补肾益精方促进自身骨髓干细胞修复干性年龄相关性黄斑变性的作用研究

发布时间：2018-02-03 14:06

本文关键词： 干性年龄相关性黄斑变性骨髓来源干细胞补肾益精方　出处：《中国中医科学院》2017年博士论文　论文类型：学位论文

【摘要】：目的:干性年龄相关性黄斑变性(age-related macular degeneration,AMD)是发达国家65岁以上人群主要的致盲性眼病,属于视网膜退行性疾病的范畴,此类疾病的病理基础是视网膜神经元细胞的不可逆性损伤,目前临床尚无有效的治疗手段。近年细胞替代治疗方法的出现,使视网膜退行性病变的修复成为可能。最新研究发现,通过动员机体自身骨髓来源细胞(bone marrow stem cells,BMCs)亦可达到修复组织损伤的作用,相比传统的骨髓干细胞移植方法更加安全易行。因此,促进自身BMCs动员及其在病变处的归巢、定向分化很可能成为干细胞治疗的新途径。补肾益精方(BSYJ方)是我们临床常用的治疗干性AMD的基本方,前期临床观察发现其能显著改善患者视功能。中医理论中的“精”在内涵和功能上与干细胞具有相似性。基于以上原因,本研究拟探讨补肾益精方促进自身BMCs修复干性AMD损伤的作用及可能机制。方法:1.以碘酸钠诱导的干性AMD小鼠模型作为研究载体,将模型动物随机分为中药组及蒸馏水对照组(对照组),中药组每日予BSYJ方灌胃,对照组以蒸馏水灌胃,以未造模的正常动物为正常对照(正常组),常规饲养。碘酸钠造模小鼠于造模后第一天给药,给药7天、14天、28天后观察视网膜电图(electroretinogram,ERG)和组织病理学指标。2.造模、分组、给药方法同上。分别于治疗后7天、14天、28天处死动物,眼眶取血,采用流式细胞仪检测外周血液中Lin-/Sca-l+/c-kit+细胞数量。同时,用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)方法检测粒细胞集落刺激因子(granulocyte colony stimulating factor,G-CSF)、基质细胞衍生因子-lα(stromal cell derived factor-1,SDF-1α)及其受体趋化因子受体-4(CXC chemokinereceptor 4,CXCR-4)的含量。3.造模、分组、给药方法同上。造模后第1天,移植绿色荧光蛋白(green fluorescent protein,GFP)转基因小鼠(遗传背景为C57/BL6小鼠)的BMCs。每只小鼠经尾静脉注射细胞数3×106,次日开始治疗。分别于治疗后7天、14天、28天处死小鼠,眼眶取血,视网膜组织匀浆,采用ELISA方法检测外周血及视网膜组织SDF-1α、CXCR-4含量。取小鼠眼球,分离视网膜-脉络膜-巩膜复合体,放射状切开铺片,激光共聚焦显微镜下观察携带GFP的BMCs在整个视网膜的分布情况。取小鼠视网膜,采用Western-Blotting方法半定量观察视网膜细胞间黏附因子(intercellular adhesion molecule 1,ICAM-1)以及粘纤连蛋白(fibronectin,FN)的蛋白水平。4.造模、分组、给药方法同上,分别于治疗后7天、14天、28天利用RT-PCR检测视网膜睫状神经营养因子(ciliary neurotrophic factor,CNTF),成纤维细胞生长因子(basic fibroblast growth factor,bFGF),脑源性神经营养因子(brain derived neurotrhophic factor,BDNF)mRNA 表达。结果:1.与正常组比较,造模后对照组和中药组ERG各项指标均显著下降,差异有统计学意义(P0.05)。治疗7d、14d、28d后,与对照组比较,中药组明视条件ERG潜伏期a、潜伏期b、振幅b显著改善,差异具有统计学意义(P0.05)。治疗14d和28d后,与对照组比较,中药组明视条件ERG振幅a显著改善,差异具有统计学意义(P0.05)。治疗28d后,与对照组比较,中药组暗适应条件ERG振幅a、振幅b,最大混合反应振幅a、振幅b,振荡电位振幅Toal,30Hz闪烁光振幅显著改善,差异具有统计学意义(P0.05)。治疗14d和28d后,视网膜感光细胞形态和数量显著改善,差异具有统计学意义(P0.05)。2.治疗7d后,中药组外周血Lin-/Sca-1+/c-kit+细胞相对数量与对照组和正常组比较显著增高(P0.05)。治疗7d、14d和28d后,中药组外周血G-CSF、SDF-1α及其受体CXCR-4含量与对照组相比显著增加(P0.05)。3.治疗7d、14d和28d后,中药组视网膜组织归巢GFP-BMCs数量较对照组组显著增加(P≤0.01)。治疗14d和28d后,对照组外周血和中药组视网膜组织SDF-1α及CXCR-4含量较对照组显著增加(P0.01)。治疗7 d、14 d、28d后,视网膜组织ICAM-1表达显著增加(P0.05)。治疗14d和28d后,视网膜组织FN表达显著增加(P0.05)。4.治疗7d,14d和28d,与对照组比较,视网膜组织中CNTFmRNA表达均显著增加(P0.05)。治疗14d和28d后,视网膜组织BDNF和bFGF mRNA表达显著增加(P0.05)。结论:1.BSYJ方对干性AMD具有保护作用,能延缓干性AMD疾病的进程。2.BSYJ方对碘酸钠诱导干性AMD小鼠BMCs具有动员、迁移作用,这可能与增加相关细胞因子C-GSF含量,启动SDF-1α/CXCR-4信号轴有关。3.BSYJ方能促进BMCs向损伤的视网膜归巢,这可能与其能增加黏附分子ICAM-1和FN的表达有关。4.BSYJ方能延缓干性AMD病理改变,这可能与促进BMCs归巢和其旁分泌CNTF、BDNF和bFGF等神经营养因子有关。
[Abstract]:Objective: dry age-related macular degeneration (age-related macular, degeneration, AMD) is the cause of blindness in people aged over 65 in developed countries, which belongs to the category of retinal degenerative diseases, the pathological basis of this kind of disease is irreversible damage to retinal neurons, at present there is no effective clinical treatment. In recent years, cell replacement therapies so, repair of retinal degeneration as possible. The latest study found that the body through the mobilization of bone marrow cells (bone marrow stem cells, BMCs) can achieve the repair of tissue