低频振动对人脐静脉血管内皮细胞钙内流和自噬流的影响及分子机制

发布时间：2018-06-21 09:07

本文选题：低频振动 + HUVEC　；参考：《第二军医大学》2017年博士论文

【摘要】：前庭功能障碍严重影响患者的工作和生活质量,尤其是眩晕。前庭功能紊乱或障碍带来的周围型眩晕有着较高的发病率,如常见的梅尼埃病,除了前庭症状外,该病还伴有耳闷、耳鸣以及听力下降等耳蜗症状。药物治疗、手法操作、手术治疗以及各种前庭康复广为开展。低频振动作为新颖的治疗手段在门诊治疗中,改善了患者的总体感觉,控制了眩晕,减轻耳鸣和耳闷症状,并增加了患者的头部舒适感。本研究观察了低频振动对单侧前庭功能低下患者眩晕恢复的促进作用,经乳突投放低频振动,明显促进了眩晕的康复过程。为了进一部探寻低频振动在治疗眩晕症状上的作用机制,从细胞学和分子生物学上探索低频振动是如何达到控制并改变内耳前庭细胞的功能。本实验课题利用公认研究脉管系统的模型细胞,即人脐静脉血内皮细胞(HUVEC),建立其转染mRFP-GFP-LC3腺病毒和线粒体毒素干扰自噬的实验模型。观察低频振动对HUVEC细胞钙内流和自噬流的影响,并与氟桂利嗪阳性对照组比较。采用磷酸化广谱筛选抗体芯片,分析低频振动对线粒体损伤的HUVEC细胞的信号通路蛋白磷酸化水平的影响,进而探索低频振动对人脐静脉血管内皮细胞影响的分子机制。第一部分低频振动剪切应激治疗单侧前庭功能低下的疗效分析目的探讨低频振动在单侧前庭功能低下患者眩晕康复中的作用。方法共收集我院门诊的诊断为单侧前庭功能低下患者25人,均为前庭神经元炎后遗症,分为低频振动治疗组和常规前庭康复治疗组。低频振动治疗组采用经患侧乳突进行100Hz低频振动,每次30分钟,每周2次,连续治疗2周。前庭康复治疗组患者在康复科康复治疗师指导下采用TECNOBODY平衡测试及训练系统进行感觉整合训练、前庭功能训练、重心控制训练。两组患者分别于治疗前、治疗后进行DHI评分及VVAS评分。所有计数资料采用均数±标准差方式记录,采用SPSS软件进行方差分析。计量资料采用卡方检验进行统计分析,取p0.05为有统计学意义。结果按照DHI总分改善18分以上为有效,低频振动治疗组总有效率为76.9%(10/13),前庭康复组为83.3%(10/12),经卡方检验两组差异无统计学意义。结论经乳突低频振动与传统的前庭康复锻炼对眩晕症状的改善效率相当,对患者无其他不良影响。相对于前庭康复锻炼需要专业的康复师和专业的场地而言,低频振动治疗前庭功能障碍操作简便,对场地无特殊要求,技术员培训难度低。本研究结果显示,低频振动治疗前庭功能低下的短期效果明显,长期效果还需要增加样本量及进一步延长随访时间。第二部分低频振动对人脐静脉血管内皮细胞钙内流的影响目的了解低频振动在调节人脐血内皮细胞钙内流方面的作用。方法利用人脐血内皮细胞(HUEVC)作为模型细胞,用FLUO-3钙离子示踪剂显示细胞内钙离子的实时变化,用3-NP处理建立细胞损伤的模型,用低频振动对其进行治疗干预,用氟桂利嗪作为阳性对照。通过荧光显微镜结合图像分析和半定量测试,实时观察3-NP诱导钙内流的变化以及低频振动干预治疗后,细胞钙内流发生的改变。结果与正常细胞相比,3NP处理后的HUEVC细胞,绿色荧光强度明显加强,出现钙超载现象。3NP处理后的HUEVC细胞,分别施加低频振动,或者加入氟桂利嗪,在3NP+低频振动组和3NP+氟桂利嗪组,分别在1分钟,5分钟,15分钟,20分钟,30分钟,与3-NP处理后的HUVEC细胞,即造模细胞相比,在不同时间点的比较上,绿色荧光强度明显降低,钙超载水平逐步降低。通过半定量荧光值的统计分析,具有明显的统计学差异。结论低频振动可以明显降低了因受线粒体损伤而增强的钙内流。第三部分低频振动对人脐静脉血管内皮细胞自噬流的影响目的了解低频振动在调节内耳血管功能方面的可能自噬因素。方法利用人脐血内皮细胞(HUEVC)作为模型细胞,用mRFP-GFP-LC3腺病毒转染使其表达荧光标记自噬标志性的微管相关蛋白1轻链3,用3-NP处理建立细胞损伤的模型,用低频振动对其进行治疗干预,用氟桂利嗪作为阳性对照。通学过激光共聚焦显微镜,从形态学上,实时观察3-NP诱导自噬流的变化。用全自动流式细胞仪,定量观测经转染mRFP-GFP-LC3腺病毒后的HUVEC细胞自噬流的变化。结果与正常细胞相比,3NP处理减少了自噬小体(绿色)的数量,更多地与溶酶体融合(黄和红色)。在经3NP处理后,分组分别加入氟桂利嗪,或者低频振动,在3NP低频振动和氟桂利嗪均明显提高了HUVEC细胞的自噬率。定量荧光值的统计分析,具有明显的统计学差异。结论低频振动可以明显提高了因受因为线粒体损伤而降低的自噬水平。低频振动有可能通过影响细胞的自噬,而抑制过渡的炎症反应,从而缓解内耳损伤的病理过程。第四部分低频振动对人脐静脉血管内皮细胞影响的分子机制目的探索低频振动对线粒体损伤后HUVEC细胞的信号通路蛋白磷酸化水平的影响,进而研究低频振动干预线粒体损伤的人脐静脉血管内皮细胞病理过程的分子机制。方法采用信号通路磷酸化广谱筛选抗体芯片(PEX100),利用三维高分子膜专利技术,在片基上高密度结合1318种高特异抗体。对HUVEC细胞的全细胞蛋白进行检测。共分为四组,空白对照组,3-NP造模组,3-NP+氟桂利嗪组以及3-NP+低频振动组。以氟桂利嗪,钙离子阻滞剂作为阳性对照。对4组细胞分别进行检测432个信号蛋白的679个磷酸化位点。对检测出的通道进行数据分析,公式计算,组间比较,进行统计学数据分析。结果与3-NP造模组未受干预的细胞相比,低频振动组增加其磷酸化水平6倍以上的主要信号通路蛋白有:NMDAR2B(Phospho-Tyr1472),MEK1(Phospho-Ser298),Lamin A/C(Phospho-Ser392),HDAC1(Phospho-Ser421),PECAM-1(Phospho-Tyr713),P90RSK(Phospho-Thr359/Ser363),P90RSK(Phospho-Thr573),LAT(Phospho-Tyr171),PP2A-alpha(Phospho-Tyr307),Merlin(Phospho-Ser10),MKK4/SEK1(Phospho-Ser80),BLNK(Phospho-Tyr96),14-3-3 zeta/delta(Phospho-Thr232)。与3-NP造模组未受干预的细胞相比,低频振动组增加其磷酸化水平6倍以上的主要信号通路蛋白有:NMDAR2B(Phospho-Tyr1472),MEK1(Phospho-Ser298),Lamin A/C(Phospho-Ser392),HDAC1(Phospho-Ser421),PECAM-1(Phospho-Tyr713),P90RSK(Phospho-Thr359/Ser363),P90RSK(Phospho-Thr573),LAT(Phospho-Tyr171),PP2A-alpha(Phospho-Tyr307),Merlin(Phospho-Ser10),MKK4/SEK1(Phospho-Ser80),BLNK(Phospho-Tyr96),14-3-3 zeta/delta(Phospho-Thr232)。结论从诸多筛选出的信号通路中,我们可以分析出:对于HUVEC细胞,用3-NP干扰其自噬,用低频振动进行干预治疗,以氟桂利嗪作为阳性对照,增加磷酸化水平6倍以上的主要信号通路中,低频振动增强信号通路控制点下游分子磷酸化水平上,有明显的促进细胞生长,增强自噬,抑制凋亡的作用。
