發(fā)布時間：2018-04-26 06:45

  本文選題：骨髓間充質(zhì)干細胞 + NCoR��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：研究背景:骨髓間充質(zhì)干細胞(BMSCs)具有分化成不同細胞組織的能力,其中包括成骨細胞,脂肪細胞,軟骨細胞以及肌細胞。體外培養(yǎng)骨髓間充質(zhì)干細胞的相關(guān)研究中發(fā)現(xiàn),在合適的環(huán)境和條件下,骨髓間充質(zhì)干細胞能夠在體外進行34—42次的分裂增殖,增殖過程中始終保持梭形并呈漩渦狀排列。不僅如此,骨髓間充質(zhì)干細胞在冷凍保存進行復(fù)蘇后,仍然具備干細胞的特性。BMSCs不僅存在于骨髓中,還游離于骨組織外。當(dāng)其在適當(dāng)信號刺激時,可游離出骨組織,到達效應(yīng)靶器官組織中,這種生物學(xué)行為叫"歸巢"。在適當(dāng)?shù)纳锖臀锢硪蛩嘏c足夠的特異性信號傳導(dǎo)的存在,BMSCs能夠分化成所需的特定細胞組織。由于這些性質(zhì),BMSC已經(jīng)成為肌肉骨骼組織再生治療研究的主要細胞來源。BMSCs分化成骨細胞受細胞外信號和轉(zhuǎn)錄水平的級聯(lián)放大調(diào)控,這需要大量基因表達之間的精細時空調(diào)節(jié)。研究表明,原發(fā)性成骨分化誘導(dǎo)主要由Runt相關(guān)轉(zhuǎn)錄因子2(Runx2)關(guān)鍵轉(zhuǎn)錄因子調(diào)控。Runx2激活和調(diào)節(jié)成骨作為許多信號通路的靶基因,包括但不限于轉(zhuǎn)化生長因子-β(TGF-b1),骨形態(tài)發(fā)生蛋白(BMP),Winglesstype1(Wnt),Hedgehog(HH)和(Nel)樣蛋白1型(NELL-1)。誘導(dǎo)后,成骨細胞分化下游的前成骨細胞分化成成骨細胞,成骨細胞成熟和基質(zhì)礦化的過程受其他轉(zhuǎn)錄因子調(diào)節(jié),包括Osterix,BSP,OCN和OPN。這個過程也受到許多其他物理和生物因素的影響,其中大多數(shù)已經(jīng)在體外證明,并且仍然在仔細審查。影響B(tài)MSCs的增殖和成骨分化的物理因素,如高葡萄糖,已經(jīng)很好地建立并通過幾個體外研究證明。臨床證據(jù)表明,與正常人相比,糖尿病患者的骨折不愈合和延遲愈合更高,此外糖尿病患者早期骨質(zhì)疏松的發(fā)生率高。最近的體外研究已經(jīng)證明高葡萄糖微環(huán)境降低細胞BMP的表達,細胞BMP是成骨分化的終末期期間的重要調(diào)節(jié)蛋白,因此抑制該過程。進一步了解高葡萄糖對骨代謝的負(fù)面影響并設(shè)計減輕這種合并癥的方法的研究正在進行。核受體輔助抑制因子(nuclear receptor corepressor,NCoR)屬于核受體輔助抑制因子,通過與許多其它轉(zhuǎn)錄因子(包括NF-Eb,AKT和PPARα)相互作用而發(fā)揮各種抑制功能。近年來發(fā)現(xiàn)NCoR可以與HDAC結(jié)合成復(fù)合物,通過影響蛋白的轉(zhuǎn)錄,在機體代謝、炎癥以及腫瘤發(fā)生中扮演著重要角色。JinZ報道HDAC9與視黃酸和甲狀腺激素受體(SMRT)/NCoR共抑制因子的沉默介體協(xié)同抑制PPARγ活性,并將HDAC9鑒定為骨重建和骨骼穩(wěn)態(tài)的重要和生理學(xué)相關(guān)的調(diào)節(jié)劑。最近,YiQin證明NCoR在標(biāo)準(zhǔn)成骨培養(yǎng)基中通過P13K/AKT細胞信號通路調(diào)節(jié)大鼠間充質(zhì)干細胞的成骨分化。因此,在高葡萄糖微環(huán)境下,NCoR基因敲除對BMSCs的成骨分化的作用是什么?研究目的:觀察NCoRsiRNA對BMSCs在高葡萄糖微環(huán)境下增殖和成骨分化的影響。研究方法:1、根據(jù)NCoRsiRNA靶位點的選擇原則,從GenBank數(shù)據(jù)庫中擇優(yōu)選取三條siRNA序列,通過構(gòu)建干擾及過表達載體,篩選出最佳序列。2、檢測NCoR對BMSCs在高葡萄糖微環(huán)境下增殖的影響。在96孔板上按1×106/孔接種大鼠BMSCs,設(shè)置空白對照組、陰性對照組(無目標(biāo)siRNA和NCoRsiRNA組,每組設(shè)3復(fù)孔。從轉(zhuǎn)染后第1天開始,達到65%的匯合后,將細胞樣品在含有5.5,16.5,25和35mmol/L葡萄糖濃度的成骨培養(yǎng)基中培養(yǎng)。培養(yǎng)9天后,使用甲基噻唑基四唑(MTT)測定細胞增殖,并在490nm測定吸光度。3、觀察NCoR對大鼠BMSCs誘導(dǎo)成骨分化的影響。在含有25mmol/L(高葡萄糖)和5.5mmol/L葡萄糖(對照)的成骨培養(yǎng)基中分別培養(yǎng)用NCoRsiRNA或無目標(biāo)siRNA轉(zhuǎn)染的細胞21天,然后通過測量細胞ALP活性,鈣沉積RT-PCR檢測相關(guān)成骨基因;Run2,Osterix,OCN,OPN和BSP的表達,來測定成骨分化。結(jié)果:1、成功構(gòu)建了 NCoRsiRNA及無目標(biāo)siRNA質(zhì)粒載體,并篩選了最佳干擾序列,載體由廣州Cyagen生物科技公司合成。2、NCoRsiRNA可使BMSCs的增殖降低。用NCoRsiRNA或無目標(biāo)siRNA轉(zhuǎn)染的BMSCs在含有四個不同葡萄糖濃度(5.5,16.5,25和35mmol/L)的成骨培養(yǎng)基中培養(yǎng)。與對照組相比,第1d三組細胞增殖差異不大。從第3d開始,在所用葡萄糖濃度,NCoRsiRNA組細胞數(shù)量及增殖明顯低于對照組。提示NCoR可促進BMSCs增殖。3、NCoRsiRNA可在高葡萄糖下促進BMSCs的成骨分化。與對照組相比,通過ALP-ELISA試劑盒和鈣診斷試劑盒測定的,NCoRsiRNA組細胞的ALP活性以及礦化(鈣沉淀)在25mmol/L(高葡萄糖)和5.5mmol/L葡萄糖下都明顯增加。此外,實時RT-PCR對成骨分化相關(guān)的五個基因Run2,Osterix,OCN,OPN和BSP的檢測顯示,與對照組相比,NCoRsiRNA組Run2,Osterix,OCN,OPN和BSP表達都顯著增加(P0.01)。提示NCoR負(fù)性調(diào)控大鼠BMSCs的成骨分化。