【摘要】：研究背景:高脂血癥是動脈粥樣硬化、骨質(zhì)疏松、高血壓、冠心病、腦卒中等疾病發(fā)生、發(fā)展的重要危險因素,是目前影響人民群眾身體健康的高發(fā)疾病之一。研究表明高脂血癥對骨愈合、骨礦化、骨密度等諸多骨代謝過程有不良影響,高脂血癥是引起骨質(zhì)疏松的危險因素之一。在骨髓間充質(zhì)干細(xì)胞(bone marrow stromal stem cells,BMSCs)向成骨細(xì)胞定向分化中Wnt信號通路起關(guān)鍵性作用。而最近幾年大量的研究表明,成骨的分化過程中微小RNA(microRNAs,miRNAs)發(fā)揮了重要的生物學(xué)功能,例如維持骨代謝的平衡。本課題組前期實驗證實高脂飼料喂養(yǎng)的大鼠種植體周圍骨質(zhì)疏松,骨小梁稀疏,排列紊亂;高脂飼料喂養(yǎng)的大鼠種植體周圍Ca/P原子個數(shù)百分比低于普通飼料喂養(yǎng)的大鼠;熒光定量PCR和Western blot結(jié)果顯示Dvl2基因、Dvl2蛋白的表達(dá)受到抑制,表明高脂血癥在一定程度上干擾高脂血癥大鼠種植體的早期骨結(jié)合。Wnt/β-catenin信號通路中的Dvl2在高脂血癥患者中具體如何發(fā)揮作用值得探討。Wnt信號通路與miRNAs存在大量的交叉信號分子,其作用靶點與具體作用機(jī)制都值得深入探討研究。實驗?zāi)康?(1)觀察高脂環(huán)境下大鼠骨髓基質(zhì)干細(xì)胞(BMSCs)成骨分化過程中Runx2、ALP、SP7、PPAR-γ、Dvl2及靶向調(diào)控Dvl2相關(guān)microRNAs的mRNA表達(dá)。(2)利用靶基因預(yù)測軟件、雙熒光素酶報告基因篩選及鑒定出靶向調(diào)節(jié)Dvl2的microRNAs。(3)探討microRNAs靶向調(diào)節(jié)Dvl2對BMSCs成骨分化的影響。實驗方法:(1)大鼠骨髓間充質(zhì)干細(xì)胞獲取傳代培養(yǎng),培養(yǎng)第三代時行流式細(xì)胞檢測,分別成骨成脂誘導(dǎo)28天后茜素紅和油紅O染色,觀察成骨成脂分化能力,鑒定BMSCs,以便用于后續(xù)實驗。用高脂培養(yǎng)基和普通培養(yǎng)基成骨誘導(dǎo)BMSCs誘導(dǎo)28天時分別行茜素紅和油紅O染色觀察成骨分化情況,誘導(dǎo)3、5、7、14、21天,利用RT-PCR檢測細(xì)胞內(nèi)成骨相關(guān)基因Runx2、ALP、SP7,成脂相關(guān)基因PPAR-γ,Wnt信號通路中Dvl2,與靶向調(diào)控Dvl2相關(guān)的miR-21-5p、miR-29c-3p、miR-138-5p、miR-351-5pmRNA的表達(dá)。(2)靶向調(diào)控Dvl2相關(guān)的microRNAs采用靶基因預(yù)測軟件Target Scan、MicroRNA.org、miRDB、Microcosm Targets 等四種 miRNA 數(shù)據(jù)庫在線分析軟件進(jìn)行生物信息學(xué)預(yù)測,以及文獻(xiàn)查詢,得到可能與Dvl2基因3'UTR區(qū)作用的 miRNAs 為:miR-21-5p、miR-29c-3p、miR-138-5p、miR-351-5p。通過體外轉(zhuǎn)染不同濃度(10nm、30nm、50nm、100nm)梯度的FAM-siRNA,6h后鏡下觀察轉(zhuǎn)染效率和CCK8檢測其細(xì)胞增殖篩選合適的轉(zhuǎn)染濃度。利用篩選濃度體外轉(zhuǎn)染 miR-21-5p、miR-29c-3p、miR-138-5p、miR-351-5p 的模擬物(mimics)和抑制物(inhibitor),48h后利用RT-PCR檢測轉(zhuǎn)染效率,實現(xiàn)Dvl2的過表達(dá)和低表達(dá),Western blot檢測BMSCs中Dvl2的表達(dá)差異,找出引起Dvl2變化最明顯的miRNA。構(gòu)建雙熒光素酶報告基因質(zhì)粒載體,分別用 miR-21-5p、miR-29c-3p、miR-138-5p、miR-351-5p 的模擬物與質(zhì)粒載體共轉(zhuǎn)293t細(xì)胞,利用多功能酶標(biāo)儀檢測熒光素酶活性,驗證miRNAs與Dvl2的靶向調(diào)控作用。(3)高脂環(huán)境下體外轉(zhuǎn)染miR-29c-3p mimics和inhibitor后成骨誘導(dǎo)BMSCs 3、7天,利用Western bolt檢測成骨相關(guān)基因Runx2、ALP的表達(dá)情況。實驗結(jié)果:(1)流式細(xì)胞儀檢測結(jié)果顯示:第三代BMSCs CD44陽性細(xì)胞比率是96.7%,CD45陽性細(xì)胞比率是2.8%,CD90陽性細(xì)胞比率是95.9%。間充質(zhì)干細(xì)胞表面標(biāo)志抗原表達(dá)陽性,驗證此細(xì)胞為BMSCs,純度較高。成骨誘導(dǎo)28天茜素紅染色、油紅O染色結(jié)果顯示BMSCs具有向成骨成脂方向分化能力。培養(yǎng)28天茜素紅染色見高脂組較對照組鈣化結(jié)節(jié)少,油紅O染色串珠樣脂滴高脂組多于對照組,RT-PCR結(jié)果顯示成骨指標(biāo)Runx2、SP7、ALPmRNA表達(dá)高脂組較對照組低,成脂指標(biāo)PPAR-γ mRNA表達(dá)高脂組較對照組高,差異有統(tǒng)計學(xué)意義(P0.05)。miR-21-5p、miR-29c-3p高脂組較對照組均降低,在3、5、7、14天時miR-138-5pmRNA表達(dá)量對照組與高脂組無明顯差異,在21天時miR-138-5pmRNA表達(dá)量對照組明顯高于高脂組,差異有顯著意義(P0.05)。在3、5、7天時miR-351-5pmRNA表達(dá)量對照組與高脂組無明顯差異,在14、21天時miR-351-5pmRNA表達(dá)量對照組低于高脂組,差異有顯著意義。(2)熒光顯微鏡觀察濃度越高轉(zhuǎn)染效率越高,CCK8濃度越高細(xì)胞增殖越受到抑制,Real-time PCR 結(jié)果顯示,轉(zhuǎn)染 miR-21-5p、miR-29c-3p、miR-138-5p、miR-351-5p的mimics后,與對照組相比其表達(dá)量分別升高5-6倍、4倍、1000倍和800倍左右。而轉(zhuǎn)染相應(yīng)的inhibitor后,與對照組相比其表達(dá)量分別降低約3倍、10倍、10倍和5倍。