[Abstract]:Vestibular dysfunction seriously affects the work and quality of life, especially vertigo. The incidence of peripheral vertigo caused by a vestibular disorder or disorder, such as the common Meniere's disease, is accompanied by cochlear symptoms such as ear boredom, tinnitus and hearing downfall. The low frequency vibration as a novel treatment in the outpatient treatment improves the overall feeling of the patient, controls the vertigo, reduces the tinnitus and ear symptoms, and increases the patient's head comfort. This study observed the promotion of vertigo recovery from low frequency vibration to the patients with single vestibule dysfunction. In order to explore the function mechanism of low frequency vibration on the treatment of vertigo, it is explored from cytology and molecular biology how to control and change the work energy of the inner ear vestibule cells in order to find the mechanism of the low-frequency vibration in the treatment of vertigo symptoms. The model cells, the human umbilical vein endothelial cells (HUVEC), set up an experimental model to transfect the mRFP-GFP-LC3 adenovirus and the mitochondrial toxin to interfere with autophagy. The effects of low frequency vibration on the calcium flow and autophagic flow in HUVEC cells were observed and compared with the flunarizine positive control group. The effect of signal pathway protein phosphorylation level of HUVEC cells damaged by mitochondria and to explore the molecular mechanism of low frequency vibration on human umbilical vein endothelial cells. The first part of low frequency vibration shear stress treatment for unilateral vestibule dysfunction Methods a total of 25 patients with low vestibulitis in our hospital were collected, all of which were the sequelae of vestibular neuritis, which were divided into low frequency vibration treatment group and conventional vestibule rehabilitation treatment group. Low frequency vibration treatment group was treated with 100Hz low-frequency vibration of the affected side mastoid, 30 minutes each time, 2 times a week for 2 weeks. Under the guidance of rehabilitation therapist of rehabilitation department, the patients in the rehabilitation treatment group adopted the TECNOBODY balance test and training system to carry out sensory integration training, vestibule function training and center of gravity control training. The two groups of patients were treated with DHI score and VVAS score before treatment. All the counting materials were recorded with mean number of standard deviation and SPSS. Analysis of variance in the software. Statistical analysis was carried out by chi square test. The results were statistically significant. The results were more than 18 points improved according to the total