本文選題：激素性 + 股骨頭壞死��； 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：研究背景及目的激素性股骨頭壞死(steroid-induced femoral head necrosis, SFHN)是由于長期使用激素類藥物,導(dǎo)致以股骨頭血供受損,骨細(xì)胞及骨髓成份死亡,繼而導(dǎo)致股骨頭結(jié)構(gòu)改變、股骨頭塌陷、關(guān)節(jié)功能障礙為特征的代謝性疾病。大約75%的患者從診斷SFHN起3年內(nèi)便可發(fā)生股骨頭塌陷,最終超過65%的患者不得不接受髖關(guān)節(jié)置換手術(shù),給患者家庭和社會帶來了巨大經(jīng)濟(jì)負(fù)擔(dān)。探索SFHN的發(fā)病機(jī)制,從而及時(shí)有效地干預(yù)SFHN的進(jìn)展將具有極大的經(jīng)濟(jì)效益和社會效益。SFHN發(fā)病機(jī)制的研究,國內(nèi)外均未取得突破性進(jìn)展。近年來,國內(nèi)外學(xué)者對SFHN的發(fā)病機(jī)制進(jìn)行了深入的研究,并提出了眾多學(xué)說,其中包括骨內(nèi)高壓學(xué)說、凝血機(jī)制改變學(xué)說、脂肪栓塞學(xué)說、骨質(zhì)疏松學(xué)說、骨細(xì)胞凋亡學(xué)說、膜微粒學(xué)說等等。至今,SFHN具體的發(fā)病機(jī)制仍不清楚,尚需進(jìn)一步的研究。骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMSC)是骨髓中一類具有多向分化潛能并在多種疾病發(fā)生發(fā)展過程中具有重要病理生理作用的細(xì)胞。體外實(shí)驗(yàn)發(fā)現(xiàn)大劑量糖皮質(zhì)激素可通過糖皮質(zhì)激素受體(GR)和激活蛋白-1(AP-1)途徑抑制BMSC的增殖和成骨分化,相關(guān)研究也發(fā)現(xiàn)股骨頭壞死患者轉(zhuǎn)子部位成骨細(xì)胞的增殖能力下降。nicroRNA (miRNA)作為細(xì)胞內(nèi)一類重要的非編碼調(diào)控分子,它所介導(dǎo)的調(diào)控作用已經(jīng)被認(rèn)為是多細(xì)胞生物基因表達(dá)轉(zhuǎn)錄后水平的一種普遍調(diào)節(jié)方式。許多niRNA已被證明參與了骨質(zhì)形成的過程,并在其中發(fā)揮關(guān)鍵作用。BMSC結(jié)合miRNA是研究SFHN發(fā)病機(jī)制的一個(gè)新的視角,存在為SFHN提供早期診斷與治療的潛在價(jià)值。本課題將應(yīng)用niRNA芯片篩選出調(diào)控SFHN相關(guān)的miRNA;對于我們篩選出的目標(biāo)miRNA,進(jìn)一步驗(yàn)證其功能,然后應(yīng)用雙熒光素酶報(bào)告基因系統(tǒng)、siRNA等技術(shù)探討其調(diào)控機(jī)制,以期闡明激素性股骨頭壞死的發(fā)病機(jī)制。方法1、BMSC的分離、培養(yǎng)與鑒定：從3例健康者和5例激素性股骨頭壞死并接受外科手術(shù)治療的患者體內(nèi)取出骨髓,按照已建立的密度梯度離心法,獲取BMSC,并培養(yǎng)傳代,并對細(xì)胞表型、成骨等功能活性進(jìn)行了初步的檢驗(yàn)。2、差異性miRNA的篩選：在成骨分化過程中,對正常BMSC、激素性股骨頭壞死BMSC和大劑量地塞米松刺激的BMSC提取RNA,然后通過基因芯片的方法測定miRNA表達(dá)情況,并篩選出差異性miRNA.3、差異性表達(dá)miR-23a的功能驗(yàn)證：將miR-23a的mimics(?)inhibitor導(dǎo)入正常BMSC,模擬過表達(dá)miR-23a和低表達(dá)miR-23a,然后進(jìn)行成骨誘導(dǎo),在3天、6天、9天和12天進(jìn)行堿性磷酸酶染色、茜素紅染色、Real Time PCR、western blot及ALP活性檢測,進(jìn)一步驗(yàn)證候選miR-23a對成骨作用的影響。4.miR-23a抑制BMSC成骨分化的機(jī)制：首先通過專業(yè)的靶基因預(yù)測網(wǎng)站,對候選miR-23a的靶基因進(jìn)行初步的預(yù)測,然后通過雙熒光素酶實(shí)驗(yàn),驗(yàn)證靶基因與miR-23a的結(jié)構(gòu)匹配性,最后通過siRNA干擾技術(shù),進(jìn)一步驗(yàn)證靶基因。最后通過通路抑制劑干擾具體的信號通路,驗(yàn)證miR-23a信號傳導(dǎo)的具體通路。結(jié)果1、成功分離出BMSC,并通過流式細(xì)胞儀對其表型進(jìn)行了鑒定,并通過相關(guān)染色、Real Time PCR的方式鑒定了其潛在的成骨分化能力。2、大劑量地塞米松刺激BMSC和激素性股骨頭壞死B MSC中,miR-423-5p、 miR-193b*、miR-744、miR-214、miR-423-3p、miR-320a、miR-140-3p等7個(gè)miRNAs 表達(dá)下降,miR-152、miR-23a、miR-425、miR-27a、miR-221、miR-151-5p、miR-22、 miR-224*等8個(gè)miRNAs表達(dá)上調(diào).3、選取miR-23a進(jìn)行功能試驗(yàn)。細(xì)胞水平、蛋白水平和mRNA水平顯示,miR-23a抑制成骨,結(jié)果與上述芯片結(jié)果相一致。4、通過TargetScan.miRBase等靶基因?qū)I(yè)預(yù)測網(wǎng)站,預(yù)測8個(gè)候選靶基因；然后通過雙熒光素酶報(bào)告實(shí)驗(yàn),最終確定miR-23a靶基因?yàn)長RP5。最終確定miR23a通過Wnt通路抑制BMSC成骨分化。結(jié)論1、篩選出SFHN特異性miRNA表達(dá)譜。2.miR-23a靶基因?yàn)長RP5,通過Wnt通路抑制人BMSC成骨分化。
[Abstract]:Background and objective steroid-induced femoral head necrosis (SFHN) is a metabolic disease characterized by the damage of the blood supply of the femoral head, the death of bone cells and bone marrow components, and a metabolic disease characterized by structural changes of the femoral head, collapse of the femoral head, and joint dysfunction. About 75% of these are due to the long-term use of hormone drugs. The patients can collapse of the femoral head within 3 years from the diagnosis of SFHN, and in the end more than 65% of the patients have to accept hip replacement, which has brought a huge economic burden to the family and society. To explore the pathogenesis of SFHN and thus effectively intervene in the progress of SFHN in time will have great economic and social benefit.SFHN pathogenesis. In recent years, domestic and foreign scholars have not made a breakthrough at home and abroad. In recent years, domestic and foreign scholars have conducted in-depth studies on the pathogenesis of SFHN, and put forward numerous theories, including the theory of intraosseous hypertension, the theory of change of coagulation mechanism, the theory of fat embolism, the theory of osteoporosis, the theory of bone cell apoptosis, the theory of membrane particles and so on. So far, the specific SFHN is specific. The pathogenesis is still unclear, and further research is needed. Bone marrow mesenchymal stem cells (BMSC) is a kind of cells with multiple differentiation potential in