MiR-29a-3p对高脂血症大鼠种植体骨整合的影响

发布时间：2018-05-24 16:35

本文选题：高脂血症 + BMSCs　；参考：《山东大学》2017年硕士论文

【摘要】：背景高脂血症在人群中的患病率高,可以引起骨质疏松,而且可以影响种植体骨整合,其对骨代谢的不良影响已经越来越多的被发现。本小组前期研究发现,高脂血症大鼠种植周围骨小梁参数降低,骨体积分数减少,骨接触率降低,骨整合不良,且高脂血症大鼠种植体周围骨组织中多个分子表达发生变化,这些分子多涉及Wnt通路。Wnt通路中这些发生变化的分子也可能引起骨整合不良。MicroRNA(miRNA,微小RNA)在细胞分化、组织发育的不同阶段具有特异性的表达,主要参与基因转录后的调节。其中microRNA-29家族是一类非常重要的microRNA基因家族,在肿瘤、癌症,糖尿病、骨质疏松以及心血管疾病等多种疾病中发挥作用。目的本实验旨在探究miR-29a-3p对高脂环境下大鼠骨髓间充质细胞成骨分化和高脂血症大鼠种植体骨整合的影响。方法1.体外实验1.1大鼠BMSCs的获取及培养提取大鼠BMSCs,培养传代,第三代细胞进行成骨和成脂诱导,分别进行茜素红和油红O染色,鉴别大鼠BMSCs,用于后续实验。1.2大鼠BMSCs成骨分化过程中miR-29a-3p表达变化实验分为高脂组和普通组,高脂组用高脂成骨诱导液诱导,普通组用传统成骨诱导液进行成骨诱导。成骨诱导3、5、7和14天RT-PCR检测miR-29a-3p的表达量。1.3 miR-29a-3p对高脂环境下大鼠BMSCs成骨分化的作用利用miR-29a-3p mmics和miR-29a-3p inhibitor过表达或者低表达大鼠骨髓间充质干细胞中的miR-29a-3p,RT-PCR和Western Blotting检测高脂成骨诱导液成骨诱导过程中大鼠BMSCs中成骨标志基因ALP、Runx2的表达变化。2.体内实验2.1建立高脂血症大鼠动物模型成年雄性Wistar大鼠随机分组,高脂组高脂饮食连续饲喂6周,建立高脂血症大鼠动物模型,普通组给予普通饮食。2.2种植体周围骨组织中miR-29a-3p表达量。两组大鼠双侧股骨植入种植体,术后5、10、15和20天处死,提取种植体周围1mm内骨组织的总RNA,用荧光定量PCR技术检测miR-29a-3p的表达变化2.3 miR-29a-3p在高脂血症大鼠种植体骨整合中的作用实验组利用慢病毒载体过表达高脂血症大鼠体内的miR-29a-3p,对照组注射阴性对照注射液。RT-PCR和Western Blotting检测大鼠种植体周围骨组织中成骨标志基因ALP、Runx2的表达变化。结果1.大鼠BMSCs成骨诱导过程中,miR-29a-3p表达量5天比3天高、7天比5天高,14天比7天表达量下降,高脂组和普通组表达趋势相同。3、5、7和14天高脂组miR-29a-3p的表达量均低于普通组(p0.05)。2.过表达大鼠骨髓间充质干细胞中的miR-29a-3p,成骨标志性基因ALP、Runx2表达量升高(p0.05);低表达大鼠骨髓间充质干细胞中的miR-29a-3p后,ALP、Runx2 表达减少(p0.05)。3.大鼠种植体植入后,miR-29a-3p的表达量10天比5天高,15天与10天相比表达减少,20天比15天表达减少,高脂组和普通组表达趋势相同,高脂组miR-29a-3p表达量明显低于普通组(p0.05)。4.利用慢病毒载体过表达高脂血症大鼠体内的miR-29a-3p后,种植体周围骨组织中成骨标志基因ALP、Runx2的表达升高(p0.05)。结论1.miR-29a-3p对高脂环境下大鼠骨髓间充质干细成骨分化过程起正向调节作用。2.miR-29a-3p能够促进高脂血症大鼠种植体周围成骨标志基因的表达,可能有利于骨结合的形成。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.6;R589.2

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