本文選題：股骨頭壞死　切入點：過氧化物酶體增殖子活化受體-γ　出處：《鄭州大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：背景與目的:激素性股骨頭壞死(osteonecrosis of the femoral head,ONFH)有極高的致殘率,其預(yù)防和治療已經(jīng)成為世界性的難題。激素性O(shè)NFH發(fā)生可能是由于激素的作用改變了細(xì)胞中的一些mi RNA表達(dá),影響了相應(yīng)靶向基因調(diào)控作用,從而使正常骨髓間充質(zhì)干細(xì)胞(Bone marrow mesenhymal stem cells,BMSCs)的分化受到影響,造成了大量脂肪細(xì)胞的生成,和成骨細(xì)胞形成的大量減少,影響了正常骨細(xì)胞的修復(fù)。因此促進(jìn)成骨細(xì)胞的分化和減少成脂細(xì)胞的分化是我們預(yù)防和治療激素性O(shè)NFH最要的環(huán)節(jié)。以往的研究通過單一基因的改變來預(yù)防骨壞死,而本研究主要是利用mi RNA具有多個靶向位點,篩選出具有抑制成脂肪分化同時具有促進(jìn)成骨分化的Micro RNA-27a基因,采用目前較成熟的RNA干擾(RNA interference,RNAi)技術(shù),通過將雙向調(diào)控基因Micro RNA-27a電轉(zhuǎn)入大鼠BMSCs中,運(yùn)用細(xì)胞學(xué)、生物化學(xué)和分子生物學(xué)等多種的檢測手段和方法檢測該基因?qū)τ诔晒呛统芍挠绊憽Ｔ趪鴥?nèi)外的報道中,尚未有mi RNA雙靶向基因調(diào)控預(yù)防激素性O(shè)NFH。材料與方法:選用2~3月齡的SD大鼠,體重約為180~200g,處死后取骨髓細(xì)胞,培養(yǎng)并制備大鼠BMSCs。將實驗分為4組,Micro RNA-27a組(mi RNA1,M):將具有雙向靶向作用的Micro RNA-27a電轉(zhuǎn)入大鼠BMSCs中并且加入激素誘導(dǎo);陰性對照組(Negative Control,NC):將無效序列的基因電轉(zhuǎn)入大鼠BMSCs中并在培養(yǎng)基中加入激素誘導(dǎo);激素模型組(Model,M):將BMSCs單純的給予激素誘導(dǎo);正常對照組(Normal,N):無需特殊因素處理;對于激素誘導(dǎo)的各組細(xì)胞中保持地塞米松濃度為10-7mol/L,按時更換細(xì)胞液并定期觀察細(xì)胞的形態(tài)及。在激素誘導(dǎo)第3天和第7天時,采用實時熒光定量-聚合酶鏈反應(yīng)(RT-q PCR)技術(shù)分別測定各實驗組中的PPARγm RNA、BMP m RNA的相對表達(dá)量。在激素誘導(dǎo)14天時,采取ELISA試劑盒測定各實驗組中的骨鈣素分泌量和甘油三脂的含量。在誘導(dǎo)14天時,對各組細(xì)胞進(jìn)行免疫組化,記錄并分析各組PPARγ、BMP蛋白表達(dá)水平。誘導(dǎo)21天時用油紅O方法對各組細(xì)胞進(jìn)行細(xì)胞染色,用倒置顯微鏡觀察并記錄脂肪細(xì)胞的數(shù)量。結(jié)果1各實驗組PPARγm RNA和BMP m RNA表達(dá)檢測結(jié)果:實驗第3天和第7天,測得陰性對照組和激素模型組中的PPARγm RNA呈高表達(dá),與正常對照組相比較,差異值有統(tǒng)計學(xué)意義(P0.05)。Micro RNA-27a組中的PPARγm RNA呈低表達(dá),與正常對照組無統(tǒng)計學(xué)差異(P0.05)。陰性對照組和激素模型組中的BMP m RNA呈低表達(dá),與正常對照組差異均有統(tǒng)計學(xué)意義(P0.05)。Micro RNA-27a組中的BMP m RNA呈高表達(dá),表達(dá)水平與正常對照組無統(tǒng)計學(xué)差異。(P0.05)。2各實驗組BMSCs脂肪染色和脂肪細(xì)胞計數(shù)結(jié)果:實驗第21天,進(jìn)行油紅O染色并計數(shù),與正常對照組相比較,激素模型組以及陰性對照組中脂滴數(shù)量升高增多,兩者的差異有統(tǒng)計學(xué)意義(P0.05)。Micro RNA-27a組中脂滴量稍稍多于正常對照組,所檢測的差異值均無統(tǒng)計學(xué)意義(P0.05);且脂滴量明顯低于陰性對照組和激素模型組,差異均有統(tǒng)計學(xué)意義(P0.05)。3各實驗組骨鈣素含量和甘油三酯含量檢測結(jié)果:實驗第14天,與激素模型組和陰性對照組與與正常對照組相比較,骨鈣素含量降低,甘油三脂含量升高,差異均有統(tǒng)計學(xué)意義(P0.05)。Micro RNA-27a組檢測的骨鈣素含量稍低于正常對照組,甘油三脂含量稍高于正常對照組,差異值均無統(tǒng)計學(xué)意義(P0.05)。4各實驗組免疫組化測定PPARγ以及BMP-2蛋白表達(dá)MIOD結(jié)果:實驗14天,通過免疫組化測得陰性對照組和激素模型組中的PPARγ表達(dá)量高,與正常對照組差異均有統(tǒng)計學(xué)意義(P0.05)。Micro RNA-27a組中的PPARγ表達(dá)量低,與正常對照組間的差異無統(tǒng)計學(xué)意義(P0.05)。陰性對照組和激素模型組中的BMP表達(dá)低,與正常對照組差異均有統(tǒng)計學(xué)意義(P0.05)。Micro RNA-27a中的BMP表達(dá)量高,表達(dá)水平與正常對照組無統(tǒng)計學(xué)差異(P0.05)。結(jié)論1.Micro RNA-27a能抑制成脂基因的表達(dá),促進(jìn)成骨基因的表達(dá)。2.依據(jù)Micro RNA-27a的多向靶向調(diào)控特性來預(yù)防并干預(yù)激素性O(shè)NFH,為預(yù)防激素性O(shè)NFH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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R681.8

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