本文選題：微小核糖核酸 + 單核細(xì)胞��； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：一、背景與目的動(dòng)脈粥樣硬化被認(rèn)為有炎癥因素參與其病理過程,而其炎癥過程與單核細(xì)胞和單核細(xì)胞來源巨噬細(xì)胞的招募、活化以及單核細(xì)胞的分化相關(guān),且與動(dòng)脈粥樣硬化斑塊穩(wěn)定性及動(dòng)脈粥樣硬化性疾病的預(yù)后相關(guān)。然而,由于單核細(xì)胞半衰期較短,如果缺少自噬,循環(huán)血中的單核細(xì)胞將會(huì)自然凋亡。目前已知主要的可誘導(dǎo)單核細(xì)胞自噬的刺激物主要有粒細(xì)胞巨噬細(xì)胞刺激因子(GM-CSF)和巨噬細(xì)胞刺激因子(M-CSF)。目前認(rèn)為自噬是一個(gè)普遍存在于真核細(xì)胞中、可通過雙層膜結(jié)構(gòu)的囊泡降解或回收不需要或無功能的細(xì)胞組分以維持細(xì)胞穩(wěn)態(tài)、抗衰老以及細(xì)胞發(fā)育的過程。其過程中,微管相關(guān)蛋白輕鏈3 (LC3)以及泛素樣結(jié)合蛋白p62/SQSTM1均為重要標(biāo)志蛋白,故常作為檢測自噬活性的常用指標(biāo)之一。短鏈非編碼RNA中微小核糖核酸(microRNA, miRNA, miR-)可對廣泛細(xì)胞各種生物學(xué)活動(dòng)進(jìn)行轉(zhuǎn)錄后調(diào)控。故我們設(shè)計(jì)本研究旨在探討miRNA在單核細(xì)胞自噬中的表達(dá)情況及其調(diào)控作用。我們通過將THP-1人單核細(xì)胞置于M-CSF刺激環(huán)境下,誘導(dǎo)單核細(xì)胞發(fā)生自噬,檢測miRNA表達(dá)情況,篩查表達(dá)變化最為顯著的miRNA,并進(jìn)一步將其在單核細(xì)胞中過表達(dá)來觀察細(xì)胞自噬活性的變化。進(jìn)而探討該miRNA調(diào)控M-CSF誘導(dǎo)的THP-1人單核細(xì)胞自噬的潛在靶基因。二、研究方法(一)THP-1人單核細(xì)胞的培養(yǎng)THP-1人單核細(xì)胞培養(yǎng)條件為完全1640培養(yǎng)基(90%RPMI-1640+10%胎牛血清+1%P/S)。誘導(dǎo)自噬條件為在完全1640培養(yǎng)液中加入M-CSF至100ng/ml。(二)流式細(xì)胞檢測THP-1人單核細(xì)胞按上述方法培養(yǎng),CYTO-ID自噬檢測試劑盒中綠色染劑染色后用流式細(xì)胞儀的綠(FL1)通道分析樣本。(三)蛋白免疫印跡(Western blot)單核細(xì)胞樣品采用HelixGenRIPA裂解液提取蛋白,對LC3-Ⅰ、LC3-Ⅱ、p62/SQSTMI以及GAPDH等蛋白表達(dá)情況進(jìn)行檢測。一抗?jié)舛热缦?anti-LC3(1:1000)、anti-p62 (1:1000)、anti-GAPDH (1:1000)。二抗?jié)舛热缦?anti-rabbit(1:10000)、anti-mouse (1:5000)。蛋白條帶應(yīng)用 BIORADFlour-SMuiltilmager 成像系統(tǒng)檢測。(四)透射電子顯微鏡單核細(xì)胞經(jīng)M-CSF處理后使用多聚甲醛固定液4℃固定6h以上后送檢,電鏡下觀察到雙層膜結(jié)構(gòu)囊泡狀的自噬體作為自噬定性檢測標(biāo)準(zhǔn)。(五)Agilent miRNA 芯片應(yīng)用mirVana RNA提取試劑盒提取total RNA,芯片雜交后應(yīng)用Aglient G2505C掃描儀中選擇相應(yīng)的參數(shù)掃描。(六)實(shí)時(shí)定量聚合酶鏈反應(yīng)(qRT-PCR)應(yīng)用miRcute miRNA提取分離試劑盒提取THP-1人單核細(xì)胞的miRNA,應(yīng)用miRcute增強(qiáng)型miRNAcDNA第一鏈合成試劑盒進(jìn)行反轉(zhuǎn)錄,miRcute增強(qiáng)型miRNA熒光定量檢測試劑盒進(jìn)行qRT-PCR,使用Roche LightCycler分析儀進(jìn)行檢測分析,以U6作為內(nèi)參計(jì)算ACt值,以2-△△Ct法計(jì)算目標(biāo)miRNA相對表達(dá)水平。(七)腺病毒構(gòu)建及細(xì)胞轉(zhuǎn)染構(gòu)建重組腺病毒并擴(kuò)增、測定滴度,繼而通過腺病毒將miR-301b-3p轉(zhuǎn)染入單核細(xì)胞。采用qRT-PCR方法檢測轉(zhuǎn)染效率。(八)雙熒光素酶報(bào)告基因分別將MYB和CYLD的mRNA 3'UTR融合至熒光素酶質(zhì)粒中,再將質(zhì)粒分別與miR-301b-3p類似物共轉(zhuǎn)染至293T細(xì)胞中,24 h后裂解細(xì)胞,應(yīng)用雙熒光素酶報(bào)告系統(tǒng)試劑盒測定熒光素酶活性并定量分析。(九)統(tǒng)計(jì)學(xué)分析計(jì)量資料以平均數(shù)±標(biāo)準(zhǔn)差表示,每組的實(shí)驗(yàn)數(shù)據(jù)重復(fù)至少3次。兩組間比較采用兩獨(dú)立樣本的t檢驗(yàn),多個(gè)組間比較采用單因素ANOVA分析。以P0.05作為顯著性差異標(biāo)準(zhǔn)。三、結(jié)果(一)M-CSF誘導(dǎo)THP-1人單核細(xì)胞自噬M-CSF誘導(dǎo)處理后,應(yīng)用CYTO-ID自噬檢測試劑盒對THP-1人單核細(xì)胞進(jìn)行流式細(xì)胞檢測顯示處理6 h時(shí)細(xì)胞自噬水平顯著升高;Western blot顯示LC3II/1比值顯著高于對照組、p62/SQSTM1顯著低于對照組,應(yīng)用巴弗洛霉素A1抑制溶酶體活性后,相同處理?xiàng)l件下LC3Ⅱ/Ⅰ比值進(jìn)一步增高、p62/SQSTM1則高于對照組;透射電子顯微鏡觀察到M-CSF處理6 h時(shí)單核細(xì)胞內(nèi)形成雙側(cè)膜結(jié)構(gòu)自噬體,而對照組則未觀察到該現(xiàn)象。上述結(jié)果均證實(shí)在M-CSF處理6 h后單核細(xì)胞自噬被顯著激活。