score of DHI. The total effective rate of the low frequency vibration treatment group was 76.9% (10/13), the vestibular rehabilitation group was 83.3% (10/12), and there was no significant difference between the two groups through the chi square test. Conclusion the low frequency of the mastoid process was low. The effect of vibration and traditional vestibular rehabilitation exercise on the improvement of vertigo symptoms is equivalent, and there is no other adverse effect on the patients. Compared with the professional rehabilitative and professional field, the low frequency vibration treatment is easy to operate, no special requirements on the site and low difficulty for the technician. The short-term effect of low frequency vibration in the treatment of vestibule dysfunction is obvious. The long-term effect needs to increase the sample size and further extend the follow-up time. Second the effect of low frequency vibration on the calcium influx of human umbilical vein endothelial cells in order to understand the role of low frequency vibration in regulating the calcium influx of human umbilical cord blood. Umbilical cord blood endothelial cells (HUEVC) were used as model cells, using FLUO-3 calcium tracer to display the real time changes of intracellular calcium ions. The model of cell damage was established by 3-NP treatment. It was treated with low frequency vibration, and flunarizine was used as the positive control. The real-time observation of 3-N by fluorescence microscope combined with image analysis and semi quantitative test. P induced changes in calcium influx and changes in intracellular calcium influx after low-frequency vibration intervention. Results compared with normal cells, the green fluorescence intensity of HUEVC cells treated by 3NP was obviously enhanced. The HUEVC cells treated with calcium overload,.3NP treated HUEVC cells, or flunarizine respectively, were added to the 3NP+ low frequency vibration group. And 3NP+ flunarizine group, 1 minutes, 5 minutes, 15 minutes, 20 minutes, 30 minutes respectively, compared with the 3-NP treated HUVEC cells, that is, compared with the model cells, the green fluorescence intensity decreased obviously and the calcium overload level decreased gradually. Frequency vibration can obviously reduce the calcium influx enhanced by mitochondrial damage. Third the effect of partial low frequency vibration on the autophagy of human umbilical vein endothelial cells in order to understand the possible autophagy factors in regulating the function of the inner ear vessels by low frequency vibration. Methods using human umbilical cord blood endothelial cells (HUEVC) as model cells, mRFP-GFP-L C3 adenovirus transfection makes it express the autophagy marker of microtubule related protein 1 light chain 3. The model of cell damage is established by 3-NP treatment. It is treated with low frequency vibration, and flunarizine is used as the positive control. The laser confocal microscope