子受體IL-2R Gamma基因進(jìn)行負(fù)調(diào)控發(fā)揮作用。
[Abstract]:Objective to detect the expression patterns of peripheral blood mononuclear cells (Peripheral blood mononuclear cells, PBMCs) in patients with non segmental vitiligo (Non-Segmental Vitiligo, NSV) using miRCURY LNATM microRNA Array microarray technology, and to screen their differential expression by real-time quantitative fluorescence spectrometry. R technique was used to verify the differential expression of miRNAs. In vitro culture of vitiligo patients PBMCs, miRNA was extracted to detect the direct effect of thymosin T alpha 1 on the changes of miRNA expression. The target gene prediction database was used to select the interleukins 2 receptor as the target gene regulated by miR-3940-5p, and the lentivirus vector was constructed to inhibit miR-3940-5p expression and infected people. Skin T cell line HuT78 cells, further study the target regulation of miR-3940-5p on the immune related genes of vitiligo, explore the mechanism of differential expression of miRNAs and T alpha in non segmental vitiligo. Methods first part: collect 5 patients with progressive non segmental vitiligo and 5 healthy people with matched age and sex in peripheral venous blood. To isolate mononuclear cells and extract total RNA. with seventh generation miRCURYTMLNA array (v.18.0) (Exiqon) chip hybridization technique, the difference analysis of the expression lineage of miRNAs in NSV patients and healthy people PBMCs RNA was analyzed. The cluster analysis was carried out by MEV software (v4.6, TIGR), and 32 cases were collected and 18 healthy human peripheral blood were collected and isolated. Nuclear cell total RNA, stem ring primer reverse transcriptase synthesis of cDNA, miRNA differential expression verification by real-time fluorescence quantitative PCR; cultured peripheral blood mononuclear cells from patients with vitiligo in vitro, different concentrations of thymosin alpha into cell suspension, 72h after 72h centrifugation to collect cells, and miRNA for RT-qPCR analysis to detect the relative miRNAs The direct effect of thymosin alpha 1 on the differential miRNAs expression in PBMCs of patients with vitiligo. The second part: the application of the biological information database miRDB and Targetscan predicted that miRNA-3940-5p may regulate the target gene IL-2RG. related to the autoimmune disease of vitiligo to construct a low expression of miR-3940-5p lentivirus vector, and transfection To the human T lymphocyte strain HuT78 cells, the lentivirus negative control group was set up. The downregulation of miR-3940-5p expression was to observe the cell transfection rate of green fluorescent protein expression positive by using pre designed antisense RNA sequence combined with miR-3940-5p precursor and cloned to GV159 carrier Hl-MCS-CMV-EGFP to observe the transfection rate of green fluorescent protein expression positive. The content and expression level of IL-2R Gamma protein in the cultured lymphocyte supernatant and cells were detected by ELISA and Western Blot techniques. The effect of miRNA-3940-5p inhibition expression on the target gene IL-2RG was analyzed. The miRNA expression profile was analyzed comprehensively. The results showed that 4 miRNAs expressions were significantly different in the PBMCs miRNA expression profiles of NSV patients, in which miR-224-3p, miR-2682-3p and miR-4712-3p expressions were significantly up-regulated (P0.05) and miR-3940-5p expression decreased significantly (P0.05). Two, using stem ring primers for reverse transcription. SYBR Green PCR technology analyzed the expression level of these four miRNAs in a large number of patients. The results showed that miR-224-3p, miR-4712-3p expression was up and miR-3940-5p expression was down regulated in NSV patients' PBMCs, and the difference was statistically significant (P0.05), which was in accordance with the results of miRNA microarray data analysis. Microarray detection technology and real time The two methods of fluorescence quantitative PCR showed a highly positive correlation (correlation coefficient r=1.000, p=0.005). But compared with healthy people, the expression level of miR-2682-3p increased but the difference was not statistically significant (P0.05). Three, the morphology of peripheral blood mononuclear cells in the patients cultured in vitro under the inverted phase contrast microscope was round and bright. Floating distribution. Compared with the blank control group, the number and aggregation of the cells in the experimental group were significantly increased after adding different concentrations of thymosin A1 (50100ug/ml). The difference was statistically significant compared with that before treatment (P0.05). After 72h thymosin alpha 1 treatment, 4 differential miRNAs expression levels were detected by PCR, and compared with that of the untreated control group. The expression of miR-224-3p, miR-2682-3p and miR-4712-3p decreased in PBMC, but the expression of miR-3940-5p increased, but there was no significant difference between the two concentrations (P0.05). Four. In vitro, the experimental results showed that the low expression of the target gene miRNA-3940-5p in the experimental group could promote the proliferation of T cells compared with the HuT78 cells in the negative control group of the slow virus. The content of IL-2RG in the cell supernatant increased significantly, and the expression of IL-2RG protein in the experimental group was up regulated (P0.05). Conclusion 1, 4 miRNAs in the miRNA expression profiles of peripheral blood mononuclear cells in patients with non segmental vitiligo were expressed differently, of which 3 miRNAs expressions were up, 1 miRNA expressed down regulated.2, and real-time fluorescent quantitative PCR technique detection It was confirmed that the abnormal expression of these four kinds of miRNAs was consistent with the results of microarray microarray analysis. The interference of thymosin alpha 1 on the expression profiles of miRNAs in peripheral blood mononuclear cells of patients with vitiligo was directly affected by.4. MiRNA-3940-5p could be transfected into human T lymphocyte lines in human skin T lymphocyte lines and could be transfected to humans by.LV-hsa-miR-3940-5p-inhibit on. T lymphocyte HuT78 cell.5 and miR-3940-5p play a negative role in regulating the cytokine receptor IL-2R Gamma gene.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R758.41

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