【摘要】：第一章人視網(wǎng)膜色素上皮細(xì)胞單層屏障模型的建立 目的：體外培養(yǎng)人源的視網(wǎng)膜色素上皮細(xì)胞系A(chǔ)RPE-19,探索并建立具有穩(wěn)定跨上皮電阻(TER)的人視網(wǎng)膜色素上皮細(xì)胞單層屏障模型,明確其表達(dá)穩(wěn)定TER值的時間區(qū)間。 方法：體外培養(yǎng)人源的視網(wǎng)膜色素上皮細(xì)胞系A(chǔ)RPE-19,觀察其生長繁殖規(guī)律,探索其最佳貼壁生長條件。以細(xì)胞角蛋白和S-100蛋白為標(biāo)志物,利用免疫細(xì)胞化學(xué)法鑒定ARPE-19細(xì)胞系的來源和純度。接種ARPE-19細(xì)胞于Millcell-PET插入式培養(yǎng)池內(nèi)的微孔濾膜表面,待細(xì)胞生長融合為單層后,降低培養(yǎng)液血清濃度至1%繼續(xù)培養(yǎng),連續(xù)檢測并記錄上皮細(xì)胞單層的跨上皮電阻(TER)值。 結(jié)果：體外培養(yǎng)的ARPE-19細(xì)胞生長及傳代良好,能夠順利貼壁并整齊排列,呈典型的鋪路石樣單層融合結(jié)構(gòu)。全細(xì)胞系表達(dá)細(xì)胞角蛋白及S-100蛋白陽性率均達(dá)100%,無雜細(xì)胞污染細(xì)胞系。微孔濾膜表面培養(yǎng)ARPE-19細(xì)胞生長良好,跨上皮電阻(TER)值隨培養(yǎng)時間的延長逐漸增大,約在培養(yǎng)開始后第12天基本穩(wěn)定,且達(dá)50Ω/cm2以上。 結(jié)論：ARPE-19細(xì)胞體外培養(yǎng)成功,具有良好的生長和傳代能力。其細(xì)胞純度高,能較好的保持視網(wǎng)膜色素上皮細(xì)胞固有的特征。接種于微孔濾膜表面后,在1%血清濃度培養(yǎng)條件下可以形成具有穩(wěn)定跨上皮電阻(TER)的上皮細(xì)胞單層結(jié)構(gòu),并可以至少在培養(yǎng)開始后28天內(nèi)保持其屏障功能特性。為下一步的干預(yù)研究奠定了模型基礎(chǔ)。 第二章全反式視黃酸對人視網(wǎng)膜色素上皮屏障功能的影響 目的：檢測不同濃度干預(yù)條件下,全反式視黃酸(atRA)對體外培養(yǎng)的人視網(wǎng)膜色素上皮細(xì)胞單層屏障模型的跨上皮電阻(TER)及大分子物質(zhì)通透性的改變,并以此評價其對上皮屏障功能的影響。 方法：建立具有穩(wěn)定跨上皮電阻(TER)值的人視網(wǎng)膜色素上皮細(xì)胞單層屏障模型。以濃度為10-6M、10-7M、10-8M、0M的全反式視黃酸(atRA)分組干預(yù)屏障模型,檢測并記錄培養(yǎng)開始后第4天、8天、12天、16天、20天、24天和28天的跨上皮電阻(TER)值。選擇培養(yǎng)開始后第24天為檢測時間點(diǎn),以辣根過氧化物酶(HRP)為示蹤劑,通過化學(xué)顯色后對其吸光度的測定,反應(yīng)干預(yù)條件下上皮細(xì)胞單層屏障對大分子物質(zhì)的通透性。 結(jié)果：濃度為10-8M的全反式視黃酸(atRA)干預(yù)后跨上皮電阻(TER)值略有升高,隨后回歸穩(wěn)定,與對照組比較差異不明顯；濃度為10-7M的atRA干預(yù)后TER值升高明顯,且穩(wěn)定于較高水平,與對照組比較有統(tǒng)計學(xué)意義；濃度為10-6M的atRA干預(yù)后,TER值一過性升高,隨后迅速下降,并維持在較低水平。濃度為10-8M和10-7M的atRA可以維持上皮細(xì)胞單層對大分子物質(zhì)的低通透性,以10-7M濃度條件下的atRA作用最為明顯。 結(jié)論：全反式視黃酸(atRA)能夠影響ARPE-19細(xì)胞單層的屏障功能。濃度為10-7M及10-8M的atRA能有效地增強(qiáng)細(xì)胞單層的跨上皮電阻(TER)值,同時相應(yīng)地降低其對大分子物質(zhì)的通透性。濃度為10-6M的atRA能夠破壞視網(wǎng)膜色素上皮細(xì)胞單層的屏障功能。 第三章全反式視黃酸對人視網(wǎng)膜色素上皮細(xì)胞間緊密連接相關(guān)蛋白的影響 目的：檢測全反式視黃酸(atRA)在影響上皮細(xì)胞單層屏障功能過程中對細(xì)胞間緊密連接(TJ)結(jié)構(gòu)相關(guān)蛋白ZO-1、Occludin和Claudin-1表達(dá)的影響。 方法：建立具有穩(wěn)定跨上皮電阻(TER)的人視網(wǎng)膜色素上皮細(xì)胞單層屏障模型。以濃度為10-6M、10-7M、10-8M、0M的全反式視黃酸(atRA)分組干預(yù)屏障模型。利用Western Blot技術(shù)檢測接種后第16及第24天各組細(xì)胞間緊密連接(TJ)結(jié)構(gòu)相關(guān)蛋白ZO-1、Occludin和Claudin-1表達(dá)水平的變化。同時選擇接種后第24天為檢測時間點(diǎn),利用免疫熒光技術(shù)觀察各組細(xì)胞間TJ結(jié)構(gòu)完整性的改變。 結(jié)果：在濃度為10-7M的全反式視黃酸(atRA)干預(yù)下,培養(yǎng)開始后第24天,細(xì)胞間緊密連接(TJ)結(jié)構(gòu)相關(guān)蛋白中的ZO-1表達(dá)明顯上調(diào),與對照組比較有統(tǒng)計學(xué)意義；Occludin的表達(dá)輕度上調(diào),與對照組比較差異無統(tǒng)計學(xué)意義；Claudin-1的表達(dá)在各干預(yù)組均無明顯改變。免疫熒光結(jié)果與Western Blot結(jié)果相符。 結(jié)論：一定濃度的全反式視黃酸(atRA)可以影響ARPE-19細(xì)胞間緊密連接相關(guān)蛋白的表達(dá)。其中以ZO-1改變最為明顯,Claudin-1無明顯改變。atRA對人視網(wǎng)膜色素上皮細(xì)胞單層屏障通透性的影響可能是通過改變細(xì)胞間緊密連接結(jié)構(gòu)相關(guān)蛋白的表達(dá)來實(shí)現(xiàn)的。.
