成骨片调节microRNA在激素性股骨头缺血坏死差异表达的初步研究

发布时间：2018-06-10 15:24

本文选题：生脉成骨片 + 微小RNA　；参考：《广州中医药大学》2016年博士论文

【摘要】：目的：1.探讨中药袁氏生脉成骨片治疗激素性股骨头缺血坏死(Steroid-induced Osteonecrosis of Femoral Head SONFH)的临床疗效。2.探索microRNA在SONFH患者外周血中的差异表达,分析差异表达的microRNA可能的调控作用。3.寻找中药干预SONFH兔模型中microRNA的差异表达,分析中药调控microRNA表达治疗SONFH可能的机理。方法：1.观察26例SONFH患者口服袁氏生脉成骨片每日3次,每次6粒,共服3个月,记录并比较SONFH患者服用药物前、服药1个月、服药2个月和服药3个月患侧髋关节Harris评分、VAS评分,收集数据进行统计学分析。评价服用袁氏生脉成骨片对SONFH患者髋关节Harris评分、VAS评分的影响。2.选取2014年7月～2015年2月广州中医药大学第一附属医院关节科门诊就诊的ARCOⅡ期SONFH患者6例(观察组)及同期广州中医药大学在校健康志愿者6例作为对照组。采集两组研究对象血浆,采用microRNA Real-time PCR芯片筛选差异表达的microRNA。根据TargetScan、mirBase及miRanda三个数据库预测差异表达microRNA的靶基因并进行生物信息学分析。3.选取普通级健康成年新西兰兔,体重2.5～3.0kg,雌雄各半,共30只。以随机数字表法分为中药组、对照组和空白组,各10只。中药组和对照组每只每日以内毒素10ug/kg为总量,经耳缘静脉分2次进行注射,共注射2日,于第3次注射内毒素时以甲强龙40mg/kg行臀肌注射,而后每间隔24h以甲强龙40mg/kg注射1次,共注射甲强龙3次。空白组于相同时间点注射等量生理盐水,注射期间各组兔均臀肌注射青霉素4万单位／只,预防感染。中药组于最后一次注射甲强龙时以袁氏生脉成骨片0.4片/kg灌胃,对照组及空白组以相同计量生理盐水灌胃,灌胃8周。于造模4周时,中药组与对照组各随机选取1只兔,取股骨头样品行HE染色,观察造模成功与否。灌胃完成后收集兔血浆采用高通量测序法测定其microNRA相对表达量,筛选出观察组、对照组较空白组差异表达的microRNA,获取此调控途径中的关键microRNA进行生物信息学分析。结果：1.口服袁氏生脉成骨片前、服药1个月、服药2个月、服药3个月患侧髋关节Harris评分分别为38.17±4.02,39.83±3.97、42.67±2.25和44.33±3.20,患侧髋关节VAS评分分别为6.83±0.41、6.33±0.52、5.00±0.89和4.33±0.52,服药前与服药2、3个月相比较均具有统计学差异。2.SONFH患者血浆中采用microRNA Real-time PCR芯片检测752个microRNA,两组差异表达的microRNA18个,其中观察组上调13个,下调5个。共筛选出交集靶基因887个。GO分析显示,观察组较对照组差异表达microRNA调控的靶基因功能主要包括解剖结构形态、间充质细胞增殖、细胞的代谢过程等,Pathway分析显示靶基因明显富集于多条信号通路。3.在SONFH兔模型实验中采用miRDeep2.0进行microRNA预测,共预测出887个可能microRNA。对照组与空白组之间存在差异表达的microRNA,经同源性分析筛选出与之对应的人类microRNA:hsa-miR-941和hsa-miR-187。并且hsa-miR-941和hsa-miR-187表达量在空白组、对照组和中药组间呈现先升高再回落的表达趋势。hsa-miR-187可调控细胞凋亡和血管生成。MAPK信号通路在中药组与空白组差异表达的microRNA靶基因中富集明显。结论：1.袁氏生脉成骨片可有效改善ARCOⅡ期SONFH患者患侧髋关节Harris评分和VAS评分。2.本研究发现SONFH患者血浆中microRNA较正常人出现差异表达,说明这些差异表达的microRNA可能作为筛选SONFH的生物学标志物。3.袁氏生脉成骨片通过影响microRNA的差异表达,调控多条信号通路,抑制细胞凋亡、促进血管生成可能是其治疗SONFH的作用机制。
[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.
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R274.9

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