本文選題：長(zhǎng)鏈非編碼RNA + 血管炎癥��； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：目的:體外觀察血管內(nèi)皮細(xì)胞(endothelial cells,ECs)和血管平滑肌細(xì)胞(vascular smooth muscle cell,VSMC)在腫瘤壞死因子α(tumor necrosis factor-α,TNF-α)刺激下,細(xì)胞內(nèi)長(zhǎng)鏈非編碼RNA心肌梗死相關(guān)轉(zhuǎn)錄本(long non-coding RNA myocardial infarction associated transcript,LncRNA-MIAT)的表達(dá)變化,并探討LncRNA-MIAT對(duì)ECs和VSMC炎癥的調(diào)控作用。方法:以TNF-α誘導(dǎo)ECs和VSMC炎癥狀態(tài),采用Western Blot、PCR及q RT-PCR方法分別檢測(cè)ECs和VSMC炎癥狀態(tài)下細(xì)胞間黏附分子-1(intercellular adhesion molecule-1,ICAM-1)、血管細(xì)胞粘附分子-1(vascular cell adhesion molecule-1,VCAM-1)和LncRNA-MIAT的表達(dá)情況。應(yīng)用LncRNA-MIAT的小干擾核糖核酸(si RNA MAIT)轉(zhuǎn)染ECs和VSMC,觀察LncRNA-MIAT敲低對(duì)ICAM-1和VCAM-1表達(dá)的影響。通過(guò)劃痕實(shí)驗(yàn),觀察ECs和VSMC的遷移功能。結(jié)果:1.(1)內(nèi)皮細(xì)胞PCR結(jié)果:與空白對(duì)照相比,加入lncRNA-MIAT引物的條帶明顯,提示ECs中表達(dá)LncRNA-MIAT,不同引物濃度的條帶亮度的比較差異不明顯,提示當(dāng)引物濃度增加時(shí),LncRNA-MIAT表達(dá)量增加不明顯。(2)平滑肌細(xì)胞PCR結(jié)果:與空白對(duì)照相比,加入lncRNA-MIAT引物的條帶明顯,提示VSMC中表達(dá)LncRNA-MIAT,不同引物濃度的條帶亮度的比較差異不明顯,提示當(dāng)引物濃度增加時(shí),LncRNA-MIAT表達(dá)量增加不明顯。2.(1)內(nèi)皮細(xì)胞q RT-PCR結(jié)果:隨著TNF-α刺激時(shí)間延長(zhǎng)和刺激濃度增高,LncRNA-MIAT表達(dá)呈逐漸上升趨勢(shì)。TNF-α刺激24 h、48 h其他刺激時(shí)長(zhǎng)比較,lncRNA-MIAT表達(dá)量增多;TNF-α刺激濃度1.000 ng/ml、10.000 ng/ml與其他刺激濃度比較,lncRNA-MIAT表達(dá)量均增多,差異均具有統(tǒng)計(jì)學(xué)意義。(2)平滑肌細(xì)胞q RT-PCR結(jié)果:隨著TNF-α刺激時(shí)間延長(zhǎng)和刺激濃度增高,LncRNA-MIAT表達(dá)呈逐漸上升趨勢(shì)。刺激12 h、24 h、48h其他刺激時(shí)長(zhǎng)比較,lncRNA-MIAT表達(dá)量增多;刺激濃度10.00 ng/ml、20.000 ng/ml與其他刺激濃度比較,lncRNA-MIAT表達(dá)量均增多,差異均具有統(tǒng)計(jì)學(xué)意義。3.(1)內(nèi)皮細(xì)胞RNA干擾結(jié)果:si RNA MAIT 48h與si RNA Scramble比較,lncRNA-MIAT表達(dá)量明顯下降,差異具有統(tǒng)計(jì)學(xué)意義;si RNA MAIT 48 h與si RNA MAIT 24 h相比,lncRNA-MIAT表達(dá)量下降,差異具有統(tǒng)計(jì)學(xué)意義。(2)平滑肌細(xì)胞RNA干擾結(jié)果:si RNA MAIT 24h與si RNA Scramble比較,lncRNA-MIAT表達(dá)量明顯下降,差異具有統(tǒng)計(jì)學(xué)意義;si RNA MAIT 48 h與si RNA Scramble比較,lncRNA-MIAT表達(dá)量明顯下降,差異具有統(tǒng)計(jì)學(xué)意義。4.(1)內(nèi)皮細(xì)胞中ICAM-1的Western Blot結(jié)果:濃度為1.000 ng/ml的TNF-α刺激ECs后,ICAM-1的表達(dá)與空白對(duì)照相比顯著增高,差異有統(tǒng)計(jì)學(xué)意義。應(yīng)用si RNA MAIT轉(zhuǎn)染ECs 48 h后,應(yīng)用濃度為1.000 ng/ml TNF-α刺激ECs后,與陽(yáng)性對(duì)照相比,ICAM-1表達(dá)量被明顯抑制,差異有統(tǒng)計(jì)學(xué)意義;與空白對(duì)照相比,差異無(wú)統(tǒng)計(jì)學(xué)意義。(2)內(nèi)皮細(xì)胞中VCAM-1的Western Blot結(jié)果:濃度為1.000 ng/ml的TNF-α刺激ECs后,VCAM-1的表達(dá)與空白對(duì)照相比顯著增高,差異有統(tǒng)計(jì)學(xué)意義。應(yīng)用si RNA MAIT轉(zhuǎn)染ECs 48 h后,應(yīng)用濃度為1.000 ng/ml TNF-α刺激ECs后,與陽(yáng)性對(duì)照相比,VCAM-1表達(dá)量被明顯抑制,差異有統(tǒng)計(jì)學(xué)意義;與空白對(duì)照相比,差異無(wú)統(tǒng)計(jì)學(xué)意義。(3)平滑肌細(xì)胞中ICAM-1的Western Blot結(jié)果:濃度為10.00 ng/ml的TNF-α刺激VSMC后,ICAM-1的表達(dá)與空白對(duì)照相比顯著增高,差異有統(tǒng)計(jì)學(xué)意義。應(yīng)用si RNA MAIT轉(zhuǎn)染VSMC 48 h后,應(yīng)用濃度為10.00 ng/ml TNF-α刺激VSMC后,與陽(yáng)性對(duì)照相比,ICAM-1表達(dá)被抑制,差異有統(tǒng)計(jì)學(xué)意義;與空白對(duì)照相比,差異無(wú)統(tǒng)計(jì)學(xué)意義。5.(1)內(nèi)皮細(xì)胞劃痕實(shí)驗(yàn)結(jié)果:濃度為1.000 ng/ml TNF-α刺激ECs培養(yǎng)24h后,與空白對(duì)照相比,ECs遷移距離延長(zhǎng),差異有統(tǒng)計(jì)學(xué)意義。si RNA MAIT敲低后的ECs加入濃度為1.000ng/ml TNF-α刺激ECs培養(yǎng)24h后,與空白對(duì)照相比,ECs遷移明顯被抑制,差異有統(tǒng)計(jì)學(xué)意義。(2)平滑肌細(xì)胞劃痕實(shí)驗(yàn)結(jié)果:濃度為10.00 ng/ml TNF-α刺激VSMC培養(yǎng)24h后,與空白對(duì)照相比,細(xì)胞遷移距離明顯延長(zhǎng),差異有統(tǒng)計(jì)學(xué)意義。si RNA MAIT敲低后的VSMC加入濃度為10.00 ng/ml TNF-α刺激培養(yǎng)24h后,與空白對(duì)照相比,VSMC遷移明顯被抑制,差異有統(tǒng)計(jì)學(xué)意義。結(jié)論:LncRNA-MIAT可能參與血管的炎癥反應(yīng),并可能起到促炎作用。
