發(fā)布時間：2018-05-24 16:35

  本文選題：高脂血癥 + BMSCs��； 參考：《山東大學》2017年碩士論文

【摘要】：背景高脂血癥在人群中的患病率高,可以引起骨質(zhì)疏松,而且可以影響種植體骨整合,其對骨代謝的不良影響已經(jīng)越來越多的被發(fā)現(xiàn)。本小組前期研究發(fā)現(xiàn),高脂血癥大鼠種植周圍骨小梁參數(shù)降低,骨體積分數(shù)減少,骨接觸率降低,骨整合不良,且高脂血癥大鼠種植體周圍骨組織中多個分子表達發(fā)生變化,這些分子多涉及Wnt通路。Wnt通路中這些發(fā)生變化的分子也可能引起骨整合不良。MicroRNA(miRNA,微小RNA)在細胞分化、組織發(fā)育的不同階段具有特異性的表達,主要參與基因轉(zhuǎn)錄后的調(diào)節(jié)。其中microRNA-29家族是一類非常重要的microRNA基因家族,在腫瘤、癌癥,糖尿病、骨質(zhì)疏松以及心血管疾病等多種疾病中發(fā)揮作用。目的本實驗旨在探究miR-29a-3p對高脂環(huán)境下大鼠骨髓間充質(zhì)細胞成骨分化和高脂血癥大鼠種植體骨整合的影響。方法1.體外實驗1.1大鼠BMSCs的獲取及培養(yǎng)提取大鼠BMSCs,培養(yǎng)傳代,第三代細胞進行成骨和成脂誘導,分別進行茜素紅和油紅O染色,鑒別大鼠BMSCs,用于后續(xù)實驗。1.2大鼠BMSCs成骨分化過程中miR-29a-3p表達變化實驗分為高脂組和普通組,高脂組用高脂成骨誘導液誘導,普通組用傳統(tǒng)成骨誘導液進行成骨誘導。成骨誘導3、5、7和14天RT-PCR檢測miR-29a-3p的表達量。1.3 miR-29a-3p對高脂環(huán)境下大鼠BMSCs成骨分化的作用利用miR-29a-3p mmics和miR-29a-3p inhibitor過表達或者低表達大鼠骨髓間充質(zhì)干細胞中的miR-29a-3p,RT-PCR和Western Blotting檢測高脂成骨誘導液成骨誘導過程中大鼠BMSCs中成骨標志基因ALP、Runx2的表達變化。2.體內(nèi)實驗2.1建立高脂血癥大鼠動物模型成年雄性Wistar大鼠隨機分組,高脂組高脂飲食連續(xù)飼喂6周,建立高脂血癥大鼠動物模型,普通組給予普通飲食。2.2種植體周圍骨組織中miR-29a-3p表達量。兩組大鼠雙側(cè)股骨植入種植體,術(shù)后5、10、15和20天處死,提取種植體周圍1mm內(nèi)骨組織的總RNA,用熒光定量PCR技術(shù)檢測miR-29a-3p的表達變化2.3 miR-29a-3p在高脂血癥大鼠種植體骨整合中的作用實驗組利用慢病毒載體過表達高脂血癥大鼠體內(nèi)的miR-29a-3p,對照組注射陰性對照注射液。RT-PCR和Western Blotting檢測大鼠種植體周圍骨組織中成骨標志基因ALP、Runx2的表達變化。結(jié)果1.大鼠BMSCs成骨誘導過程中,miR-29a-3p表達量5天比3天高、7天比5天高,14天比7天表達量下降,高脂組和普通組表達趨勢相同。3、5、7和14天高脂組miR-29a-3p的表達量均低于普通組(p0.05)。2.過表達大鼠骨髓間充質(zhì)干細胞中的miR-29a-3p,成骨標志性基因ALP、Runx2表達量升高(p0.05);低表達大鼠骨髓間充質(zhì)干細胞中的miR-29a-3p后,ALP、Runx2 表達減少(p0.05)。3.大鼠種植體植入后,miR-29a-3p的表達量10天比5天高,15天與10天相比表達減少,20天比15天表達減少,高脂組和普通組表達趨勢相同,高脂組miR-29a-3p表達量明顯低于普通組(p0.05)。4.利用慢病毒載體過表達高脂血癥大鼠體內(nèi)的miR-29a-3p后,種植體周圍骨組織中成骨標志基因ALP、Runx2的表達升高(p0.05)。結(jié)論1.miR-29a-3p對高脂環(huán)境下大鼠骨髓間充質(zhì)干細成骨分化過程起正向調(diào)節(jié)作用。2.miR-29a-3p能夠促進高脂血癥大鼠種植體周圍成骨標志基因的表達,可能有利于骨結(jié)合的形成。
[Abstract]:Background hyperlipidemia has a high prevalence in the population, which can cause osteoporosis and can affect bone integration. The adverse effects on bone metabolism have been more and more found. Earlier studies in this group found that the parameters of the bone trabecula in the hyperlipidemic rats were lower, bone volume fraction, bone contact rate decreased, and bone integration. The expression of multiple molecules in the bone tissue around the implants of hyperlipidemia rats, these molecules are mostly involved in Wnt pathway.Wnt pathway, and these molecules may also cause bone malintegration.MicroRNA (miRNA, small RNA) in cell differentiation, different stages of tissue development with specific expression, mainly involved in the gene. Post transcriptional regulation. In which the microRNA-29 family is a very important family of microRNA genes that play a role in a variety of diseases, such as cancer, cancer, diabetes, osteoporosis, and cardiovascular disease. The purpose of this experiment was to explore miR-29a-3p for the cultivation of bone marrow mesenchymal cells and hyperlipidemia rats in rats with high lipid environment. Method 1. in vitro experiment 1. in vitro, 1.1 rats were obtained and cultured in vitro. The rats were cultured and cultured to extract BMSCs, culture generation and third generation of cells to induce osteogenesis and lipid induction. Alizarin red and oil red O were used to identify the rat BMSCs. The experiment of miR-29a-3p expression in the process of BMSCs osteogenesis in.1.2 rats was divided into high fat. Group and general group, high fat group was induced by high lipo osseous induction, common group was induced by traditional osteogenic inducer. 