本文關(guān)鍵詞：石斛酚、丁香酸與人晶狀體上皮細胞相互作用機制研究　出處：《廣州中醫(yī)藥大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 石斛酚 丁香酸 晶狀體上皮細胞 相互作用 細胞硬度 細胞流動性

【摘要】：目的:研究石斛酚(gigantol)、丁香酸(syringic acid)與人晶狀體上皮細胞(human lens epithelial cells,HLECs)相互作用機制,從單個活細胞的力學形態(tài)方面闡明石斛抗糖尿病性白內(nèi)障(diabetic cataract,DC)的作用機制。方法:1.采用透射電鏡(transmission electron microscope,TEM)檢測石斛酚、丁香酸作用于高濃度葡萄糖(50mM)造模之后的HLECs的細胞內(nèi)超微結(jié)構(gòu),對其細胞內(nèi)的細胞核、線粒體、內(nèi)質(zhì)網(wǎng)等結(jié)構(gòu)變化進行詳細地考察;2.在活細胞條件下,利用原子力顯微鏡(atomic force microscopy,AFM)對石斛酚、丁香酸作用于高葡萄糖造模之后的單個HLECs細胞表面形貌變化進行成像,并通過單個活細胞的表面形貌計算藥物作用之后的細胞表面粗糙度與細胞硬度等細胞力學形態(tài)性質(zhì)的改變;3.利用表面增強拉曼光譜(surface-enhanced Raman spectroscopy,SERS),分別對石斛酚、丁香酸作用于高葡萄糖造模時間24h與48h之后的HLECs細胞膜磷脂結(jié)構(gòu)C-C鏈的鏈內(nèi)與鏈間的結(jié)構(gòu)變化進行考察,并通過拉曼光譜相對強度的變化計算石斛酚、丁香酸對HLECs細胞膜流動性的具體影響;4.利用細胞免疫熒光的檢測方法,通過激光共聚焦掃描顯微鏡考察石斛酚、丁香酸作用于葡萄糖造模之后HLECs細胞的骨架蛋白F-actin的影響。結(jié)果:1.通過TEM實驗考察結(jié)果顯示:在正常組中,HLECs細胞內(nèi)的結(jié)構(gòu)呈現(xiàn)出正常細胞形態(tài),細胞核的染色質(zhì)均勻分布,并且線粒體嵴清晰、完整;在模型組中,利用高濃度葡萄糖對HLECs造模之后,HLECs內(nèi)的細胞結(jié)構(gòu)表現(xiàn)出明顯病理特征,細胞質(zhì)里面出現(xiàn)大量空泡現(xiàn)象,細胞核內(nèi)的染色質(zhì)發(fā)生明顯沉積,顏色加深,而細胞器線粒體則表現(xiàn)出明顯的腫脹現(xiàn)象,其線粒體嵴發(fā)生斷裂,及紋路模糊的現(xiàn)象,內(nèi)質(zhì)網(wǎng)也呈現(xiàn)出明顯的腫脹現(xiàn)象;而通過藥物石斛酚、丁香酸作用于之后,HLECs細胞內(nèi)結(jié)構(gòu)的病理程度得到相應(yīng)地改善,可以發(fā)現(xiàn)細胞核中染色質(zhì)聚集現(xiàn)象減輕,分布較均勻,而線粒體與內(nèi)質(zhì)網(wǎng)的腫脹程度也得到相應(yīng)地減輕,線粒體嵴恢復至完整狀態(tài),并且石斛酚藥物組對高濃度葡萄糖造模之后HLECs細胞內(nèi)結(jié)構(gòu)病理變化的減輕程度優(yōu)于丁香酸藥物組。2.通過AFM在活細胞條件下對HLECs的細胞表面形貌進行考察之后,結(jié)果顯示:在正常組中,細胞高度為3.5±0.35μm,細胞表面粗糙度Ra為112±3.25nm,Rq 為 155±2.98nm,細胞硬度為 5.6±0.56KPa;在模型組中,HLECs細胞高度與細胞硬度發(fā)生了明顯地變化,細胞高度為1.8±0.25μm,細胞表面粗糙度Ra為144±2.16nm,Rq為176±2.67nm,細胞硬度為11.6±0.91KPa,細胞高度明顯降低,細胞粗糙度與細胞硬度明顯升高,與正常組相比,差異具有統(tǒng)計學意義,P0.01;石斛酚藥物組,細胞高度為3.1±0.15μm,細胞表面粗糙度Ra為114±3.45nm,Rq為135±5.12nm,細胞硬度為7.5±0.45KPa,與模型組相比,細胞高度明顯上升,細胞粗糙度與細胞硬度明顯降低,差異具有統(tǒng)計學意義,P0.01;丁香酸藥物組中,細胞高度為 2.8±0.25μm,Ra 為 132±4.12nm,Rq 為 161±3.91nm,細胞硬度為8.9±0.45KPa,與模型組相比,細胞高度明顯上升,細胞表面粗糙度與細胞硬度降低,差異具有統(tǒng)計學意義,P0.05;石斛酚與丁香酸藥物聯(lián)合組中,3.8±0.43μm,細胞表面粗糙度Ra為104±4.21nm,Rq為121±2.76nm,細胞硬度為7.51±0.31KPa,與模型組相比,細胞高度升高,粗糙度與細胞硬度降低,差異具有統(tǒng)計學意義,P0.01。此結(jié)果顯示出石斛酚、丁香酸可以在一定程度上恢復高糖造模之后的HLECs的細胞表面粗糙度、細胞硬度等細胞力學形態(tài)性質(zhì);3.通過拉曼光譜實驗,結(jié)果顯示:在模型中,HLECs的細胞膜磷脂結(jié)構(gòu)中的C-C鏈內(nèi)的縱向有序性參數(shù)Strans和橫向相互作用參數(shù)Slat較正常組升高了 25%和25.9%,細胞膜的流動性降低,差異具有統(tǒng)計學意義,P0.01;在石斛酚組中,Strans與Slat較模型組降低了 16%和20%,細胞膜的流動性上升,差異具有統(tǒng)計學意義,P0.01;在丁香酸組中,Strans與Slat較模型組降低了 2.9%和3%;石斛酚與丁香酸聯(lián)合組中,Strans與Slat較模型組降低了 5%和20.8%,細胞膜的流動性上升,差異具有統(tǒng)計學意義,P0.01。由此,藥物石斛酚、丁香酸可以在一定程度上有效地升高HLECs的細胞流動性。4.通過激光共聚焦對細胞免疫熒光檢測之后,結(jié)果顯示:在正常組中,細胞骨架蛋白F-actin的排列有序,靠近細胞膜;在模型組中,骨架蛋白F-actin的排列表現(xiàn)出彌散,雜亂現(xiàn)象,在細胞膜附近的比列減少;而在石斛酚、丁香酸藥物組,使HLECs的細胞骨架蛋白F-actin的表達與排列有序程度得到一定程度上的恢復,顯示出藥物石斛酚與丁香酸可以在一定程度上恢復細胞HLECs的細胞骨架的有序性。結(jié)論:石斛酚、丁香酸能有效地降低高濃度葡萄糖所致的HLECs細胞內(nèi)結(jié)構(gòu)的病理損傷,以及高糖造模之后的HLECs細胞力學形態(tài)的變化。在此過程中石斛酚、丁香酸通過影響HLECs的細胞膜脂質(zhì)結(jié)構(gòu)的C-C鏈及細胞骨架結(jié)構(gòu)的有序度達到增強HLECs的細胞流動性,進一步有效降低HLECs細胞膜表面粗糙度和細胞硬度,由此達到改善細胞力學形態(tài)作用。由此石斛酚與丁香酸可以通過改變HLECs的細胞力學形態(tài)進一步發(fā)揮抗糖尿病性白內(nèi)障的藥理作用,在單細胞動態(tài)成像水平上為石斛抗糖尿病性白內(nèi)障提供基礎(chǔ)資料。