[Abstract]:Chapter 1 Establishment of a monolayer barrier model for human retinal pigment epithelial cells
AIM: To culture human retinal pigment epithelial cell line ARPE-19 in vitro and establish a single layer barrier model of human retinal pigment epithelial cells (RPE-19) with stable transepithelial resistance (TER).
METHODS: Human retinal pigment epithelial cell line ARPE-19 was cultured in vitro to observe its growth and reproduction, and to explore the optimal conditions for adherent growth. On the surface of the microporous membrane, after the cells grew and fused into a single layer, the serum concentration of the culture medium was reduced to 1% and the transepithelial resistance (TER) of the epithelial cells was continuously measured and recorded.
Results: The growth and passage of ARPE-19 cells in vitro were good, and the cells could adhere to the wall smoothly and arrange orderly, showing a typical paving stone-like monolayer fusion structure. R) value increased gradually with the prolongation of culture time, and was basically stable about 12 days after the beginning of culture, and reached more than 50_/cm 2.
CONCLUSION: ARPE-19 cells have been successfully cultured in vitro and possess good growth and passage abilities. The purity of ARPE-19 cells is high and it can maintain the intrinsic characteristics of RPE cells. In order to maintain the barrier function at least 28 days after the culture start, a model was established for further intervention study.
The second chapter is the effect of all trans retinoic acid on the barrier function of human retinal pigment epithelium.
AIM: To investigate the effects of all-trans retinoic acid (ATRA) on the transepithelial resistance (TER) and macromolecule permeability of cultured human retinal pigment epithelial cells (RPE) in vitro.
METHODS: A human retinal pigment epithelial cell monolayer barrier model with stable transepithelial resistance (TER) values was established. All-trans retinoic acid (ATRA) at concentrations of 10-6M, 10-7M, 10-8M, and 0M was used as an intervention barrier model. The transepithelial resistance (TER) values of 4, 8, 12, 16, 20, 24 and 28 days after culture were measured and recorded. Horseradish peroxidase (HRP) was used as a tracer to determine the absorbance of HRP after chemical coloration. The permeability of epithelial cell monolayer barrier to macromolecular substances was measured under the condition of reaction intervention.
Results: The transepithelial resistance (TER) of 10-8M ATRA increased slightly, and then returned to stable level, which was not significantly different from that of the control group. The atRA concentration of 10-8M and 10-7M could maintain the low permeability of epithelial monolayer to macromolecule substances, especially at 10-7M.