結(jié)論:我們利用RNA干擾技術(shù)成功設(shè)計并構(gòu)建了 NCoR基因特異性的小干擾siRNA,以及設(shè)計并構(gòu)建了無目標(biāo)siRNA。經(jīng)證實,基因沉默的效果良好。我們成功將NCoRsiRNA和無目標(biāo)siRNA轉(zhuǎn)染至大鼠骨髓間充質(zhì)干細胞內(nèi),并證實轉(zhuǎn)染效率較高,然后在四個不同葡萄糖濃度下(5.5,16.5,25和35mmol/L)的成骨培養(yǎng)基中培養(yǎng)。然后檢測BMSCs的增殖發(fā)現(xiàn),NCoRsiRNA組BMSCs的增殖明顯下降,提示NCoR可促進BMSCs的增殖。沉默NCoR時,在正常(5.5mmol/L)和高糖(25mmol/L)濃度下,大鼠BMSCs誘導(dǎo)成骨分化,成骨細胞分化標(biāo)志基因Run2,Osterix,OCN,OPN和BSP的表達明顯增強。提示NCoR負(fù)性調(diào)控大鼠BMSCs的成骨分化。結(jié)合糖濃度分析,NCoR siRNA可中和高糖對大鼠BMSCs成骨分化的抑制作用。綜合既往研究,NCoR siRNA能改善機體胰島素敏感性,NCoR可作為潛在靶點,在糖尿病病人中改善機體胰島素敏感性,降糖的同時,保護糖尿病患者骨質(zhì)疏松。
[Abstract]:Background: bone marrow mesenchymal stem cells (BMSCs) have the ability to differentiate into different cellular tissues, including osteoblasts, adipocytes, chondrocytes, and myocytes. In the study of bone marrow mesenchymal stem cells in vitro, bone marrow mesenchymal stem cells can carry out 34 - 42 in the suitable environment and conditions. In addition, after the resuscitation of cryopreservation, bone marrow mesenchymal stem cells still possess the characteristics of stem cells, not only in the bone marrow, but also free from the bone tissue. When the stem cells are stimulated by proper signal,.BMSCs can free the bone tissue and reach the effect target. In the official organization, this biological behavior is called "homing". In the presence of adequate biological and physical factors and sufficient specific signal transduction, BMSCs can differentiate into specific cellular tissues required. Because of these properties, BMSC has become a major cell source for the study of musculoskeletal tissue regeneration,.BMSCs differentiation into osteoblasts from cells. Cascade amplification regulation of signal and transcriptional levels requires fine space-time regulation between a large number of gene expressions. Studies have shown that primary osteogenic differentiation is induced by Runt related transcription factor 2 (Runx2) key transcription factors to regulate.Runx2 activation and regulation of bone formation as a target gene for many signaling pathways, including but not limited to transformation growth factors. Sub beta (TGF-b1), bone morphogenetic protein (BMP), Winglesstype1 (Wnt), Hedgehog (HH) and (Nel) like protein 1 (NELL-1). After induction, osteoblasts are differentiated into osteoblasts downstream of osteoblast differentiation, and the process of osteoblast maturation and matrix mineralization is regulated by other transcription factors, including Osterix, BSP, OCN, and OPN.. The effects of many other physical and biological factors, most of which have been proven in vitro, are still under scrutiny. Physical factors affecting the proliferation and osteogenesis of BMSCs, such as Hyperglucose, have been well established and proved by several in vitro studies. Clinical evidence suggests that the fracture nonunion of diabetic