[Abstract]:Objective: To observe in vitro endothelial cells (ECs) and vascular smooth muscle cells (vascular smooth muscle cell, VSMC) under the stimulation of tumor necrosis factor alpha (tumor necrosis factor- alpha, TNF- alpha). The expression changes of IPT, LncRNA-MIAT, and the regulatory role of LncRNA-MIAT on the inflammation of ECs and VSMC. Methods: TNF- alpha induced ECs and VSMC inflammatory states. 1 (vascular cell adhesion molecule-1, VCAM-1) and LncRNA-MIAT expression. Use LncRNA-MIAT's small interfering ribonucleic acid (Si RNA MAIT) transfection ECs and VSMC. The bands with lncRNA-MIAT primers were obvious, suggesting that LncRNA-MIAT was expressed in ECs. The difference of band brightness of different primers was not obvious. It suggested that when the concentration of primers increased, the expression of LncRNA-MIAT was not obvious. (2) PCR results of smooth muscle cells: compared with empty white, the bands with lncRNA-MIAT primers were obvious, suggesting that the bands of lncRNA-MIAT primers were obvious, suggesting that the bands of lncRNA-MIAT primers were obvious. The expression of LncRNA-MIAT in VSMC was not obvious in the band brightness of different primers. It was suggested that when the primer concentration increased, the increase of LncRNA-MIAT expression was not obvious in.2. (1) endothelial cells Q RT-PCR results: with the prolongation of TNF- alpha stimulation time and the increase of stimulation concentration, the expression of LncRNA-MIAT showed a gradual increase of.TNF- alpha stimulation 24 h, and 48 h. Compared with the time of stimulation, the expression of lncRNA-MIAT increased, the concentration of TNF- alpha was 1 ng/ml, and the expression of lncRNA-MIAT was increased with the concentration of 10 ng/ml. (2) the Q RT-PCR results of smooth muscle cells: the expression of LncRNA-MIAT increased with the prolongation of the time of TNF- alpha stimulation and the increase of the concentration of stimulation. Trend. Compared with 12 h, 24 h, and 48h other stimuli, the expression of lncRNA-MIAT increased, the concentration of stimulation was 10 ng/ml, 20 ng/ml was compared with other stimuli, lncRNA-MIAT expression increased, and the difference was statistically significant.3. (1) of RNA interference in endothelial cells: Si RNA MAIT. The difference has statistical significance, Si RNA MAIT 48 h and Si RNA MAIT 24 h, lncRNA-MIAT expression decreased, and the difference has statistical significance. (2) RNA