本文關(guān)鍵詞： 干性年齡相關(guān)性黃斑變性 骨髓來(lái)源干細(xì)胞 補(bǔ)腎益精方　出處：《中國(guó)中醫(yī)科學(xué)院》2017年博士論文　論文類型：學(xué)位論文

【摘要】：目的:干性年齡相關(guān)性黃斑變性(age-related macular degeneration,AMD)是發(fā)達(dá)國(guó)家65歲以上人群主要的致盲性眼病,屬于視網(wǎng)膜退行性疾病的范疇,此類疾病的病理基礎(chǔ)是視網(wǎng)膜神經(jīng)元細(xì)胞的不可逆性損傷,目前臨床尚無(wú)有效的治療手段。近年細(xì)胞替代治療方法的出現(xiàn),使視網(wǎng)膜退行性病變的修復(fù)成為可能。最新研究發(fā)現(xiàn),通過動(dòng)員機(jī)體自身骨髓來(lái)源細(xì)胞(bone marrow stem cells,BMCs)亦可達(dá)到修復(fù)組織損傷的作用,相比傳統(tǒng)的骨髓干細(xì)胞移植方法更加安全易行。因此,促進(jìn)自身BMCs動(dòng)員及其在病變處的歸巢、定向分化很可能成為干細(xì)胞治療的新途徑。補(bǔ)腎益精方(BSYJ方)是我們臨床常用的治療干性AMD的基本方,前期臨床觀察發(fā)現(xiàn)其能顯著改善患者視功能。中醫(yī)理論中的“精”在內(nèi)涵和功能上與干細(xì)胞具有相似性�；谝陨显�,本研究擬探討補(bǔ)腎益精方促進(jìn)自身BMCs修復(fù)干性AMD損傷的作用及可能機(jī)制。方法:1.以碘酸鈉誘導(dǎo)的干性AMD小鼠模型作為研究載體,將模型動(dòng)物隨機(jī)分為中藥組及蒸餾水對(duì)照組(對(duì)照組),中藥組每日予BSYJ方灌胃,對(duì)照組以蒸餾水灌胃,以未造模的正常動(dòng)物為正常對(duì)照(正常組),常規(guī)飼養(yǎng)。碘酸鈉造模小鼠于造模后第一天給藥,給藥7天、14天、28天后觀察視網(wǎng)膜電圖(electroretinogram,ERG)和組織病理學(xué)指標(biāo)。2.造模、分組、給藥方法同上。分別于治療后7天、14天、28天處死動(dòng)物,眼眶取血,采用流式細(xì)胞儀檢測(cè)外周血液中Lin-/Sca-l+/c-kit+細(xì)胞數(shù)量。同時(shí),用酶聯(lián)免疫吸附試驗(yàn)(enzyme linked immunosorbent assay,ELISA)方法檢測(cè)粒細(xì)胞集落刺激因子(granulocyte colony stimulating factor,G-CSF)、基質(zhì)細(xì)胞衍生因子-lα(stromal cell derived factor-1,SDF-1α)及其受體趨化因子受體-4(CXC chemokinereceptor 4,CXCR-4)的含量。3.造模、分組、給藥方法同上。造模后第1天,移植綠色熒光蛋白(green fluorescent protein,GFP)轉(zhuǎn)基因小鼠(遺傳背景為C57/BL6小鼠)的BMCs。每只小鼠經(jīng)尾靜脈注射細(xì)胞數(shù)3×106,次日開始治療。分別于治療后7天、14天、28天處死小鼠,眼眶取血,視網(wǎng)膜組織勻漿,采用ELISA方法檢測(cè)外周血及視網(wǎng)膜組織SDF-1α、CXCR-4含量。取小鼠眼球,分離視網(wǎng)膜-脈絡(luò)膜-鞏膜復(fù)合體,放射狀切開鋪片,激光共聚焦顯微鏡下觀察攜帶GFP的BMCs在整個(gè)視網(wǎng)膜的分布情況。取小鼠視網(wǎng)膜,采用Western-Blotting方法半定量觀察視網(wǎng)膜細(xì)胞間黏附因子(intercellular adhesion molecule 1,ICAM-1)以及粘纖連蛋白(fibronectin,FN)的蛋白水平。4.造模、分組、給藥方法同上,分別于治療后7天、14天、28天利用RT-PCR檢測(cè)視網(wǎng)膜睫狀神經(jīng)營(yíng)養(yǎng)因子(ciliary neurotrophic factor,CNTF),成纖維細(xì)胞生長(zhǎng)因子(basic fibroblast growth factor,bFGF),腦源性神經(jīng)營(yíng)養(yǎng)因子(brain derived neurotrhophic factor,BDNF)mRNA 表達(dá)。結(jié)果:1.與正常組比較,造模后對(duì)照組和中藥組ERG各項(xiàng)指標(biāo)均顯著下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。治療7d、14d、28d后,與對(duì)照組比較,中藥組明視條件ERG潛伏期a、潛伏期b、振幅b顯著改善,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。治療14d和28d后,與對(duì)照組比較,中藥組明視條件ERG振幅a顯著改善,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。治療28d后,與對(duì)照組比較,中藥組暗適應(yīng)條件ERG振幅a、振幅b,最大混合反應(yīng)振幅a、振幅b,振蕩電位振幅Toal,30Hz閃爍光振幅顯著改善,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。治療14d和28d后,視網(wǎng)膜感光細(xì)胞形態(tài)和數(shù)量顯著改善,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。2.治療7d后,中藥組外周血Lin-/Sca-1+/c-kit+細(xì)胞相對(duì)數(shù)量與對(duì)照組和正常組比較顯著增高(P0.05)。治療7d、14d和28d后,中藥組外周血G-CSF、SDF-1α及其受體CXCR-4含量與對(duì)照組相比顯著增加(P0.05)。3.治療7d、14d和28d后,中藥組視網(wǎng)膜組織歸巢GFP-BMCs數(shù)量較對(duì)照組組顯著增加(P≤0.01)。治療14d和28d后,對(duì)照組外周血和中藥組視網(wǎng)膜組織SDF-1α及CXCR-4含量較對(duì)照組顯著增加(P0.01)。治療7 d、14 d、28d后,視網(wǎng)膜組織ICAM-1表達(dá)顯著增加(P0.05)。治療14d和28d后,視網(wǎng)膜組織FN表達(dá)顯著增加(P0.05)。4.治療7d,14d和28d,與對(duì)照組比較,視網(wǎng)膜組織中CNTFmRNA表達(dá)均顯著增加(P0.05)。治療14d和28d后,視網(wǎng)膜組織BDNF和bFGF mRNA表達(dá)顯著增加(P0.05)。結(jié)論:1.BSYJ方對(duì)干性AMD具有保護(hù)作用,能延緩干性AMD疾病的進(jìn)程。2.BSYJ方對(duì)碘酸鈉誘導(dǎo)干性AMD小鼠BMCs具有動(dòng)員、遷移作用,這可能與增加相關(guān)細(xì)胞因子C-GSF含量,啟動(dòng)SDF-1α/CXCR-4信號(hào)軸有關(guān)。3.BSYJ方能促進(jìn)BMCs向損傷的視網(wǎng)膜歸巢,這可能與其能增加黏附分子ICAM-1和FN的表達(dá)有關(guān)。4.BSYJ方能延緩干性AMD病理改變,這可能與促進(jìn)BMCs歸巢和其旁分泌CNTF、BDNF和bFGF等神經(jīng)營(yíng)養(yǎng)因子有關(guān)。
[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.

【學(xué)位授予單位】：中國(guó)中醫(yī)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R774.5

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