石斛酚、丁香酸与人晶状体上皮细胞相互作用机制研究

发布时间：2017-12-27 02:07

本文关键词：石斛酚、丁香酸与人晶状体上皮细胞相互作用机制研究　出处：《广州中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:研究石斛酚(gigantol)、丁香酸(syringic acid)与人晶状体上皮细胞(human lens epithelial cells,HLECs)相互作用机制,从单个活细胞的力学形态方面阐明石斛抗糖尿病性白内障(diabetic cataract,DC)的作用机制。方法:1.采用透射电镜(transmission electron microscope,TEM)检测石斛酚、丁香酸作用于高浓度葡萄糖(50mM)造模之后的HLECs的细胞内超微结构,对其细胞内的细胞核、线粒体、内质网等结构变化进行详细地考察;2.在活细胞条件下,利用原子力显微镜(atomic force microscopy,AFM)对石斛酚、丁香酸作用于高葡萄糖造模之后的单个HLECs细胞表面形貌变化进行成像,并通过单个活细胞的表面形貌计算药物作用之后的细胞表面粗糙度与细胞硬度等细胞力学形态性质的改变;3.利用表面增强拉曼光谱(surface-enhanced Raman spectroscopy,SERS),分别对石斛酚、丁香酸作用于高葡萄糖造模时间24h与48h之后的HLECs细胞膜磷脂结构C-C链的链内与链间的结构变化进行考察,并通过拉曼光谱相对强度的变化计算石斛酚、丁香酸对HLECs细胞膜流动性的具体影响;4.利用细胞免疫荧光的检测方法,通过激光共聚焦扫描显微镜考察石斛酚、丁香酸作用于葡萄糖造模之后HLECs细胞的骨架蛋白F-actin的影响。结果:1.通过TEM实验考察结果显示:在正常组中,HLECs细胞内的结构呈现出正常细胞形态,细胞核的染色质均匀分布,并且线粒体嵴清晰、完整;在模型组中,利用高浓度葡萄糖对HLECs造模之后,HLECs内的细胞结构表现出明显病理特征,细胞质里面出现大量空泡现象,细胞核内的染色质发生明显沉积,颜色加深,而细胞器线粒体则表现出明显的肿胀现象,其线粒体嵴发生断裂,及纹路模糊的现象,内质网也呈现出明显的肿胀现象;而通过药物石斛酚、丁香酸作用于之后,HLECs细胞内结构的病理程度得到相应地改善,可以发现细胞核中染色质聚集现象减轻,分布较均匀,而线粒体与内质网的肿胀程度也得到相应地减轻,线粒体嵴恢复至完整状态,并且石斛酚药物组对高浓度葡萄糖造模之后HLECs细胞内结构病理变化的减轻程度优于丁香酸药物组。2.通过AFM在活细胞条件下对HLECs的细胞表面形貌进行考察之后,结果显示:在正常组中,细胞高度为3.5±0.35μm,细胞表面粗糙度Ra为112±3.25nm,Rq 为 155±2.98nm,细胞硬度为 5.6±0.56KPa;在模型组中,HLECs细胞高度与细胞硬度发生了明显地变化,细胞高度为1.8±0.25μm,细胞表面粗糙度Ra为144±2.16nm,Rq为176±2.67nm,细胞硬度为11.6±0.91KPa,细胞高度明显降低,细胞粗糙度与细胞硬度明显升高,与正常组相比,差异具有统计学意义,P0.01;石斛酚药物组,细胞高度为3.1±0.15μm,细胞表面粗糙度Ra为114±3.45nm,Rq为135±5.12nm,细胞硬度为7.5±0.45KPa,与模型组相比,细胞高度明显上升,细胞粗糙度与细胞硬度明显降低,差异具有统计学意义,P0.01;丁香酸药物组中,细胞高度为 2.8±0.25μm,Ra 为 132±4.12nm,Rq 为 161±3.91nm,细胞硬度为8.9±0.45KPa,与模型组相比,细胞高度明显上升,细胞表面粗糙度与细胞硬度降低,差异具有统计学意义,P0.05;石斛酚与丁香酸药物联合组中,3.8±0.43μm,细胞表面粗糙度Ra为104±4.21nm,Rq为121±2.76nm,细胞硬度为7.51±0.31KPa,与模型组相比,细胞高度升高,粗糙度与细胞硬度降低,差异具有统计学意义,P0.01。此结果显示出石斛酚、丁香酸可以在一定程度上恢复高糖造模之后的HLECs的细胞表面粗糙度、细胞硬度等细胞力学形态性质;3.通过拉曼光谱实验,结果显示:在模型中,HLECs的细胞膜磷脂结构中的C-C链内的纵向有序性参数Strans和横向相互作用参数Slat较正常组升高了 25%和25.9%,细胞膜的流动性降低,差异具有统计学意义,P0.01;在石斛酚组中,Strans与Slat较模型组降低了 16%和20%,细胞膜的流动性上升,差异具有统计学意义,P0.01;在丁香酸组中,Strans与Slat较模型组降低了 2.9%和3%;石斛酚与丁香酸联合组中,Strans与Slat较模型组降低了 5%和20.8%,细胞膜的流动性上升,差异具有统计学意义,P0.01。由此,药物石斛酚、丁香酸可以在一定程度上有效地升高HLECs的细胞流动性。4.通过激光共聚焦对细胞免疫荧光检测之后,结果显示:在正常组中,细胞骨架蛋白F-actin的排列有序,靠近细胞膜;在模型组中,骨架蛋白F-actin的排列表现出弥散,杂乱现象,在细胞膜附近的比列减少;而在石斛酚、丁香酸药物组,使HLECs的细胞骨架蛋白F-actin的表达与排列有序程度得到一定程度上的恢复,显示出药物石斛酚与丁香酸可以在一定程度上恢复细胞HLECs的细胞骨架的有序性。结论:石斛酚、丁香酸能有效地降低高浓度葡萄糖所致的HLECs细胞内结构的病理损伤,以及高糖造模之后的HLECs细胞力学形态的变化。在此过程中石斛酚、丁香酸通过影响HLECs的细胞膜脂质结构的C-C链及细胞骨架结构的有序度达到增强HLECs的细胞流动性,进一步有效降低HLECs细胞膜表面粗糙度和细胞硬度,由此达到改善细胞力学形态作用。由此石斛酚与丁香酸可以通过改变HLECs的细胞力学形态进一步发挥抗糖尿病性白内障的药理作用,在单细胞动态成像水平上为石斛抗糖尿病性白内障提供基础资料。
[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
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R776.1

【参考文献】