轉(zhuǎn)染miR-21-5p、miR-29c-3p、miR-138-5p、miR-351-5p 的 mimics 和 inhibitor 及 NC48h 后,RT-PCR 結(jié)果顯示,轉(zhuǎn)染 miR-21-5p 和 miR-29c-3p mimics 后,與 mimics nc 組相比 Dvl2的表達(dá)降低,轉(zhuǎn)染 miR-138-5p 和 miR-351-5p mimics 后,與 mimics nc 組相比 Dvl2 的表達(dá)升高。轉(zhuǎn)染 miR-21-5p、miR-29c-3p、miR-138-5p、miR-351-5p inhibitor后與inhibitor nc組相比Dvl2的表達(dá)都有不同程度的升高。Western blot結(jié)果顯示,miR-29c-3pmimics/inhibitor轉(zhuǎn)染細(xì)胞后,Dvl2表達(dá)差異最明顯。(3)Western blot 結(jié)果顯示轉(zhuǎn)染 miR-29c-3p mimics 后 Runx2 和 ALP 的蛋白表達(dá)水平均明顯比mimics NC組增高,轉(zhuǎn)染inhibitor后Runx2和ALP的蛋白表達(dá)水平均明顯比inhibitor NC組降低。結(jié)論:(1)高脂環(huán)境下BMSCs向成骨細(xì)胞分化減少,miR-21-5p、miR-29c-3p、miR-138-5p、miR-351-5p參與調(diào)控高脂環(huán)境下骨代謝和成骨細(xì)胞的生物學(xué)活性。(2)經(jīng)雙熒光素酶報告基因篩選及鑒定出靶向調(diào)節(jié)Dvl2的miRNA是miR-29c-3p,而miR-21-5p、miR-138-5p、miR-351-5p對Dvl2的負(fù)性調(diào)節(jié)作用弱。(3)miR-29c-3p通過靶向調(diào)節(jié)Dvl2間接促進(jìn)BMSCs向成骨分化、礦化能力。
[Abstract]:Background: Hyperlipemia is an important risk factor for the occurrence and development of atherosclerosis, osteoporosis, hypertension, coronary heart disease, and stroke. It is one of the most frequent diseases that affect the health of the people. The results show that the hyperlipoidemia has an adverse effect on bone metabolism, bone mineralization, bone mineral density and other bone metabolism, and it is one of the risk factors for osteoporosis. Bone marrow mesenchymal stem cells (BMSCs) play a critical role in the directional differentiation of bone marrow mesenchymal stem cells (BMSCs) into the osteoblast-oriented differentiation. In recent years, a large number of studies have shown that microRNAs (microRNAs, miRNAs) play an important biological function in the differentiation of osteogenesis, such as maintaining a balance of bone metabolism. In the early stage of the research group, the rats with high-fat diet were proved to be loose, the bone trabecula is sparse and the arrangement is disordered; the percentage of Ca/ P atoms in the peripheral Ca/ P atoms in the rat implant fed by the high-fat feed is lower than that of the normal feed-fed rats; and the fluorescence quantitative PCR and the Western blot result show that the Dvl2 gene, The expression of the Dvl2 protein is inhibited, indicating that the hyperlipidemia can interfere with the early bone combination of the hyperlipidemic rat implant to a certain extent. The role of Dvl2 in the Wnt/ HCO3-cattenin signal pathway in patients with hyperlipidemia is worth exploring. The Wnt signaling pathway and miRNAs have a large number of cross-signal molecules, and the action target and the specific action mechanism of the Wnt signal pathway are worth exploring deeply. Objective: (1) To observe the expression of Runx2, ALP, SP7, PPAR-1, Dvl2 and targeted regulation of Dvl2-related microRNAs in rat bone marrow stromal stem cells (BMSCs) in high-fat environment. And (2) screening and identifying