injury, compared with the traditional methods of bone marrow stem cell transplantation is safe and easy. Therefore, to promote their own BMCs mobilization and homing in lesions that is likely to become the new way of differentiation of stem cell therapy. BSYJF (BSYJ) is commonly used in our clinical treatment of dry AMD the The basic prescription, found it can improve visual function in patients with early clinical observation. The theory of traditional Chinese medicine in the "fine" in connotation and function of stem cells is similar. Based on the above reasons, this paper intends to discuss the BSYJF promote BMCs repair dry AMD injury and its possible mechanism. Methods: 1. to iodate sodium induced dry AMD mice as the research carrier, the animal models were randomly divided into Chinese medicine group and distilled water group (control group), Chinese medicine group BSYJ treated by intragastric administration, the control group with distilled water, the normal animal models for non normal control (normal group), conventional breeding sodium iodate. Mice model of rats in the first day after Drug Administration for 7 days, 14 days, 28 days after the observation of electroretinogram (electroretinogram, ERG) and tissue pathology index.2. model, grouping, administration methods. After the treatment respectively for 7 days, 14 days, 28 days of animal, Orbital blood, the number of Lin-/Sca-l+/c-kit+ cells by flow cytometry in peripheral blood. At the same time, by enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA) method for detection of granulocyte colony-stimulating factor (granulocyte colony, stimulating factor, G-CSF), stromal cell derived factor -l alpha (stromal cell derived factor-1, SDF-1 alpha) and its receptor -4 chemokine receptor (CXC chemokinereceptor 4, CXCR-4) the content of.3. modeling, grouping, administration method ibid. First days after modeling, transplantation of green fluorescent protein (green fluorescent, protein, GFP) transgenic mice (C57/BL6 mice for genetic background) BMCs. per mice by tail vein injection cell number 3 * 106, the next day to begin treatment. After the treatment respectively for 7 days, 14 days, 28 days, the mice were sacrificed, orbital blood, retinal tissue homogenate, ELISA method was used to detect the peripheral blood and retina SDF-1 Alpha, the content of CXCR-4. The mice eyeball, isolated retina choroidal sclera complex, radial incision flatmount was observed under laser confocal microscope with GFP BMCs distribution in the entire retina. The mouse retina, using Western-Blotting method and semi quantitative observation of retinal cell adhesion factor intercellular (adhesion molecule 1, ICAM-1 fibronectin (fibronectin) and viscose, FN) the protein level of.4. model, packet delivery method ibid, respectively in 7 days after treatment, 14 days, 28 days by RT-PCR detection of retinal ciliary neurotrophic factor (ciliary neurotrophic, factor, CNTF), fibroblast growth factor (basic fibroblast growth factor, bFGF), brain-derived neurotrophic factor (brain derived, neurotrhophic factor, BDNF) expression