CONCLUSION: All-trans retinoic acid (atRA) can affect the barrier function of ARPE-19 cell monolayer. AtRA at concentrations of 10-7M and 10-8M can effectively enhance the transepithelial resistance (TER) of ARPE-19 cell monolayer, and decrease the permeability of ATRA to macromolecules. AtRA at concentrations of 10-6M can destroy the barrier function of retinal pigment epithelial cell monolayer. Yes.
The third chapter is the effect of all trans retinoic acid on tight junction proteins in human retinal pigment epithelial cells.
AIM: To investigate the effects of all-trans retinoic acid (atRA) on the expression of tight junction (TJ) structure-related proteins ZO-1, Occludin and Claudin-1 in epithelial cell monolayer barrier.
METHODS: A human retinal pigment epithelial cell monolayer barrier model with stable transepithelial resistance (TER) was established. All-trans retinoic acid (ATRA) at concentrations of 10-6M, 10-7M, 10-8M, and 0M was used as an intervention barrier model. At the same time, 24 days after inoculation were selected as the detection time point, and the structural integrity of TJ was observed by immunofluorescence technique.
Results: The expression of ZO-1 in TJ structure-related protein was significantly up-regulated 24 days after culture with 10-7M ATRA, and the expression of Occludin was slightly up-regulated, but there was no significant difference between the two groups. No significant changes were found in the intervention group. Immunofluorescence results were consistent with the results of Western Blot.
CONCLUSION: All-trans retinoic acid (atRA) at a certain concentration can affect the expression of tight junction-related proteins in ARPE-19 cells, especially ZO-1 and Claudin-1. The effect of atRA on the permeability of the monolayer barrier of human retinal pigment epithelial cells may be mediated by altering the tight junction-related proteins between cells. Expression to achieve.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R774.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 劉雙珍,蔣莉,王劍峰;視黃酸對培養(yǎng)的雞鞏膜成纖維細(xì)胞增殖及超微結(jié)構(gòu)的影響[J];國際眼科雜志;2005年01期

2 劉雙珍,王華,蔣晶晶,王平寶,吳小影,譚星平,夏朝華;VIPR2在形覺剝奪性近視眼中的動態(tài)表達(dá)[J];中南大學(xué)學(xué)報(醫(yī)學(xué)版);2005年04期

3 王劍鋒;劉雙珍;吳文燦;;外源性視黃酸對雞后鞏膜基質(zhì)MMP-2/TIMP-2表達(dá)作用的動態(tài)觀察[J];眼科研究;2005年06期

4 劉雙珍;王潔月;魏欣;吳小影;譚星平;;白化豚鼠視網(wǎng)膜多巴胺的表達(dá)及其與高度近視的關(guān)系探討[J];眼科研究;2008年10期

5 汪芳潤;近視眼研究的現(xiàn)狀與存在問題[J];中華眼科雜志;2003年06期

6 胡誕寧,StevenA.McCormick;視網(wǎng)膜色素上皮—脈絡(luò)膜在近視發(fā)病中的作用[J];眼視光學(xué)雜志;2000年04期

7 李瑾;瞿小妹;褚仁遠(yuǎn);;全反視黃酸在豚鼠近視形成中的作用[J];眼視光學(xué)雜志;2006年02期

，

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