is used to observe the changes of autophagic flow induced by 3-NP in real time. Automatic flow cytometer, quantitative observation of autophagic changes in HUVEC cells after transfection of mRFP-GFP-LC3 adenovirus. Results compared with normal cells, 3NP treatment reduced the number of autophagosomes (green), more with lysosomes (yellow and red). After 3NP treatment, the group added flunarizine, or low frequency vibration, at low frequency 3NP. Vibration and flunarizine significantly increased the autophagy rate of HUVEC cells. Statistical analysis of quantitative fluorescence values showed significant statistical differences. Conclusion low frequency vibration can significantly increase the level of autophagy reduced by mitochondrial damage. Low frequency vibration may affect the autophagy of the cell and inhibit the transition inflammation. The molecular mechanism of the effect of low frequency vibration on human umbilical vein endothelial cells in fourth parts to explore the effect of low frequency vibration on the level of signaling pathway protein phosphorylation in HUVEC cells after mitochondrial damage, and then to study the human umbilical vein endothelial cell disease with low frequency vibration interfering with cord injury. The molecular mechanism of the process. Methods using the signal pathway phosphorylation broad-spectrum screening antibody chip (PEX100), using the three dimensional polymer membrane patent technology, the high density combined with 1318 high specific antibodies on the substrate, the whole cell protein of HUVEC cells was detected. It was divided into four groups, the blank control group, the 3-NP model group, the 3-NP+ flunarizine group and the 3-NP + low frequency vibration group. With flunarizine and calcium ion blocker as positive control, 679 phosphorylation sites of 432 signal proteins were detected in 4 groups of cells. Data analysis of the detected channels, formula calculation, comparison between groups, statistical data analysis. The results were compared with the untreated cells in the 3-NP model module. NMDAR2B (Phospho-Tyr1472), MEK1 (Phospho-Ser298), Lamin A/C (Phospho-Ser392), HDAC1 (Phospho-Ser421), PECAM-1 (Phospho-Tyr713), PECAM-1 (Phospho-Tyr713). Spho-Ser10), MKK4/SEK1 (Phospho-Ser80), BLNK (Phospho-Tyr96), 14-3-3 zeta/delta (Phospho-Thr232). O-Ser421), PECAM-1 (Phospho-Tyr713), P90RSK (Phospho-Thr359/Ser363), P90RSK (Phospho-Thr573), LAT (Phospho-Tyr171), PP2A-alpha (Phospho-Tyr307). For HUVEC cells, interfering with autophagy by 3-NP and interfering with low frequency vibration with flunarizine as positive control and increasing the level of phosphorylation at more than 6 times the phosphorylation level of low frequency vibration enhanced signal channel control point, there is an obvious promotion of cell growth, enhancement of autophagy and inhibition of apoptosis. Use.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R764.3

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