發(fā)布時(shí)間：2018-06-10 15:24

  本文選題：生脈成骨片 + 微小RNA��； 參考：《廣州中醫(yī)藥大學(xué)》2016年博士論文

【摘要】：目的：1.探討中藥袁氏生脈成骨片治療激素性股骨頭缺血壞死(Steroid-induced Osteonecrosis of Femoral Head SONFH)的臨床療效。2.探索microRNA在SONFH患者外周血中的差異表達(dá),分析差異表達(dá)的microRNA可能的調(diào)控作用。3.尋找中藥干預(yù)SONFH兔模型中microRNA的差異表達(dá),分析中藥調(diào)控microRNA表達(dá)治療SONFH可能的機(jī)理。方法：1.觀察26例SONFH患者口服袁氏生脈成骨片每日3次,每次6粒,共服3個(gè)月,記錄并比較SONFH患者服用藥物前、服藥1個(gè)月、服藥2個(gè)月和服藥3個(gè)月患側(cè)髖關(guān)節(jié)Harris評(píng)分、VAS評(píng)分,收集數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析。評(píng)價(jià)服用袁氏生脈成骨片對(duì)SONFH患者髖關(guān)節(jié)Harris評(píng)分、VAS評(píng)分的影響。2.選取2014年7月～2015年2月廣州中醫(yī)藥大學(xué)第一附屬醫(yī)院關(guān)節(jié)科門診就診的ARCOⅡ期SONFH患者6例(觀察組)及同期廣州中醫(yī)藥大學(xué)在校健康志愿者6例作為對(duì)照組。采集兩組研究對(duì)象血漿,采用microRNA Real-time PCR芯片篩選差異表達(dá)的microRNA。根據(jù)TargetScan、mirBase及miRanda三個(gè)數(shù)據(jù)庫預(yù)測(cè)差異表達(dá)microRNA的靶基因并進(jìn)行生物信息學(xué)分析。3.選取普通級(jí)健康成年新西蘭兔,體重2.5～3.0kg,雌雄各半,共30只。以隨機(jī)數(shù)字表法分為中藥組、對(duì)照組和空白組,各10只。中藥組和對(duì)照組每只每日以內(nèi)毒素10ug/kg為總量,經(jīng)耳緣靜脈分2次進(jìn)行注射,共注射2日,于第3次注射內(nèi)毒素時(shí)以甲強(qiáng)龍40mg/kg行臀肌注射,而后每間隔24h以甲強(qiáng)龍40mg/kg注射1次,共注射甲強(qiáng)龍3次�？瞻捉M于相同時(shí)間點(diǎn)注射等量生理鹽水,注射期間各組兔均臀肌注射青霉素4萬單位／只,預(yù)防感染。中藥組于最后一次注射甲強(qiáng)龍時(shí)以袁氏生脈成骨片0.4片/kg灌胃,對(duì)照組及空白組以相同計(jì)量生理鹽水灌胃,灌胃8周。于造模4周時(shí),中藥組與對(duì)照組各隨機(jī)選取1只兔,取股骨頭樣品行HE染色,觀察造模成功與否。灌胃完成后收集兔血漿采用高通量測(cè)序法測(cè)定其microNRA相對(duì)表達(dá)量,篩選出觀察組、對(duì)照組較空白組差異表達(dá)的microRNA,獲取此調(diào)控途徑中的關(guān)鍵microRNA進(jìn)行生物信息學(xué)分析。結(jié)果：1.口服袁氏生脈成骨片前、服藥1個(gè)月、服藥2個(gè)月、服藥3個(gè)月患側(cè)髖關(guān)節(jié)Harris評(píng)分分別為38.17±4.02,39.83±3.97、42.67±2.25和44.33±3.20,患側(cè)髖關(guān)節(jié)VAS評(píng)分分別為6.83±0.41、6.33±0.52、5.00±0.89和4.33±0.52,服藥前與服藥2、3個(gè)月相比較均具有統(tǒng)計(jì)學(xué)差異。2.SONFH患者血漿中采用microRNA Real-time PCR芯片檢測(cè)752個(gè)microRNA,兩組差異表達(dá)的microRNA18個(gè),其中觀察組上調(diào)13個(gè),下調(diào)5個(gè)。共篩選出交集靶基因887個(gè)。GO分析顯示,觀察組較對(duì)照組差異表達(dá)microRNA調(diào)控的靶基因功能主要包括解剖結(jié)構(gòu)形態(tài)、間充質(zhì)細(xì)胞增殖、細(xì)胞的代謝過程等,Pathway分析顯示靶基因明顯富集于多條信號(hào)通路。3.在SONFH兔模型實(shí)驗(yàn)中采用miRDeep2.0進(jìn)行microRNA預(yù)測(cè),共預(yù)測(cè)出887個(gè)可能microRNA。對(duì)照組與空白組之間存在差異表達(dá)的microRNA,經(jīng)同源性分析篩選出與之對(duì)應(yīng)的人類microRNA:hsa-miR-941和hsa-miR-187。并且hsa-miR-941和hsa-miR-187表達(dá)量在空白組、對(duì)照組和中藥組間呈現(xiàn)先升高再回落的表達(dá)趨勢(shì)。hsa-miR-187可調(diào)控細(xì)胞凋亡和血管生成。MAPK信號(hào)通路在中藥組與空白組差異表達(dá)的microRNA靶基因中富集明顯。結(jié)論：1.袁氏生脈成骨片可有效改善ARCOⅡ期SONFH患者患側(cè)髖關(guān)節(jié)Harris評(píng)分和VAS評(píng)分。2.本研究發(fā)現(xiàn)SONFH患者血漿中microRNA較正常人出現(xiàn)差異表達(dá),說明這些差異表達(dá)的microRNA可能作為篩選SONFH的生物學(xué)標(biāo)志物。3.袁氏生脈成骨片通過影響microRNA的差異表達(dá),調(diào)控多條信號(hào)通路,抑制細(xì)胞凋亡、促進(jìn)血管生成可能是其治療SONFH的作用機(jī)制。
[Abstract]:Objective: 1. to explore the clinical effect of Yuan's raw vein osteogenesis tablets on hormone induced osteonecrosis of the femoral head (Steroid-induced Osteonecrosis of Femoral Head SONFH).2. explore the differential expression of microRNA in the peripheral blood of patients with SONFH, and analyze the possible regulating role of the differentially expressed microRNA in the microRNA. The differential expression of oRNA was used to analyze the possible mechanism of microRNA expression in the treatment of SONFH. Methods: 1. to observe 26 cases of SONFH patients taking oral Yuan Shisheng vein osteogenesis tablets 3 times a day, 6 capsules each time for 3 months, recording and comparing SONFH patients before taking drugs for 1 months, taking medicine for 2 months and taking medicine 3 months for the side hip joint Harris score and VAS score. Collect data for statistical analysis. Evaluate the effect of Yuen Sheng Mai osteogenesis on the Harris score of the hip joint of SONFH patients and the effect of VAS score on 6 cases of ARCO II SONFH patients in the First Affiliated Hospital of Guangzhou University of Chinese Medicine from July 2014 to February 2015 (observation group) and Guangzhou University of Chinese Medicine at the same time. 6 subjects were selected as the control group. The plasma of two groups of subjects were collected, and the differentially expressed microRNA. was screened by microRNA Real-time PCR chip. The target genes of microRNA were predicted by the three databases of TargetScan, mirBase and miRanda, and the healthy adult New