(二)miRNA表達(dá)情況Agilent miRNA芯片檢測結(jié)果提示與對照組相比,M-CSF處理6 h時(shí)單核細(xì)胞表達(dá) miRNA 中 miR-1249-3p、miR-301b-3p、miR-4653-3p、miR-513a-5p、miR-6734-5p存在顯著差異,進(jìn)一步應(yīng)用qRT-PCR檢測驗(yàn)證miR-301b-3p表達(dá)顯著增高。(三)過表達(dá)miR-301b-3p促進(jìn)M-CSF誘導(dǎo)的單核細(xì)胞自噬用miR-301b-3p轉(zhuǎn)染單核細(xì)胞,48 h時(shí)qRT-PCR檢測顯示miR-301b-3p表達(dá)量與對照組相比顯著增高,提示轉(zhuǎn)染有效。M-CSF誘導(dǎo)單核細(xì)胞發(fā)生自噬,繼而應(yīng)用Western blot檢測顯示miR-301b-3p過表達(dá)后LC311/I1比值顯著增高、p62/SQSTM1顯著降低,提示過表達(dá)miR-301b-3p進(jìn)一步促進(jìn)M-CSF誘導(dǎo)的單核細(xì)胞自噬。(四)靶基因預(yù)測及驗(yàn)證應(yīng)用TargetScan、microRNAorg等數(shù)據(jù)庫檢索預(yù)測篩選顯示MYB、CYLD可能為miR-301b-3p的潛在靶基因,其3'UTR含有miR-301b-3p可能的結(jié)合位點(diǎn)且保守性較好,故本研究應(yīng)用熒光素酶報(bào)告基因方法驗(yàn)證MYB與CYLD是否為miR-301b-3p的靶基因。結(jié)果顯示過表達(dá)miR-301b-3p可顯著抑制MYB-3'UTR與CYLD-3'URT的熒光素酶表達(dá),提示MYB與CYLD為miR-301b-3p的直接靶基因。四、結(jié)論(一)M-CSF能夠誘導(dǎo)THP-1人單核細(xì)胞發(fā)生自噬,在誘導(dǎo)6 h后自噬活性水平顯著增高。(二)在M-CSF能夠誘導(dǎo)THP-1人單核細(xì)胞自噬時(shí),miR-301b-3p表達(dá)顯著增高。(三)過表達(dá)miR-301b-3p能促進(jìn)M-CSF誘導(dǎo)的單核細(xì)胞自噬。(四)miR-301b-3p靶向作用于MYB與CYLD,可能通過該途徑實(shí)現(xiàn)促進(jìn)M-CSF誘導(dǎo)的單核細(xì)胞自噬。
[Abstract]:Background and objective atherosclerosis is considered to be involved in the pathological process of inflammation, which is associated with the recruitment and activation of monocyte and monocyte derived macrophages, and the differentiation of monocyte, and is related to the stability of atherosclerotic plaque and the prognosis of atherosclerotic disease. Monocyte half-life is short. If autophagy is short of autophagy, mononuclear cells in circulating blood will be spontaneously apoptotic. At present, the main inducible stimulants for autophagy are granulocyte macrophage stimulating factor (GM-CSF) and macrophage stimulating factor (M-CSF). It is often used as one of the commonly used indicators for the detection of autophagic activity in the process of maintaining cell homeostasis, aging and cell development by degradation or recovery of non functional or non functional cell components through the bilayer membrane vesicles. In the process, the light chain 3 (LC3) and the ubiquitin like binding protein p62/SQSTM1 are all important markers. MicroRNA, miRNA (miRNA, miR-) in short chain non coding RNA can be regulated after transcriptional regulation of various biological activities in a wide range of cells. Therefore, we designed this study to explore the expression and regulation of miRNA in autophagy in monocyte. We induce mononuclear cells to induce mononuclear cells by placing THP-1 human mononuclear cells in a M-CSF stimulus environment. Autophagy, detection of miRNA expression, screening the most significant changes in the expression of miRNA, and further overexpression in mononuclear cells to observe the changes in cell autophagy. Further explore the potential target gene for the regulation of M-CSF induced autophagy in THP-1 human mononuclear cells. Two, the study method (1) THP-1 human mononuclear cell culture