interference results of smooth muscle cells: Si RNA Compared with amble, the expression of lncRNA-MIAT decreased significantly, and the difference was statistically significant.4. (1) the Western Blot result of ICAM-1 in endothelial cells: the expression of TNF- a with a concentration of 1 ng/ml was significantly higher than that of the blank control, and the difference was statistically significant. The application concentration was 1. After TNF- alpha was stimulated by ECs, the expression of ICAM-1 was significantly inhibited and the difference was statistically significant. The difference was not statistically significant compared with the blank control. (2) the Western Blot result of VCAM-1 in endothelial cells: the expression of VCAM-1 was significantly higher than that of the blank control, and the difference was statistically significant compared with that of the blank control. Significance. After the application of Si RNA MAIT to ECs 48 h, the concentration was 1 ng/ml TNF- A and ECs was stimulated, and the amount of VCAM-1 expression was significantly inhibited and the difference was statistically significant. The difference was not statistically significant compared with the blank control. (3) Western Blot results in the ICAM-1 of the smooth muscle cells: the concentration of 10 After MC, the expression of ICAM-1 was significantly higher than that of the blank control. The difference was statistically significant after the transfection of VSMC 48 h with Si RNA MAIT, with a concentration of 10 ng/ml TNF- a VSMC, the ICAM-1 expression was inhibited and the difference was statistically significant; there was no significant difference between the endothelial cells of.5. (1) compared with the blank control. The result of the mark experiment: after the concentration of 1 ng/ml TNF- alpha stimulated ECs culture 24h, compared with the blank control, the migration distance of ECs was prolonged, the difference was statistically significant after.Si RNA MAIT was knocked down, the concentration of ECs added to 1.000ng/ml TNF- alpha stimulated ECs culture, compared with the blank control, the migration movement was obviously inhibited, and the difference was statistically significant. (2) smooth muscle The result of cell scratch test: after the concentration of 10 ng/ml TNF- alpha stimulated VSMC culture 24h, the cell migration distance was obviously prolonged compared with the blank control, the difference was statistically significant after.Si RNA MAIT knockout concentration was 10 ng/ml TNF- alpha stimulation 24h, and the VSMC migration was obviously suppressed compared with the blank. The difference was statistically significant. Conclusion: LncRNA-MIAT may play an important role in the inflammatory reaction of blood vessels and may play a proinflammatory role.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R54

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