the microRNAs targeting the Dvl2 by using the target gene prediction software and the double-luciferase reporter gene. (3) To investigate the effect of microRNAs targeting regulation of Dvl2 on the osteogenic differentiation of BMSCs. Methods: (1) The rat bone marrow-derived mesenchymal stem cells were collected and cultured, and the flow cytometry was carried out in the third generation. After 28 days of formation of the bone marrow-derived mesenchymal stem cells, the bone marrow-derived mesenchymal stem cells were cultured for 28 days, respectively, and stained with red and red O-red O, and the osteogenic differentiation ability was observed, and BMSCs were identified for subsequent experiments. The osteogenesis and differentiation of BMSCs were observed with high-fat medium and normal medium for 28 days, and the osteogenic differentiation was observed in 3,5,7,14 and 21 days by RT-PCR, and the expression of Dvl2, ALP, SP7, lipoid-related gene PPAR-1 and Wnt in Wnt signaling pathway was detected by RT-PCR. The expression of miR-21-5p, miR-29c-3p, miR-138-5p, and miR-351-5pmRNA associated with targeted regulation of Dvl2. (2) The target gene prediction software, Target Scan, MicroRNA.org, miRDB, Microcosm Targets and other four miRNA database on-line analysis software is used for bioinformatics prediction and the literature query to obtain the miRNAs that can function with the Dvl2 gene 3 'UTR region as the miR-21-5p, the miR-29c-3p, the miR-138-5p, and the miR-351-5p. The transfection efficiency and the cell proliferation of CCK8 were detected by transfecting the FAM-siRNA with different concentration (10 nm,30 nm,50 nm,100 nm) in vitro. Transfection of miR-21-5p, miR-29c-3p, miR-138-5p, miR-351-5p and the inhibitor in vitro with the screening concentration, the transfection efficiency was detected by RT-PCR after 48 h, the overexpression and low expression of Dvl2 were achieved, and the expression of Dvl2 in BMSCs was detected by Western blot, and the most obvious miRNAs were found. The plasmid vector of the two-luciferase reporter gene was constructed, and the 293T cells were co-transferred with the plasmid vector by the miRNAs-21-5p, the miR-29c-3p, the miR-138-5p, and the miR-351-5p, and the activity of the luciferase was detected by the multi-function microplate reader to verify the targeting and control effects of miRNAs and Dvl2. (3) The expression of bone-related gene Runx2 and ALP was detected by Western blot. Results: (1) The results of flow cytometry showed that the ratio of CD44 positive cells in third generation BMSCs was 96.7%, the ratio of CD45 positive cells was 2.8%, and the ratio of CD90 positive cells was 95.9%. The surface marker antigen of the mesenchymal stem cells is positive, and the cell is BMSCs, and the purity is high. The results showed that BMSCs had the ability to differentiate into the direction