MircoRNA-27a靶向调控BMSCs分化预防激素性股骨头坏死的实验研究

发布时间：2018-03-05 16:51

本文选题：股骨头坏死　切入点：过氧化物酶体增殖子活化受体-γ　出处：《郑州大学》2015年硕士论文　论文类型：学位论文

【摘要】：背景与目的:激素性股骨头坏死(osteonecrosis of the femoral head,ONFH)有极高的致残率,其预防和治疗已经成为世界性的难题。激素性ONFH发生可能是由于激素的作用改变了细胞中的一些mi RNA表达,影响了相应靶向基因调控作用,从而使正常骨髓间充质干细胞(Bone marrow mesenhymal stem cells,BMSCs)的分化受到影响,造成了大量脂肪细胞的生成,和成骨细胞形成的大量减少,影响了正常骨细胞的修复。因此促进成骨细胞的分化和减少成脂细胞的分化是我们预防和治疗激素性ONFH最要的环节。以往的研究通过单一基因的改变来预防骨坏死,而本研究主要是利用mi RNA具有多个靶向位点,筛选出具有抑制成脂肪分化同时具有促进成骨分化的Micro RNA-27a基因,采用目前较成熟的RNA干扰(RNA interference,RNAi)技术,通过将双向调控基因Micro RNA-27a电转入大鼠BMSCs中,运用细胞学、生物化学和分子生物学等多种的检测手段和方法检测该基因对于成骨和成脂的影响。在国内外的报道中,尚未有mi RNA双靶向基因调控预防激素性ONFH。材料与方法:选用2~3月龄的SD大鼠,体重约为180~200g,处死后取骨髓细胞,培养并制备大鼠BMSCs。将实验分为4组,Micro RNA-27a组(mi RNA1,M):将具有双向靶向作用的Micro RNA-27a电转入大鼠BMSCs中并且加入激素诱导;阴性对照组(Negative Control,NC):将无效序列的基因电转入大鼠BMSCs中并在培养基中加入激素诱导;激素模型组(Model,M):将BMSCs单纯的给予激素诱导;正常对照组(Normal,N):无需特殊因素处理;对于激素诱导的各组细胞中保持地塞米松浓度为10-7mol/L,按时更换细胞液并定期观察细胞的形态及。在激素诱导第3天和第7天时,采用实时荧光定量-聚合酶链反应(RT-q PCR)技术分别测定各实验组中的PPARγm RNA、BMP m RNA的相对表达量。在激素诱导14天时,采取ELISA试剂盒测定各实验组中的骨钙素分泌量和甘油三脂的含量。在诱导14天时,对各组细胞进行免疫组化,记录并分析各组PPARγ、BMP蛋白表达水平。诱导21天时用油红O方法对各组细胞进行细胞染色,用倒置显微镜观察并记录脂肪细胞的数量。结果1各实验组PPARγm RNA和BMP m RNA表达检测结果:实验第3天和第7天,测得阴性对照组和激素模型组中的PPARγm RNA呈高表达,与正常对照组相比较,差异值有统计学意义(P0.05)。Micro RNA-27a组中的PPARγm RNA呈低表达,与正常对照组无统计学差异(P0.05)。阴性对照组和激素模型组中的BMP m RNA呈低表达,与正常对照组差异均有统计学意义(P0.05)。Micro RNA-27a组中的BMP m RNA呈高表达,表达水平与正常对照组无统计学差异。(P0.05)。2各实验组BMSCs脂肪染色和脂肪细胞计数结果:实验第21天,进行油红O染色并计数,与正常对照组相比较,激素模型组以及阴性对照组中脂滴数量升高增多,两者的差异有统计学意义(P0.05)。Micro RNA-27a组中脂滴量稍稍多于正常对照组,所检测的差异值均无统计学意义(P0.05);且脂滴量明显低于阴性对照组和激素模型组,差异均有统计学意义(P0.05)。3各实验组骨钙素含量和甘油三酯含量检测结果:实验第14天,与激素模型组和阴性对照组与与正常对照组相比较,骨钙素含量降低,甘油三脂含量升高,差异均有统计学意义(P0.05)。Micro RNA-27a组检测的骨钙素含量稍低于正常对照组,甘油三脂含量稍高于正常对照组,差异值均无统计学意义(P0.05)。4各实验组免疫组化测定PPARγ以及BMP-2蛋白表达MIOD结果:实验14天,通过免疫组化测得阴性对照组和激素模型组中的PPARγ表达量高,与正常对照组差异均有统计学意义(P0.05)。Micro RNA-27a组中的PPARγ表达量低,与正常对照组间的差异无统计学意义(P0.05)。阴性对照组和激素模型组中的BMP表达低,与正常对照组差异均有统计学意义(P0.05)。Micro RNA-27a中的BMP表达量高,表达水平与正常对照组无统计学差异(P0.05)。结论1.Micro RNA-27a能抑制成脂基因的表达,促进成骨基因的表达。2.依据Micro RNA-27a的多向靶向调控特性来预防并干预激素性ONFH,为预防激素性ONFH提供了新的方法和思路。
[Abstract]:Background and objective: steroid induced avascular necrosis of femoral head (osteonecrosis of the femoral head, ONFH) has a high disability rate, its prevention and treatment has become a worldwide problem. The occurrence of ONFH may be due to steroid hormones changed Mi expression in RNA cells, affecting the corresponding target gene regulation thus, normal bone marrow mesenchymal stem cells (Bone marrow mesenhymal stem cells, BMSCs) differentiation affected, resulting in a large number of fat cells, and reduce the amount of osteoblast formation, the repair effect of normal bone cells. Thus promote osteoblast differentiation and adipogenic differentiation is reduced the prevention and treatment of steroid induced ONFH to link. Most previous studies changed by a single gene to prevent bone necrosis, the purpose of this study is to use mi RNA with multiple targets, and screened with. At the same time, adipose differentiation can promote the formation of Micro RNA-27a gene differentiation, using the mature RNA interference (RNA interference RNAi) technology, the bidirectional regulation of gene Micro RNA-27a into rat BMSCs, using cytology, detection means and methods of Biochemistry and molecular biology of the gene to bone and adipogenic effects. Reports at home and abroad, yet mi RNA dual targeting gene regulation to prevent steroid induced ONFH. materials and methods: SD rats were 2~3 months old, weighing about 180~200g, bone marrow cells were taken, cultured and prepared BMSCs. rats were divided into 4 groups, Micro group RNA-27a (MI RNA1 M): with bidirectional targeting Micro RNA-27a electroporated into hormone induction and adding BMSCs in rats; negative control group (Negative, Control, NC): invalid gene electric into the rat BMSCs sequence and in culture Adding hormone induction medium; hormone model group (Model, M): BMSCs pure hormone induction; the normal control group (Normal, N): there is no special