3,5,7 and 14 days RT-PCR were used to detect miR-29a-3p expression by osteogenesis and.1.3 miR-29a-3p on BMSCs osteogenesis in hyperlipidemic rats using miR-29a-3p MMICs and miR-29a-3p inhibitor over expression or low table MiR-29a-3p, RT-PCR and Western Blotting in rat bone marrow mesenchymal stem cells (MSCs) were used to detect the osteogenic marker gene ALP in rat BMSCs during the induction of osteogenesis induced by high lipogenic bone marrow, and the expression of Runx2 was changed in.2. in vivo 2.1 to establish a rat model of hyperlipidemia rats randomly divided into adult male rats, and the high fat diet was continuous in the high fat group. The animal model of hyperlipidemia rats was established for 6 weeks. The expression of miR-29a-3p in the bone tissue around the common diet.2.2 implant was given in the ordinary group. The two groups of rats were implanted with the implants, 5,10,15 and 20 days after the operation. The total RNA of the bone tissue around the 1mm was extracted. The expression of miR-29a-3p was detected by the fluorescence quantitative PCR technique 2.3. The effect of miR-29a-3p on the implant bone integration in hyperlipidemia rats by using lentivirus vector over expression of miR-29a-3p in hyperlipidemia rats. The control group was injected with negative control injection.RT-PCR and Western Blotting to detect the osteogenic marker gene ALP and Runx2 expression in the bone tissue around the implant. Results 1. rat BMS In the process of Cs osteogenesis, the expression of miR-29a-3p was higher than that of 3 days, 7 days, 5 days, 14 days and 7 days, the expression of miR-29a-3p in the high fat group and the common group.3,5,7 and 14 day high fat group were lower than the miR-29a-3p in the normal group (P0.05).2. overexpressed rat bone marrow mesenchymal stem cells, ALP, Runx2 Expression increased (P0.05); after miR-29a-3p in bone marrow mesenchymal stem cells of low expression rat, ALP, Runx2 expression decreased (P0.05).3. rat implant implantation, miR-29a-3p expression was 10 days higher than that of 5 days, 15 days compared with 10 days, 20 days was less than 15 days, high fat group and ordinary group expression trend was the same, high fat group miR-29a-3p table The amount of P0.05.4. was significantly lower than that of the common group (P0.05).4. using lentivirus vector over expression of hyperlipidemia rats. The osteogenic marker gene in the bone tissue around the implant was ALP, and the expression of Runx2 increased (P0.05). Conclusion 1.miR-29a-3p plays a positive regulatory role in the differentiation process of bone marrow mesenchymal stem osteogenesis in hyperlipidemic rats. P can promote the expression of osteogenic marker genes in the periosseous tissue of hyperlipidemic rats, and may be beneficial to the formation of osseointegration.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R783.6;R589.2

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本文編號：1929795