of osteogenesis. The results of RT-PCR showed that the expression of Runx2, SP7 and ALPmRNA in the high-fat group was lower than that in the control group, and the expression of PPAR-mRNA in the high-fat group was higher than that of the control group. The difference was significant (P0.05). The miR-21-5p, miR-29c-3p high-fat group and the control group were decreased, and the expression of miR-138-5 pmRNA in the 3,5,7 and 14 days was not significantly different from that in the high-fat group, and the expression of miR-138-5 pmRNA in the control group was significantly higher than that of the control group at 21 days (P0.05). At 3,5 and 7 days, the expression of miR-351-5pmRNA in the control group was not significantly different from that of the high-fat group, and the expression of miR-351-5pmRNA in the control group was lower than that of the control group at 14 and 21 days, and the difference was significant. (2) The higher the observation concentration of the fluorescence microscope, the higher the efficiency of the transfection, the higher the concentration of the CCK8, the higher the proliferation of the cells. The real-time PCR results showed that the expression of the miR-21-5p, the miR-29c-3p, the miR-138-5p, and the miR-351-5p increased by 5-6 times,4-fold,1000-fold and 800-fold, respectively, as compared to the control group. Compared with the control group, the expression was about 3-fold,10-fold,10-fold and 5-fold, respectively. The expression of Dvl2 in miR-21-5p, miR-29c-3p, miR-138-5p, miR-351-5p, miR-138-5p, miR-351-5p was reduced after transfection of miR-21-5p and miR-29c-3p misitics, and the expression of Dvl2 was increased compared to the mimics nc group after the transfection of miR-138-5p and miR-351-5p mmics. The expression of Dvl2 in miR-21-5p, miR-29c-3p, miR-138-5p, and miR-351-5p inhimitor was increased in different degrees after transfection. The results of Western blot showed that the expression of Dvl2 was the most obvious after the transfection of the cells with miR-29c-3pmomics/ inhitor. (3) The results of Western blot show that the level of protein expression of both Runx2 and ALP after transfection of miR-29c-3p mmics is significantly higher than that of the mimics NC group. Conclusion: (1) In high-fat environment, BMSCs differentiated into osteoblasts, miR-21-5p, miR-29c-3p, miR-138-5p, and miR-351-5p participate in the control of the biological activity of bone metabolism and osteoblast in high-fat environment. (2) The miRNAs targeted to the regulation of Dvl2 are miR-29c-3p, and miR-21-5p, miR-138-5p, and miR-351-5p are weak in negative regulation of Dvl2. (3) miR-29c-3p indirectly promotes the differentiation and mineralization of BMSCs by targeting and regulating Dvl2.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R589.2;R580

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