[Abstract]:Objective: To study the Dendrobium phenol (gigantol), Ding Xiangsuan (syringic acid) and human lens epithelial cells (human lens epithelial cells, HLECs) interaction mechanism, from a single live cell to clarify the mechanical state of Dendrobium anti diabetic cataract (diabetic cataract DC) mechanism. Methods: 1. using transmission electron microscopy (transmission electron microscope, TEM) detection of gigantol was syringic acid in high concentration of glucose (50mM) rats after HLECs cell ultrastructure of nucleus, mitochondria and endoplasmic reticulum for changes in the structure of cells in a detailed study in living cells under the condition of 2.; by using atomic force microscopy (atomic, force microscopy, AFM) imaging of single HLECs cell surface morphology changes after gigantol was syringic acid in high glucose model, and through the single living cell surface morphology after the drug effect calculation of surface roughness and hardness of cell cell cell morphology mechanical properties change; 3. the use of surface enhanced Raman spectroscopy (surface-enhanced Raman spectroscopy, SERS), HLECs cells of gigantol was syringic acid in high glucose modeling time 24h and 48h respectively. The structure of C-C membrane phospholipid chain in the structure change and inter chain is discussed, and the specific effects of the flow of gigantol was syringic acid on HLECs cell membrane by changing the relative intensity of the Raman calculation; 4. were detected by immunofluorescence methods, by confocal laser scanning microscopy study gigantol was syringic acid after effect in the model glucose affect HLECs cell skeleton protein F-actin. Results: 1. through TEM experimental investigation results show that: in the normal group, the structure of HLECs cells showed normal cell morphology, chromatin nucleus distribution, and mitochondrial cristae were clear and complete; in the model group, the HLECs model with high concentration of glucose, cell structure in HLECs showed obvious pathology characteristics of cytoplasmic vacuoles inside phenomenon, chromatin within the nucleus was deposited, the color deepened, and the mitochondria showed swelling phenomenon obviously, the mitochondrial cristae fracture, and texture fuzzy phenomenon, the endoplasmic reticulum also showed obvious swelling; and by drugs of Dendrobium phenol, clove the role of acid in HLECs cells, the pathological degree of the structure has been accordingly improved, can be found in nuclear chromatin aggregation reduced, more uniform distribution, and the mitochondria and endoplasmic reticulum swelling The degree of mitochondrial crest returned to the intact state, and the degree of structural pathological changes in HLECs cells after the establishment of high concentration glucose was better than that of the syringic acid group. After that, 2. by AFM in living cells under the condition of cell surface morphology of the HLECs study results showed that: in the normal group, the cell height was 3.5 + 0.35 m, the cell surface roughness Ra is 112 + 3.25nm, Rq + 2.98nm cells was 155, the hardness was 5.6 + 0.56KPa; in the model group, HLECs cell height and hardness of cells changed significantly, cell height was 1.8 + 0.25 m, the cell surface roughness Ra is 144 + 2.16nm, Rq + 2.67nm cells was 