【作者】 马修彬；

【导师】 陈松；

【作者基本信息】 天津医科大学 ， 眼科学， 2008， 硕士

【摘要】 目的:蓝光光照可以引起人视网膜色素上皮(retinal pigment epithelium,RPE)细胞发生视网膜光损伤。研究发现,紫外光(ultraviolet,UV)可以引起牛RPE细胞复制衰老。蓝光和紫外光对RPE细胞有类似的损伤,本研究目的在于观察蓝光能否诱导人RPE细胞发生复制衰老及蓝光对细胞内趋化因子信使核糖核酸(messenger ribonucleic acid,mRNA)表达的影响,探讨复制衰老与人RPE细胞内趋化因子的关系,为探讨蓝光损伤、复制衰老及炎症在老年性黄斑变性(age-related macular degeneration,AMD)发病机制方面提供理论根据。方法:1.取意外死亡的健康成年男性眼球,作角膜移植后,采用胰蛋白酶消化法培养人原代RPE细胞,观察细胞的生长状况及特性,免疫组化鉴定细胞来源。2.将细胞分成实验组和对照组,实验组细胞培养瓶置于蓝光发光二极管(lightemitting diode,LED)光源下照射,对照组用黑纸完全包裹培养瓶,继续培养。3.将各组细胞的第3代到第5代,消化后调整细胞密度为2×10~5个/ml,接种于24孔板培养24小时,进行衰老相关-β半乳糖苷酶(senescence associatedbeta-galactosidae,SA-β-Gal)活性的检测。高倍镜下观察细胞内是否有蓝色颗粒的表达,随机选取5个视野计数阳性细胞数目。4.将各组细胞的第3代到第5代,消化后调整细胞密度为1×10~6个/ml,接种于6孔板培养24小时,半定量逆转录聚合酶联反应(reversedtranscript-polymerase chain reaction,RT-PCR)法检测细胞内趋化因子mRNA的表达情况。5.采用SPSS 13.0统计软件,所有数据用均数±标准差((?)±s)表示,经正态性检验和方差齐性检验,方差齐后进行多因素方差分析,并用LSD-t检验对各组均数之间进行两两比较。复制衰老和细胞内趋化因子的表达之间的关系用Pearson相关关系分析,P＜0.05具有统计学意义。结果:1.体外培养的人原代RPE细胞生长良好,附壁后呈圆形,或扁平多角形,富含色素颗粒,传代稳定,传代后色素颗粒明显减少,免疫组化鉴定抗角蛋白抗原阳性。2.随着蓝光光照度的增加,复制衰老阳性细胞数目逐渐增多,不同光照度之间差异有统计学意义(P=0.0031);在不同的光照度之间,LSD-t两两比较发现,除中光照与高光照组相比,差异没有统计学意义(P=0.094)外,其余每两组之间的差异均具有统计学意义(P＜0.05);随着传代次数增加,复制衰老阳性细胞数目逐渐增多,不同代次数之间差异具有统计学意义(P=0.0057);在不同的传代次数之间,LSD-t两两比较发现,除第4代和第5代之间差异没有统计学意义(p=0.059)外,其余每两组之间的差异均具有统计学意义(P＜0.05)。3.通过半定量RT-PCR法检测,随光照度增加,细胞内趋化因子的mRNA表达增加,不同光照度之间差异有统计学意义(P＜0.01);在不同的光照度之间,LSD-t两两比较发现,除中光照与高光照组相比差别没有统计学意义(P＞0.05)外,其余每两组之间的差异均具有统计学意义(P＜0.05)。细胞内趋化因子的mRNA表达随传代次数增加而表达增加,不同传代次数之间差异具有统计学意义(P＜0.01);在不同的传代次数之间,LSD-t两两比较发现,除第4代和第5代之间差异没有统计学意义(P＞0.05)外,其余每两组之间的差异均具有统计学意义(P＜0.05)。4.随着人RPE细胞复制衰老的增加,细胞内趋化因子的表达增加,复制衰老和趋化因子白介素8(interleukin-8,IL-8)之间相关具有统计学意义(r=0.736,P=0.0087);复制衰老和趋化因子单核细胞趋化蛋白1(monocytechemoattractant protein-1,MCP-1)之间相关具有统计学意义(r=0.682,P=0.015)。结论:1.应用胰蛋白酶消化法,成功的培养了人原代RPE细胞,经免疫组化鉴定,可以作为人RPE细胞来源,细胞生长良好,可以正常传代,而且培养的人原代RPE细胞形态和数量基本可以满足实验的需要。2.本研究应用SA-β-Gal染色的方法,证实了蓝光可以诱导体外培养人RPE细胞复制衰老,并且光照度和传代次数可促进细胞复制衰老。3.本研究用半定量RT-PCR法检测了人RPE细胞内趋化因子mRNA的表达情况,证实了蓝光可以促进人RPE细胞内趋化因子的表达,并且光照度和传代次数增加,可以促进细胞内趋化因子的表达。4.本研究证实了人RPE细胞复制衰老的增加,可以促进细胞内趋化因子的表达,因此,当人RPE细胞发生复制衰老时,细胞内趋化因子的mRNA表达上调。5.本研究率先将蓝光、RPE细胞复制衰老、和趋化因子结合起来,对于蓝光诱导人RPE细胞复制衰老的机制研究提供理论支持,对于蓝光诱导细胞内趋化因子的表达提供理论支持,对于人RPE复制衰老细胞内趋化因子mRNA上调提供理论支持,对于阐释蓝光光照、RPE细胞复制衰老和炎症在AMD的发病机制中的作用提供理论依据。更多还原