treatment for hormone induced factors; the cells were maintained in dexamethasone concentration is 10-7mol/L, the replacement of cell sap and regular observation of cell morphology and in time. The hormone induced for third days and seventh days, using real-time fluorescence quantitative polymerase chain reaction (RT-q PCR) were measured by the PPAR gamma m RNA in every experimental group, the relative expression of BMP m RNA. In hormone induced 14 days, the content of ELISA kit were taken in the experimental group and the osteocalcin secretion glycerin three greases. In 14 days of induction, immunohistochemical analysis of cells in each group, each group of PPAR and gamma records, the expression of BMP protein. After 21 days induction oil red O method for each group of cells stained by inverted microscope and recorded fat cells The number of cells in each experimental group. Results of the 1 PPAR m RNA and BMP m gamma RNA expression detection results: third days and seventh days, measured the negative control expression of PPAR gamma m RNA group and the hormone in the model group were compared with normal control group, the difference was statistically significant (P0.05) PPAR gamma m RNA.Micro in the RNA-27a group showed low expression, and the normal control group showed no significant difference (P0.05). BMP m RNA negative control group and the hormone in the model group showed low expression, and the normal control group had significant difference (P0.05) high expression of BMP m RNA.Micro in the RNA-27a group showed expression and the normal control group showed no significant difference (P0.05). The.2 of each experimental group BMSCs fat fat staining and cell counting results: on the twenty-first day, oil red O staining and counting, compared with normal control group, hormone group and negative control group in the number of lipid droplets increased increased, the difference between the two is Statistical significance (P0.05).Micro RNA-27a group, lipid droplet content is a little more than the normal control group, the difference value detection had no statistical significance (P0.05); and lipid droplets were significantly lower than that in the negative control group and hormone group, the differences were statistically significant (P0.05).3 test results of each experiment group osteocalcin content and triglyceride content. The fourteenth day of the experiment group and compared with the normal control group and model group and negative control hormone, osteocalcin content decreased, increased glycerin three fat content, the differences were statistically significant (P0.05).Micro group RNA-27a bone calcium content detection is slightly lower than the normal control group, glycerin three fat content is slightly higher than the normal control group, the difference value there was no statistical significance (P0.05.4) in the experimental group were determined by immunohistochemistry. The expression of BMP-2 protein and PPAR gamma MIOD results: the 14 day of the experiment to the negative control group and model group of PPAR hormone gamma table measured by immunohistochemistry As the amount is high, and the normal control group had significant difference (P0.05) PPAR gamma.Micro in RNA-27a group is low expression, no statistically significant differences between the normal control group (P0.05). The negative control group and hormone in the model group the expression of BMP is low, and the normal control group showed significant difference (P0.05.Micro) RNA-27a BMP expression levels in high expression level and the normal control group showed no significant difference (P0.05). Conclusion 1.Micro RNA-27a can inhibit the expression of lipid gene, promote osteogenic gene expression of multi target.2. according to the Micro RNA-27a to the special regulation to prevention and intervention of steroid ONFH, provides methods and ideas new for the prevention of steroid induced ONFH.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R681.8

【共引文献】