of mRNA. Results: 1. compared with the normal group, model group and traditional Chinese medicine according to the indicators of group ERG were significantly Decreased, the difference was statistically significant (P0.05). The treatment of 7D, 14d, 28d, compared with the control group, Chinese medicine group photopic condition ERG latency of a, latency of B, amplitude of B was significantly improved, the difference was statistically significant (P0.05). The treatment of 14d and 28d, compared with the control group, Chinese medicine group photopic condition ERG amplitude a significantly improved, the difference was statistically significant (P0.05). After 28d treatment, compared with control group, ERG a B amplitude, amplitude of dark adaptation of Chinese medicine group, the largest mixed reaction amplitude A, amplitude B, Toal amplitude oscillatory potentials, 30Hz flashing light amplitude significantly improved, the difference was statistically significant (P0.05) for the treatment of 14d. And 28d, retinal photoreceptor cell morphology and number were significantly improved, the difference was statistically significant (P0.05.2.) after 7d treatment, the Chinese medicine group of peripheral blood Lin-/Sca-1+/c-kit+ cells relatively and the number of control group and normal group were significantly higher (P0.05). The treatment of 7D, 14d and 28d, the Chinese medicine group Peripheral blood G-CSF, SDF-1 and its receptor alpha CXCR-4 content increased significantly compared with the control group (P0.05.3.) for the treatment of 7D, 14d and 28d, the number of Chinese medicine group retinal tissue homing GFP-BMCs increased significantly compared with control group (P = 0.01). Treatment of 14d and 28d, the control group SDF-1 alpha and CXCR-4 content in peripheral blood the Chinese medicine group and the retinal tissue increased significantly compared with the control group (P0.01). The treatment of 7 d, 14 d, 28d, retinal tissue ICAM-1 expression increased significantly (P0.05). The treatment of 14d and 28d, retinal tissue FN expression increased significantly (P0.05).4. 14d and 28d, 7d treatment, compared with control group, the retinal tissue in the expression of CNTFmRNA increased significantly (P0.05). The treatment of 14d and 28d, significantly increased the expression of BDNF and bFGF mRNA retina (P0.05). Conclusion: 1.BSYJ has protective effect on dry AMD, dry AMD can delay the disease process of.2.BSYJ with mobilization of iodine acid sodium induced dry AMD BMCs mice And the transfer function, which may be related to increased cytokine content of C-GSF, start the SDF-1 alpha /CXCR-4 signal axis.3.BSYJ can promote BMCs to damage the retinal homing, this is probably due to the increase of adhesion molecule ICAM-1 and FN on the expression of.4.BSYJ can delay the pathological change of dry AMD, which may promote the homing of BMCs and its adjacent the secretion of CNTF, BDNF and bFGF and other neurotrophic factors.

【学位授予单位】：中国中医科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R774.5

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