patients is compared with normal people. And delayed healing is higher, in addition to the high incidence of early osteoporosis in diabetic patients. Recent in vitro studies have shown that hyperglycemic microenvironment reduces the expression of cell BMP, and cell BMP is an important regulatory protein during the end stage of osteogenic differentiation, thus inhibiting the process. The study of methods designed to mitigate this complication is ongoing. Nuclear receptor corepressor (NCoR) is a nuclear receptor auxiliary inhibitor, which plays a variety of inhibitory functions by interacting with many other transcription factors (including NF-Eb, AKT and PPAR alpha). In recent years, it is found that NCoR can be combined with HDAC. Compounds, by influencing the transcription of protein, play an important role in body metabolism, inflammation, and oncology,.JinZ reports that HDAC9 is synergistic to inhibit PPAR gamma activity with retinoic acid and thyroid hormone receptor (SMRT) /NCoR co suppressor, and to identify HDAC9 as an important and physiological regulation of bone remodeling and bone homeostasis Recently, YiQin has shown that NCoR regulates the osteogenic differentiation of rat mesenchymal stem cells by P13K/AKT cell signaling pathway in the standard osteogenic medium. So, what is the effect of NCoR knockout on the osteogenic differentiation of BMSCs in the Hyperglucose microenvironment? The purpose of this study was to observe the proliferation and formation of BMSCs in the Hyperglucose microenvironment of BMSCs. The effect of bone differentiation. 1. According to the selection principle of NCoRsiRNA target site, three siRNA sequences were selected from the GenBank database, and the optimal sequence.2 was screened by constructing the interference and overexpression vector. The effect of NCoR on the proliferation of BMSCs in the hyperglycemic microenvironment was detected. The rat BMSCs was inoculated on the 96 orifice plate by 1 x 106/ holes. In the blank control group, the negative control group (no target siRNA and NCoRsiRNA group, each set 3 compound holes. After first days after transfection, the cell samples were cultured in the osteogenic medium containing 5.5,16.5,25 and 35mmol/L glucose concentration. After 9 days, the cell proliferation was measured using methyl thiazolidazole four azoles (MTT), and 490nm was measured. " The effect of NCoR on the osteogenic differentiation induced by BMSCs in rats was observed. The cells transfected with NCoRsiRNA or non target siRNA were cultured in the osteogenic medium containing 25mmol/L (high glucose) and 5.5mmol/L glucose (control) for 21 days, and then by measuring the cell ALP activity, calcium deposition RT-PCR was used to detect the related osteogenic genes; Run2, Osterix, OCN, and OCN. The expression of PN and BSP to determine osteogenic differentiation. Results: 