發(fā)布時(shí)間：2018-04-20 01:03

  本文選題：膠質(zhì)瘤 + miRNA-34a�。� 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究目的:(1)探究TPD52基因在膠質(zhì)瘤組織、正常腦組織及U87細(xì)胞中的表達(dá)水平;(2)探究干擾TPD52基因表達(dá)后對(duì)膠質(zhì)瘤U87細(xì)胞增殖、周期及凋亡的影響;(3)探究干擾TPD52基因表達(dá)后對(duì)膠質(zhì)瘤U87侵襲、遷移的影響;(4)探究mi RNA-34a在膠質(zhì)瘤組織及U87細(xì)胞中的表達(dá)水平;(5)探究mi RNA-34a對(duì)膠質(zhì)瘤細(xì)胞細(xì)胞周期的調(diào)節(jié)作用;(6)探究mi RNA-34a通過靶向TPD52調(diào)節(jié)膠質(zhì)瘤U87細(xì)胞的周期變化。研究方法:(1)收集我院12例膠質(zhì)瘤患者病例標(biāo)本,12例腦外傷患者正常腦組織病理標(biāo)本,應(yīng)用熒光定量PCR檢測(cè)TPD52 m RNA的表達(dá)量,應(yīng)用Western blot檢測(cè)TPD52蛋白的表達(dá)量。應(yīng)用熒光定量PCR檢測(cè)mi RNA-34a的表達(dá)量。(2)將攜帶著sh RNA-tpd52(抑制tpd52的核苷酸序列)、sh RNA-NC(陰性對(duì)照的核苷酸序列)的慢病毒體外轉(zhuǎn)染膠質(zhì)瘤細(xì)胞系U87。在轉(zhuǎn)染48 h后熒光顯微鏡下觀察表達(dá)GFP細(xì)胞數(shù)量,采用熒光定量PCR、Western blot分別檢測(cè)TPD52 m RNA及蛋白表達(dá)量,MTT檢測(cè)細(xì)胞增殖能力,流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期分布,Annexin V和PI雙染流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡,劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞遷移能力,Transwell實(shí)驗(yàn)檢測(cè)細(xì)胞侵襲能力。(3)將mi RNA34a模擬物及mi RNA-34a抑制物轉(zhuǎn)染入U(xiǎn)87細(xì)胞,并用熒光定量PCR檢測(cè)轉(zhuǎn)然后細(xì)胞mi RNA-34a的表達(dá)水平。查閱相關(guān)文獻(xiàn)并使用生物信息網(wǎng)分析預(yù)測(cè)TPD52的3,UTR是mi RNA-34a的作用靶點(diǎn),采用熒光素酶報(bào)告實(shí)驗(yàn)檢驗(yàn)TPD52是否為mi RNA-34a的靶基因。U87細(xì)胞經(jīng)不同因素處理后,采用熒光定量PCR檢測(cè)TPD52及相關(guān)蛋白的m RNA表達(dá)水平;采用Western blot實(shí)驗(yàn)檢測(cè)TPD52及相關(guān)蛋白的蛋白表達(dá)水平;利用流式細(xì)胞儀檢測(cè)U87細(xì)胞的細(xì)胞周期。研究結(jié)果:(1)熒光定量PCR檢測(cè)結(jié)果顯示tpd52m RNA在Ⅲ-Ⅳ級(jí)膠質(zhì)瘤組織(ΔCT0.73±0.47)、U87細(xì)胞(ΔCT0.74±0.32)中的表達(dá)水平明顯高于正常腦組織(ΔCT1.92±0.42)(P0.05);而U87細(xì)胞與Ⅲ-Ⅳ級(jí)膠質(zhì)瘤組織中tpd52m RNA表達(dá)水平差異無統(tǒng)計(jì)學(xué)意義。Western blot檢測(cè)結(jié)果顯示TPD52蛋白表達(dá)量在在Ⅲ-Ⅳ級(jí)膠質(zhì)瘤組織、U87細(xì)胞中的表達(dá)水平明顯高于正常腦組織的表達(dá)水平。(2)U87細(xì)胞慢病毒轉(zhuǎn)染率通過攜帶的GFP表達(dá)來評(píng)估,通過在熒光顯微鏡下細(xì)胞計(jì)數(shù)得出細(xì)胞轉(zhuǎn)染率90%。與sh RNA-NC組及空白對(duì)照組相比較,sh RNA-tpd52組細(xì)胞TPD52 m RNA及蛋白表達(dá)量明顯減少(P0.01),而且細(xì)胞增殖能力明顯下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05);細(xì)胞周期被阻滯在G0/G1期、細(xì)胞凋亡比例明顯增加(P0.05),細(xì)胞遷移能力明顯下降差異有統(tǒng)計(jì)學(xué)意義(P0.05);細(xì)胞侵襲能力明顯下降差異有統(tǒng)計(jì)學(xué)意義(P0.05)。(3)熒光定量PCR檢測(cè)結(jié)果顯示mi RNA-34a在Ⅲ-Ⅳ級(jí)膠質(zhì)瘤組織(ΔCT3.26±0.24)、U87細(xì)胞(ΔCT3.45±0.12)中的表達(dá)水平明顯低于正常腦組織(ΔCT1.94±0.40)(P0.05);而U87細(xì)胞與Ⅲ-Ⅳ級(jí)膠質(zhì)瘤組織中mi RNA-34a表達(dá)水平差異無統(tǒng)計(jì)學(xué)意義。(4)經(jīng)轉(zhuǎn)染后的U87細(xì)胞,經(jīng)周期實(shí)驗(yàn)發(fā)現(xiàn)轉(zhuǎn)染mi RNA-34a模擬物可明顯使U87細(xì)胞停留在S期的細(xì)胞數(shù)量比例明顯減少為16.12%,P0.05,而轉(zhuǎn)染mi RNA-34a抑制物可明顯使U87細(xì)胞停留在S期的細(xì)胞數(shù)量比例明顯增加為43.56%,P0.05。雙熒光素酶報(bào)告基因?qū)嶒?yàn)發(fā)現(xiàn)轉(zhuǎn)染mi RNA-34a模擬物可明顯使U87細(xì)胞中TPD52的3,UTR活性明顯降低為32%,P0.05,而轉(zhuǎn)染mi RNA-34a抑制物可明顯使U87細(xì)胞中TPD52的3,UTR活性明顯升高為58%,P0.05。結(jié)論:(1)TPD52在膠質(zhì)瘤組織中表達(dá)水平比正常腦組織中較高。(2)干擾TPD52基因表達(dá),可抑制膠質(zhì)瘤細(xì)胞的增殖,促進(jìn)細(xì)胞凋亡、阻滯細(xì)胞周期。(3)干擾TPD52基因表達(dá),可抑制膠質(zhì)瘤細(xì)胞遷移能力及侵襲能力。(4)mi RNA-34a在膠質(zhì)瘤組織中表達(dá)水平比正常腦組織中較低。(5)mi RNA-34a可以調(diào)節(jié)膠質(zhì)瘤細(xì)胞的細(xì)胞周期,抑制膠質(zhì)瘤細(xì)胞S期細(xì)胞比例。(6)mi RNA-34a具有直接靶向TPD52調(diào)節(jié)膠質(zhì)瘤細(xì)胞的周期變化。