176, the hardness is 11.6 + 0.91KPa, cell height significantly decreased, roughness and hardness of cell cells increased significantly compared with the normal group, with statistical the significance of differences, P0.01; Dendrobium drug group, cell height was 3.1 + 0.15 m, the cell surface roughness Ra is 114 + 3.45nm, Rq + 5.12nm cells was 135, the hardness is 7.5 + 0.45KPa, compared with the model group, the cell height increased obviously, roughness and hardness of cell cells decreased obviously, the difference Wasstatisticallysignificant, syringic acid P0.01; drug group, cell height was 2.8 + 0.25 m, Ra 132 + 4.12nm, Rq + 3.91nm cells was 161, the hardness is 8.9 + 0.45KPa, compared with the model group, the cell height increased obviously, cell surface roughness and hardness of cells decreased, with statistical difference meaning, P0.05; Dendrobium phenol and syringic acid combined drug group, 3.8 + 0.43 m, the cell surface roughness Ra is 104 + 4.21nm, Rq + 2.76nm cells was 121, the hardness is 7.51 + 0.31KPa, compared with the model group, cell height, roughness and hardness were decreased, the difference was statistically significant. P0.01. The results show that the surface of gigantol was syringic acid can be made after the recovery of high glucose to a certain extent HLECs cell roughness, hardness and mechanical properties of cell cell morphology; 3. by Raman spectroscopy experiments, the results show that: in the model, the structure of cell membrane phospholipids in HLECs C-C chain in vertical order parameter Strans and lateral interaction parameters of Slat compared to the normal group increased by 25% and 25.9%, the cell membrane fluidity decreased, the difference was statistically significant, P0.01; phenol in Dendrobium group, Strans and Slat decreased by 16% and 20% compared with the model group, the cell membrane fluidity increased, with statistical significance, the difference of P0.01 in clove; acid group, Strans and Slat decreased by 2.9% and 3% compared to the model group; Dendrobium phenol and syringic acid group, Strans and Slat decreased by 5% and 20.8% compared with the model group, the cell membrane fluidity increased, the difference has statistical significance, P0.01 . Thus, dendrobiol and syringic acid can increase the cell mobility of HLECs to a certain extent. 4. by confocal laser on after immunofluorescence test results showed: in normal group, cytoskeletal protein F-actin orderly arranged near the cell membrane; in the model group, the arrangement of cytoskeletal protein F-actin showed diffuse, chaotic phenomenon, in the vicinity of the cell membrane, and decreased; phenol, syringic acid in Dendrobium the drug group, the expression of F-actin HLECs and the ordered degree to obtain a certain degree of recovery, showing order of cytoskeletal drugs of Dendrobium phenol and syringic acid can restore HLECs cells to a certain extent. Conclusion: dendrobiol and syringic acid can effectively reduce the intracellular structure of HLECs cells caused by high concentration of glucose
【學位授予單位】：廣州中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R587.2;R776.1

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