本文選題：低頻振動 + HUVEC　； 參考：《第二軍醫(yī)大學》2017年博士論文

【摘要】：前庭功能障礙嚴重影響患者的工作和生活質(zhì)量,尤其是眩暈。前庭功能紊亂或障礙帶來的周圍型眩暈有著較高的發(fā)病率,如常見的梅尼埃病,除了前庭癥狀外,該病還伴有耳悶、耳鳴以及聽力下降等耳蝸癥狀。藥物治療、手法操作、手術(shù)治療以及各種前庭康復(fù)廣為開展。低頻振動作為新穎的治療手段在門診治療中,改善了患者的總體感覺,控制了眩暈,減輕耳鳴和耳悶癥狀,并增加了患者的頭部舒適感。本研究觀察了低頻振動對單側(cè)前庭功能低下患者眩暈恢復(fù)的促進作用,經(jīng)乳突投放低頻振動,明顯促進了眩暈的康復(fù)過程。為了進一部探尋低頻振動在治療眩暈癥狀上的作用機制,從細胞學和分子生物學上探索低頻振動是如何達到控制并改變內(nèi)耳前庭細胞的功能。本實驗課題利用公認研究脈管系統(tǒng)的模型細胞,即人臍靜脈血內(nèi)皮細胞(HUVEC),建立其轉(zhuǎn)染mRFP-GFP-LC3腺病毒和線粒體毒素干擾自噬的實驗?zāi)Ｐ汀Ｓ^察低頻振動對HUVEC細胞鈣內(nèi)流和自噬流的影響,并與氟桂利嗪陽性對照組比較。采用磷酸化廣譜篩選抗體芯片,分析低頻振動對線粒體損傷的HUVEC細胞的信號通路蛋白磷酸化水平的影響,進而探索低頻振動對人臍靜脈血管內(nèi)皮細胞影響的分子機制。第一部分低頻振動剪切應(yīng)激治療單側(cè)前庭功能低下的療效分析目的探討低頻振動在單側(cè)前庭功能低下患者眩暈康復(fù)中的作用。方法共收集我院門診的診斷為單側(cè)前庭功能低下患者25人,均為前庭神經(jīng)元炎后遺癥,分為低頻振動治療組和常規(guī)前庭康復(fù)治療組。低頻振動治療組采用經(jīng)患側(cè)乳突進行100Hz低頻振動,每次30分鐘,每周2次,連續(xù)治療2周。前庭康復(fù)治療組患者在康復(fù)科康復(fù)治療師指導下采用TECNOBODY平衡測試及訓練系統(tǒng)進行感覺整合訓練、前庭功能訓練、重心控制訓練。兩組患者分別于治療前、治療后進行DHI評分及VVAS評分。所有計數(shù)資料采用均數(shù)±標準差方式記錄,采用SPSS軟件進行方差分析。計量資料采用卡方檢驗進行統(tǒng)計分析,取p0.05為有統(tǒng)計學意義。結(jié)果按照DHI總分改善18分以上為有效,低頻振動治療組總有效率為76.9%(10/13),前庭康復(fù)組為83.3%(10/12),經(jīng)卡方檢驗兩組差異無統(tǒng)計學意義。結(jié)論經(jīng)乳突低頻振動與傳統(tǒng)的前庭康復(fù)鍛煉對眩暈癥狀的改善效率相當,對患者無其他不良影響。相對于前庭康復(fù)鍛煉需要專業(yè)的康復(fù)師和專業(yè)的場地而言,低頻振動治療前庭功能障礙操作簡便,對場地無特殊要求,技術(shù)員培訓難度低。本研究結(jié)果顯示,低頻振動治療前庭功能低下的短期效果明顯,長期效果還需要增加樣本量及進一步延長隨訪時間。第二部分低頻振動對人臍靜脈血管內(nèi)皮細胞鈣內(nèi)流的影響目的了解低頻振動在調(diào)節(jié)人臍血內(nèi)皮細胞鈣內(nèi)流方面的作用。方法利用人臍血內(nèi)皮細胞(HUEVC)作為模型細胞,用FLUO-3鈣離子示蹤劑顯示細胞內(nèi)鈣離子的實時變化,用3-NP處理建立細胞損傷的模型,用低頻振動對其進行治療干預(yù),用氟桂利嗪作為陽性對照。通過熒光顯微鏡結(jié)合圖像分析和半定量測試,實時觀察3-NP誘導鈣內(nèi)流的變化以及低頻振動干預(yù)治療后,細胞鈣內(nèi)流發(fā)生的改變。結(jié)果與正常細胞相比,3NP處理后的HUEVC細胞,綠色熒光強度明顯加強,出現(xiàn)鈣超載現(xiàn)象。