[Abstract]:Objective: (1) to explore the expression level of TPD52 gene in glioma, normal brain and U87 cells; (2) to explore the effect of TPD52 gene expression on the proliferation, cycle and apoptosis of glioma U87 cells, and (3) to explore the influence of TPD52 gene expression on the invasion and migration of glioma U87, and (4) to explore the MI RNA-34a in glioma tissue and The expression level in U87 cells; (5) explore the regulation of MI RNA-34a on the cell cycle of glioma cells; (6) explore the periodic changes of MI RNA-34a through targeted TPD52 to regulate glioma U87 cells. (1) collect 12 cases of glioma cases in our hospital, 12 cases of normal brain tissue pathology specimens of patients with traumatic brain injury, and use fluorescent quantitative PCR The expression of TPD52 m RNA was detected and the expression of TPD52 protein was detected by Western blot. The expression of MI RNA-34a was detected by fluorescence quantitative PCR. (2) the transfection of the glioma cell line with SH RNA-tpd52 (inhibition of the nucleotide sequence of tpd52) and transfected to the glioma cell line in vitro The number of GFP cells was observed under microscopical microscope. TPD52 m RNA and protein expression were detected by fluorescence quantitative PCR and Western blot respectively. MTT was used to detect cell proliferation. Flow cytometry was used to detect cell cycle distribution. Annexin V and PI double dye flow cytometry was used to detect cell apoptosis. The migration ability of cells was detected by scratch test. Transwell experiment detected cell (3) mi RNA34a mimics and MI RNA-34a inhibitors were transfected into U87 cells, and the expression level of MI RNA-34a was detected by fluorescence quantitative PCR and the expression level of MI RNA-34a was detected by using the related literature and the bioinformatics network was used to predict the 3 of TPD52. UTR was the target of MI RNA-34a. After the target gene.U87 cells of 34a were treated by different factors, the expression of M RNA expression of TPD52 and related proteins was detected by fluorescence quantitative PCR, the protein expression level of TPD52 and related proteins was detected by Western blot test, and the cell cycle of U87 cells was detected by flow cytometry. The results were as follows: (1) the fluorescence quantitative PCR detection results showed tpd52m The expression level of NA in grade III to IV glioma (delta CT0.73 + 0.47) and U87 cells (delta CT0.74 + 0.32) was significantly higher than that of normal