全反式视黄酸（atRA）对人视网膜色素上皮（RPE）屏障功能影响的研究
发布时间：2018-08-26 10:08
【摘要】：第一章人视网膜色素上皮细胞单层屏障模型的建立 目的：体外培养人源的视网膜色素上皮细胞系ARPE-19,探索并建立具有稳定跨上皮电阻(TER)的人视网膜色素上皮细胞单层屏障模型,明确其表达稳定TER值的时间区间。 方法：体外培养人源的视网膜色素上皮细胞系ARPE-19,观察其生长繁殖规律,探索其最佳贴壁生长条件。以细胞角蛋白和S-100蛋白为标志物,利用免疫细胞化学法鉴定ARPE-19细胞系的来源和纯度。接种ARPE-19细胞于Millcell-PET插入式培养池内的微孔滤膜表面,待细胞生长融合为单层后,降低培养液血清浓度至1%继续培养,连续检测并记录上皮细胞单层的跨上皮电阻(TER)值。 结果：体外培养的ARPE-19细胞生长及传代良好,能够顺利贴壁并整齐排列,呈典型的铺路石样单层融合结构。全细胞系表达细胞角蛋白及S-100蛋白阳性率均达100%,无杂细胞污染细胞系。微孔滤膜表面培养ARPE-19细胞生长良好,跨上皮电阻(TER)值随培养时间的延长逐渐增大,约在培养开始后第12天基本稳定,且达50Ω/cm2以上。 结论：ARPE-19细胞体外培养成功,具有良好的生长和传代能力。其细胞纯度高,能较好的保持视网膜色素上皮细胞固有的特征。接种于微孔滤膜表面后,在1%血清浓度培养条件下可以形成具有稳定跨上皮电阻(TER)的上皮细胞单层结构,并可以至少在培养开始后28天内保持其屏障功能特性。为下一步的干预研究奠定了模型基础。 第二章全反式视黄酸对人视网膜色素上皮屏障功能的影响 目的：检测不同浓度干预条件下,全反式视黄酸(atRA)对体外培养的人视网膜色素上皮细胞单层屏障模型的跨上皮电阻(TER)及大分子物质通透性的改变,并以此评价其对上皮屏障功能的影响。 方法：建立具有稳定跨上皮电阻(TER)值的人视网膜色素上皮细胞单层屏障模型。以浓度为10-6M、10-7M、10-8M、0M的全反式视黄酸(atRA)分组干预屏障模型,检测并记录培养开始后第4天、8天、12天、16天、20天、24天和28天的跨上皮电阻(TER)值。选择培养开始后第24天为检测时间点,以辣根过氧化物酶(HRP)为示踪剂,通过化学显色后对其吸光度的测定,反应干预条件下上皮细胞单层屏障对大分子物质的通透性。 结果：浓度为10-8M的全反式视黄酸(atRA)干预后跨上皮电阻(TER)值略有升高,随后回归稳定,与对照组比较差异不明显；浓度为10-7M的atRA干预后TER值升高明显,且稳定于较高水平,与对照组比较有统计学意义；浓度为10-6M的atRA干预后,TER值一过性升高,随后迅速下降,并维持在较低水平。浓度为10-8M和10-7M的atRA可以维持上皮细胞单层对大分子物质的低通透性,以10-7M浓度条件下的atRA作用最为明显。 结论：全反式视黄酸(atRA)能够影响ARPE-19细胞单层的屏障功能。浓度为10-7M及10-8M的atRA能有效地增强细胞单层的跨上皮电阻(TER)值,同时相应地降低其对大分子物质的通透性。浓度为10-6M的atRA能够破坏视网膜色素上皮细胞单层的屏障功能。 第三章全反式视黄酸对人视网膜色素上皮细胞间紧密连接相关蛋白的影响 目的：检测全反式视黄酸(atRA)在影响上皮细胞单层屏障功能过程中对细胞间紧密连接(TJ)结构相关蛋白ZO-1、Occludin和Claudin-1表达的影响。 方法：建立具有稳定跨上皮电阻(TER)的人视网膜色素上皮细胞单层屏障模型。以浓度为10-6M、10-7M、10-8M、0M的全反式视黄酸(atRA)分组干预屏障模型。利用Western Blot技术检测接种后第16及第24天各组细胞间紧密连接(TJ)结构相关蛋白ZO-1、Occludin和Claudin-1表达水平的变化。同时选择接种后第24天为检测时间点,利用免疫荧光技术观察各组细胞间TJ结构完整性的改变。 结果：在浓度为10-7M的全反式视黄酸(atRA)干预下,培养开始后第24天,细胞间紧密连接(TJ)结构相关蛋白中的ZO-1表达明显上调,与对照组比较有统计学意义；Occludin的表达轻度上调,与对照组比较差异无统计学意义；Claudin-1的表达在各干预组均无明显改变。免疫荧光结果与Western Blot结果相符。 结论：一定浓度的全反式视黄酸(atRA)可以影响ARPE-19细胞间紧密连接相关蛋白的表达。其中以ZO-1改变最为明显,Claudin-1无明显改变。atRA对人视网膜色素上皮细胞单层屏障通透性的影响可能是通过改变细胞间紧密连接结构相关蛋白的表达来实现的。.
[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.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1
本文编号：2204532
[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.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1
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