THP -1 human monocyte culture condition was a complete 1640 culture medium (90%RPMI-1640+10% fetal bovine serum +1%P/S). The induction of autophagy was cultured in complete 1640 culture medium by adding M-CSF to 100ng/ml. (two) flow cytometry to detect THP-1 human mononuclear cells, and the green dye in the CYTO-ID autophagy test kit was green with a flow cytometer. FL1) channel analysis samples. (three) protein immunoblotting (Western blot) mononuclear cells were used to extract protein from HelixGenRIPA lysate and detect the expression of LC3- I, LC3- II, p62/SQSTMI and GAPDH. The concentration of anti LC3- was as follows: anti-LC3 (1:1000), anti-p62 (1:1000), and two 10000), anti-mouse (1:5000). The protein strip was detected by the BIORADFlour-SMuiltilmager imaging system. (four) the mononuclear cells of the transmission electron microscope were treated with polyoxymethylene fixed solution at 4 degrees centigrade for more than 6h after M-CSF treatment, and the vesicular autophagy was observed as a qualitative test standard for autophagy under the electron microscope. (five) Agilent MiRNA chip is used to extract total RNA by mirVana RNA extraction kit. After chip hybridization, the corresponding parameter scanning is selected in Aglient G2505C scanner. (six) real-time quantitative polymerase chain reaction (qRT-PCR) is used to extract miRNA of THP-1 mononuclear cells by miRcute miRNA extraction and separation kit. Reagents were reverse transcribed, miRcute enhanced miRNA fluorescence quantitative detection kit was qRT-PCR, and Roche LightCycler analyzer was used for detection and analysis. U6 was used as the internal parameter to calculate ACt value. 2- Delta Delta Ct method was used to calculate the relative expression level of target miRNA. (seven) adenovirus construction and cell transfection construction of recombinant adenovirus and amplification, determination of drops And then transfection of miR-301b-3p into mononuclear cells by adenovirus. The transfection efficiency was detected by qRT-PCR method. (eight) double luciferase reporter gene fused mRNA 3'UTR of MYB and CYLD into luciferase plasmid, and then plasmids were co transfected with miR-301b-3p analogues to 293T cells, after 24 h, lysate cells were used, and double fluorescein was applied. The enzyme report system kits were used to determine the luciferase activity and quantitative analysis. (nine) the statistical data were expressed with mean standard deviation, and the experimental data in each group were repeated at least 3 times. The two groups were compared with two independent samples of t test, and a single factor ANOVA analysis was used in multiple groups. P0.05 was used as a significant difference standard. Three, knot Fruit (1) M-CSF induced THP-1 human mononuclear autophagic M-CSF induction treatment, the use of CYTO-ID autophagy detection