brain tissue (delta CT1.92 + 0.42) (P0.05), while there was no significant difference in the RNA expression of tpd52m RNA in the tissues of U87 cells and grade III to IV glioma..Western blot detection results showed that the expression of TPD52 protein was in grade III - IV The expression level of U87 cells in glioma tissue was significantly higher than that in normal brain tissue. (2) U87 cell lentivirus transfection rate was evaluated by the expression of GFP, and the cell transfection rate was compared with the SH RNA-NC group and the blank control group by counting the cell count under the fluorescence microscope. TPD52 M RNA and protein of SH RNA-tpd52 group cells. The expression volume decreased significantly (P0.01), and the cell proliferation ability decreased significantly (P0.05), the cell cycle was blocked in G0/G1 phase, the percentage of cell apoptosis increased significantly (P0.05), the difference of cell migration ability was statistically significant (P0.05), and the difference of cell invasion ability was statistically significant (P0.05). (3) fluoret The results of optical quantitative PCR detection showed that MI RNA-34a was in grade III to IV glioma (delta CT3.26 + 0.24), U87 cells (delta CT3.45 + 0.12) were significantly lower than normal brain tissues (delta CT1.94 + 0.40) (P0.05), but there was no significant difference in the level of MI RNA-34a in U87 cells and grade III - IV glioma tissues. (4) the transfected U87 cells, It was found that transfection of MI RNA-34a mimics could obviously reduce the number of U87 cells in S phase to 16.12%, P0.05, and MI RNA-34a inhibitor could obviously increase the number of U87 cells in S phase to 43.56%, and P0.05. double fluoropenzyme reporter gene experiment found that MI RNA-34a model was transfected. The 3 and UTR activity of TPD52 in U87 cells were obviously reduced to 32%, P0.05, and MI RNA-34a inhibitor could significantly increase the 3 and UTR activity of TPD52 in U87 cells to 58%, P0.05. conclusion: (1) TPD52 in glioma tissues was higher than that in normal brain tissue. (2) interference of the gene expression of TPD52 genes and the inhibition of glioma cells Proliferation, promoting cell apoptosis and blocking cell cycle. (3) interference with TPD52 gene expression can inhibit the migration ability and invasion ability of glioma cells. (4) the expression level of MI RNA-34a in glioma tissues is lower than that in normal brain tissue. (5) mi RNA-34a can regulate the cell cycle of glioma cells and inhibit the proportion of S cells in glioma cells. (6) M I RNA-34a has direct targeting TPD52 to regulate the cell cycle of glioma cells.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.41

【參考文獻(xiàn)】

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1 泮長存;湯R，

本文編號(hào)：1775516