【Abstract】 Objective:A blue light can induce human retinal pigment epithelium(RPE) cells to be retinal photo-damage. A study indicated that ultraviolet(UV) can make RPE cells replicative senescence. The damage of blue light is similar to UV. The aim of the experiment is to observe the effects of blue light on human RPE cells replicative senescence and expression of Chemokines messenger ribonucleic acid(mRNA) in RPE cell. We want to find the relationship between replicative senescence and Chemokines. It is to be discussed the role of blue light, RPE replicative senescence and inflammatory reaction in pathogenesis of age-related macular degeneration (AMD).Methods:1. After corneal transplantation, healthy human eyeball which came from human died by accident was used for experiments. Primary human RPE culture was harvested with trypsin digestion. Cell morphology and characterization were assessed by phase contrast microscopy. The cells were identified with immunohistochemistry.2. The cells were distributed into experiment and control groups. The experiment groups were placed under exposure of blue light source of light emitting diode(LED). The control group was encapsulated by black paper during exposure.3. All the groups from passage 3 to passage 5 were seeded at a density of 2×10~5 cells/ml into 24 well culture plate for 24h to detect senescence associated beta-galactosidae(SA-β-Gal) activity. RPE cells replicative senescence was defined of blue particles in plasma of cells. Positive cells were counted in 5 selected random high power microscope fields.4. All the cells from passage 3 to passage 5 were seeded at a density of 1×10~6 cells/ml into 6 well culture plates for 24h and followed by semiquantitative RT-PCR assay to detect expressions of Chemokines mRNA in RPE cells.5. All data was showed with mean±standard deviation (x|-±s) and was dealt by SPSS 13.0. After test of normality and homogeneity test for variance, multiple factor analysis of variance was used if equal variance assumed followed by multiple comparisons with test of LSD-t. The relationship between replicative senescence and chemukines were dealt by Pearson correlation coefficient. The mean difference was significant at the level of 0.05(which is checked by professor zheng yuezhong who is from Tianjin eye hospital).Results:1. This primary culture resulted in cells was well-preserved morphology. some cells after adhere was round or irregular. It was rich in pigment granules. The next passages were grew well and were the pigment granules decreased obviousely. The result of immunohistochemistry was positive of the antigen of anti-keratin.2. While the number of the replicative senescence cells with blue particles increased as the function of exposure intensity. The difference within the different light exposure intensity had statistics significance (P=0.0031). After two was dealt by LSD-t, we found that except for the difference between high light intensity and middle light intensity, every other two groups had statistics significance (P<0.05). Between different passages, the difference within the different passages had statistics significance (P=0.0094). Except for the difference between passage 4 and passage 5, the difference between every other two passage had statistics significance (P<0.05).3. Different intensity of blue light was detected by semiquantitative RT-PCR assay. The exprressions Chemokines mRNA in RPE cells increased as the increase of exposure intensity. The difference within the different light exposure intensity had statistics significance (P<0.01). After two was dealt by LSD-t, we found that except for the difference between high light intensity and middle light intensity, every other two groups had statistics significance (P<0.05). Between different passages, the difference within the different passages had statistics significance (P<0.01). Except for the difference between passage 4 and passage 5, the difference between every other two passage had statistics significance (P<0.05).4. The Chemokines in RPE cells were increased as the replicative senescence cells increased. The relation between replicative senescence and interleukin-8(IL-8) had statistics significance(r=0.736, P=0.0087). The relation between replicative senescence and monocyte chemoattractant protein-1 (MCP-1) had statistics significance (r=0.682, P=0.015).Conclusions:1. Primary human RPE culture is harvested with trypsin digestion. They can become the source of the human RPE cell which are checked by immunohistochemistry. The cells grow well. They can be passaged normally. The morphology and number could meet the need of the experiments.2. By SA-β-Gal staining, we confirm the replicative senescence of human RPE cells cultured in vitro are positive correlation with blue light intensity and passage times and as the intensity of blue light and the passages increase, the replicative senescence increases.3. The chomokins in human RPE cells was detected by semiquantitative RT-PCR assay. We find the blue light increases the expressions of chemokins in human RPE cells cultured in vitro. The expressions of chemokins increases with the increase of blue light intensity and passage times.4. We confirm the expressions of chemokins increases with the increase of replicative senescence cells. The chemokins mRNA in the RPE cells which become replicative senescence increases.5. These experiments firstly combine blue light, RPE cell replicative senescence, and Chemokines together and provide held evidence for blue light induced RPE cells replicative senescence, blue light induced the Chemokines in RPE cells, the Chemokines mRNA in replicative senescence cells increases and explanation of the role of the exposure of blue light, RPE cell replicative senescence, inflammatory reaction in pathogenesis of AMD.更多还原