3NP處理后的HUEVC細胞,分別施加低頻振動,或者加入氟桂利嗪,在3NP+低頻振動組和3NP+氟桂利嗪組,分別在1分鐘,5分鐘,15分鐘,20分鐘,30分鐘,與3-NP處理后的HUVEC細胞,即造模細胞相比,在不同時間點的比較上,綠色熒光強度明顯降低,鈣超載水平逐步降低。通過半定量熒光值的統(tǒng)計分析,具有明顯的統(tǒng)計學差異。結(jié)論低頻振動可以明顯降低了因受線粒體損傷而增強的鈣內(nèi)流。第三部分低頻振動對人臍靜脈血管內(nèi)皮細胞自噬流的影響目的了解低頻振動在調(diào)節(jié)內(nèi)耳血管功能方面的可能自噬因素。方法利用人臍血內(nèi)皮細胞(HUEVC)作為模型細胞,用mRFP-GFP-LC3腺病毒轉(zhuǎn)染使其表達熒光標記自噬標志性的微管相關(guān)蛋白1輕鏈3,用3-NP處理建立細胞損傷的模型,用低頻振動對其進行治療干預(yù),用氟桂利嗪作為陽性對照。通學過激光共聚焦顯微鏡,從形態(tài)學上,實時觀察3-NP誘導自噬流的變化。用全自動流式細胞儀,定量觀測經(jīng)轉(zhuǎn)染mRFP-GFP-LC3腺病毒后的HUVEC細胞自噬流的變化。結(jié)果與正常細胞相比,3NP處理減少了自噬小體(綠色)的數(shù)量,更多地與溶酶體融合(黃和紅色)。在經(jīng)3NP處理后,分組分別加入氟桂利嗪,或者低頻振動,在3NP低頻振動和氟桂利嗪均明顯提高了HUVEC細胞的自噬率。定量熒光值的統(tǒng)計分析,具有明顯的統(tǒng)計學差異。結(jié)論低頻振動可以明顯提高了因受因為線粒體損傷而降低的自噬水平。低頻振動有可能通過影響細胞的自噬,而抑制過渡的炎癥反應(yīng),從而緩解內(nèi)耳損傷的病理過程。第四部分低頻振動對人臍靜脈血管內(nèi)皮細胞影響的分子機制目的探索低頻振動對線粒體損傷后HUVEC細胞的信號通路蛋白磷酸化水平的影響,進而研究低頻振動干預(yù)線粒體損傷的人臍靜脈血管內(nèi)皮細胞病理過程的分子機制。方法采用信號通路磷酸化廣譜篩選抗體芯片(PEX100),利用三維高分子膜專利技術(shù),在片基上高密度結(jié)合1318種高特異抗體。對HUVEC細胞的全細胞蛋白進行檢測。共分為四組,空白對照組,3-NP造模組,3-NP+氟桂利嗪組以及3-NP+低頻振動組。以氟桂利嗪,鈣離子阻滯劑作為陽性對照。對4組細胞分別進行檢測432個信號蛋白的679個磷酸化位點。對檢測出的通道進行數(shù)據(jù)分析,公式計算,組間比較,進行統(tǒng)計學數(shù)據(jù)分析。結(jié)果與3-NP造模組未受干預(yù)的細胞相比,低頻振動組增加其磷酸化水平6倍以上的主要信號通路蛋白有: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)。與3-NP造模組未受干預(yù)的細胞相比,低頻振動組增加其磷酸化水平6倍以上的主要信號通路蛋白有: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)。結(jié)論從諸多篩選出的信號通路中,我們可以分析出:對于HUVEC細胞,用3-NP干擾其自噬,用低頻振動進行干預(yù)治療,以氟桂利嗪作為陽性對照,增加磷酸化水平6倍以上的主要信號通路中,低頻振動增強信號通路控制點下游分子磷酸化水平上,有明顯的促進細胞生長,增強自噬,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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R764.3

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