Zealand rabbits were selected by the bioinformatics analysis, and the weight of 2.5 ~ 3.0kg was selected. A total of 30 males and females were divided into two groups. The random number table was divided into Chinese medicine group, control group and blank group, each 10. The total amount of 10ug/kg in the traditional Chinese medicine group and the control group was injected into the ear vein 2 times for 2 days. At the time of third injections of endotoxin, the gluteal muscle was injected with methylprednisolone 40mg/kg, and then each interval 24h was a strong dragon 40mg/. Kg was injected 1 times and a total of 3 times a strong dragon was injected. The blank group injected the same amount of normal saline at the same time point. The rabbits were injected with penicillin 40 thousand units / only to prevent infection during the period of injection. The Chinese medicine group was given 0.4 slices of Yuan's raw vein osteogenesis tablet for the stomach during the last injection of a strong dragon, and the control group and the blank group were given the same measurement of physiological saline. After 8 weeks of gavage, 1 rabbits were randomly selected from the Chinese medicine group and the control group at 4 weeks. HE staining of the femoral head sample was taken to observe whether the model was successful or not. After the gastric perfusion was completed, the relative expression of microNRA was measured by high throughput sequencing in rabbit plasma, and the observation group was selected and the control group was compared with the blank group, which was expressed in microRNA, and the control route was obtained. The key microRNA in the diameter was analyzed by bioinformatics. Results: 1. before oral Yuan Shisheng vein osteogenesis, taking medicine for 1 months and taking medicine for 2 months, the Harris score of the lateral hip joint for 3 months was 38.17 + 4.02,39.83 + 3.97,42.67 + 2.25 and 44.33 + 3.20 respectively. The VAS score of the affected hip joint was 6.83 + 0.41,6.33 + 0.52,5.00 + 0.89 and 4.33 + 0.52, Before taking the medicine, there were statistically significant differences between the 2,3 months and the 2,3 months of the medicine. The plasma of.2.SONFH patients were detected by microRNA Real-time PCR chip to detect 752 microRNA, and the two groups were expressed differently, among which the observation group was up 13 and down 5. A total of 887.GO analysis of the intersection target gene showed that the observation group was more than the control group. The function of target gene regulated by NA mainly included anatomical structure, proliferation of mesenchymal cells and metabolic process of cells. Pathway analysis showed that the target gene was obviously enriched in multiple signal pathways.3. in SONFH rabbit model experiment using miRDeep2.0 for microRNA prediction, and 887 possible microRNA. control groups were predicted to exist between the blank group and the blank group. The differential expression of microRNA, the homologous analysis of the corresponding human microRNA:hsa-miR-941 and hsa-miR-187., and the expression of hsa-miR-941 and hsa-miR-187 in the blank group, the expression trend between the control group and the traditional Chinese medicine group, which first increased and then receded, can regulate the apoptosis and the angiogenic.MAPK signal pathway in the Chinese Medicine. The group was enriched with the microRNA target gene differentially expressed in the blank group. Conclusion: 1. Yuan Shi Sheng Mai osteogenic tablet can effectively improve the Harris score and VAS score of the patient's hip joint in ARCO II SONFH patients. The difference in the expression of microRNA in the plasma of SONFH patients is more than that of the normal people, indicating that the microRNA of these differentially expressed microRNA may be used as a sieve. The biological marker of SONFH.3. Yuan Shi Sheng Mai osteogenesis tablet, by influencing the differential expression of microRNA, regulates multiple signal pathways, inhibits apoptosis and promotes angiogenesis may be its mechanism for the treatment of SONFH.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R274.9

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