kit for THP-1 human mononuclear cell flow cytometry showed that the level of autophagy increased significantly at 6 h; Western blot showed that the LC3II/1 ratio was significantly higher than the control group, p62/SQSTM1 was significantly lower than the control group, using buffalamycin. After the inhibition of lysosome activity by A1, the ratio of LC3 II / I increased further under the same treatment condition, and p62/SQSTM1 was higher than that of the control group; transmission electron microscopy observed that the autophagosome was formed in the mononuclear cells when M-CSF treatment was 6 h, while the control group did not observe the phenomenon. The results all showed that the mononuclear cells from the M-CSF treatment after 6 h self were from the mononuclear cells. (two) the Agilent miRNA chip detection results of miRNA expression showed that there were significant differences in the expression of miR-1249-3p, miR-301b-3p, miR-4653-3p, miR-513a-5p, miR-6734-5p in the mononuclear cells miRNA at 6 h when compared with the control group. (three) over expression (three) overexpression. MiR-301b-3p promoted monocyte autophagy induced by M-CSF to transfect mononuclear cells with miR-301b-3p. At 48 h, qRT-PCR detection showed that the expression of miR-301b-3p was significantly higher than that of the control group, suggesting that the transfection of effective.M-CSF induced autophagy induced mononuclear cells, and then the LC311/I1 ratio was significantly increased after Western blot detection showed that miR-301b-3p overexpressed. P62/SQSTM1 was significantly reduced, suggesting that over expression of miR-301b-3p further promoted autophagy induced by M-CSF. (four) target gene prediction and verification application of TargetScan, microRNAorg and other database retrieval and prediction screening showed MYB, CYLD might be a potential target gene for miR-301b-3p, and 3'UTR contained miR-301b-3p possible binding sites and conservatism. Well, this study uses luciferase reporter gene method to verify whether MYB and CYLD are the target genes of miR-301b-3p. The results show that overexpression of miR-301b-3p can significantly inhibit the luciferase expression of MYB-3'UTR and CYLD-3'URT, suggesting that MYB and CYLD are the direct target genes of miR-301b-3p. Four, nodal (1) M-CSF can induce THP-1 mononuclear cells. Autophagy increased significantly after induction of 6 h. (two) the expression of miR-301b-3p increased significantly when M-CSF could induce autophagy in THP-1 mononuclear cells. (three) overexpression of miR-301b-3p could promote M-CSF induced autophagy in monocytes. (four) miR-301b-3p targeted MYB and CYLD, possibly through this pathway to promote M-CSF induction. Nuclear cells are autophagy.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R543.5

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