1, NCoRsiRNA and non target siRNA plasmid vectors were successfully constructed, and the optimal interference sequence was screened. The carrier was synthesized from.2 in Guangzhou Cyagen biotechnology company, NCoRsiRNA could reduce the proliferation of BMSCs. BMSCs in NCoRsiRNA or non target siRNA was contained in four different glucose concentrations ( 5.5,16.5,25 and 35mmol/L were cultured in the osteogenic medium. Compared with the control group, there was little difference in cell proliferation in the group 1D Three. From 3D, the number and proliferation of cells in the NCoRsiRNA group were significantly lower than that in the control group. It suggests that NCoR can promote BMSCs proliferation.3, NCoRsiRNA can promote the osteogenesis of BMSCs under high glucose. Compared with the ALP-ELISA kit and the calcium diagnostic kit, the ALP activity of the NCoRsiRNA group and the mineralization (calcium precipitation) were significantly increased under 25mmol/L (high glucose) and 5.5mmol/L glucose. In addition, the real-time RT-PCR for the five genes related to osteogenesis differentiation, Run2, Osterix, OCN, OPN and BSP, showed that the control group was with the control group. The expression of Run2, Osterix, OCN, OPN and BSP in the NCoRsiRNA group increased significantly (P0.01). It suggests that NCoR negatively regulates the osteogenic differentiation of BMSCs in rats. Conclusion: we successfully designed and constructed the small interference siRNA of NCoR gene by RNA interference technique, and the design and construction of a non target gene have proved that the effect of gene silencing is good. NCoRsiRNA and non target siRNA were successfully transfected into rat bone marrow mesenchymal stem cells and confirmed that the transfection efficiency was high and then cultured in the osteogenic medium of four different glucose concentrations (5.5,16.5,25 and 35mmol/L). Then the proliferation of BMSCs was detected, and the proliferation of BMSCs in the NCoRsiRNA group decreased significantly, suggesting that NCoR promoted the increase of BMSCs. At the concentration of normal (5.5mmol/L) and high glucose (25mmol/L), rat BMSCs induced osteogenic differentiation, and the expression of osteoblast differentiation marker gene Run2, Osterix, OCN, OPN and BSP increased obviously at the concentration of normal (5.5mmol/L) and high glucose (25mmol/L), suggesting that NCoR negatively regulated the osteogenic differentiation of BMSCs rats. In a comprehensive study, NCoR siRNA can improve the body's insulin sensitivity. NCoR can be a potential target for improving the body's insulin sensitivity and reducing glucose in diabetic patients, while protecting the osteoporosis in diabetic patients.

【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】：R587.1;R580

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