the bone marrow and has important pathophysiological functions in the process of various diseases. In vitro, large doses of glucocorticoids can be used through sugar skin. The hormone receptor (GR) and activation protein -1 (AP-1) pathway inhibits the proliferation and osteogenic differentiation of BMSC. Related studies have also found that the proliferation of osteoblasts in the rotors of the femoral head necrosis patients decreased.NicroRNA (miRNA) as one of the most important non coding regulatory molecules in the cell. Its regulatory role has been considered to be multicellular organisms. Many niRNA have been proved to be involved in the process of bone formation, and the key role of.BMSC binding miRNA is a new perspective in the study of the pathogenesis of SFHN, and there is a potential value for the early diagnosis and treatment of SFHN. This topic will use niRNA chip to screen out the tone. Control SFHN related miRNA; for our screened target miRNA to further verify its function, and then apply the dual luciferase reporter gene system, siRNA and other techniques to explore the mechanism of its regulation in order to elucidate the pathogenesis of steroid necrosis of the femoral head. Methods 1, BMSC isolation, culture and identification: from 3 healthy persons and 5 cases of hormone femoral head. The bone marrow was removed from the patients with necrotic and surgical treatment. BMSC was obtained according to the established density gradient centrifugation, and the generation was cultured. The functional activity of cell phenotype and osteogenesis was preliminarily tested.2, differential miRNA screening: in the process of osteogenesis, normal BMSC, Hormonal Avascular Necrosis of the femoral head BMSC and large dose RNA was extracted from BMSC stimulated by dexamethasone, then the expression of miRNA was measured by gene chip method, and the functional verification of differential expression of miRNA.3 was screened and the differential expression of miR-23a was verified: miR-23a mimics (?) inhibitor was introduced into normal BMSC to simulate overexpression miR-23a and low expression miR-23a, and then osteogenic induction was performed at 3 days, 6 days, 9 days, and 12 days of alkaline phosphatase staining, alizarin red staining, Real Time PCR, Western blot and ALP activity detection, further verify the effect of candidate miR-23a on the osteogenesis effect of.4.miR-23a inhibition of BMSC osteogenesis differentiation mechanism: first of all through professional target gene prediction site, the candidate miR-23a target gene is preliminarily predicted, and then through double The luciferase test was used to verify the structure matching of the target gene and miR-23a. Finally, the target gene was verified by siRNA interference technique. Finally, the specific pathway of miR-23a signal transduction was verified by the pathway inhibitor interference specific signal pathway. Results 1, the BMSC was successfully isolated and the phenotype was identified by flow cytometry. The potential osteogenic differentiation ability of.2 was identified by Real Time PCR, and large dose of dexamethasone stimulated BMSC and hormonal necrosis of the femoral head necrosis, B MSC, miR-423-5p, miR-193b*, miR-744, miR-214, miR-423-3p, etc. -22, miR-224* and other 8 miRNAs expressions were up regulated and selected to perform functional tests. Cell level, protein level and mRNA level showed that miR-23a inhibited osteogenesis, and the results were consistent with the results of the above chip, and 8 candidate target genes were predicted through the professional prediction of the target gene of TargetScan.miRBase and other target genes. Then the double Luciferase Report was used to report the experiment. Finally, the miR-23a target base was determined because LRP5. ultimately determined that miR23a could inhibit BMSC osteogenesis through the Wnt pathway. Conclusion 1, the SFHN specific miRNA expression spectrum.2.miR-23a target group was screened by LRP5, and the BMSC osteogenic differentiation was inhibited by Wnt pathway.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R681.8

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