铅诱导的视网膜色素上皮氧化应激应答机制及其细胞生物学特征
发布时间：2018-08-28 06:14
【摘要】：视网膜色素上皮细胞（retinal pigment epithelium, RPE）是位于神经视网膜与脉络膜血管层之间一单层细胞，由神经外胚层分化而来。正常的RPE层对于视细胞结构、生物学功能的维持以及视周期的顺利进行起着极为关键的作用。当RPE发生病变，会引起一系列视网膜疾病。目前就RPE发生病变的机制并未完全阐明，，但这些视网膜病变疾病通常都伴随有RPE细胞氧化应激（oxidativestress）的发生。这成为了视网膜病变相关疾病机制研究的一个重要突破口。目前已经知道诱发RPE产生氧化应激的因素主要有超氧阴离子自由基、蓝光辐射、致毒性物质的摄入、吸烟、肥胖等等。有研究发现重金属铅经个体吸收后在体内会诱发某些细胞或组织产生自由基，这些成分会进一步导致细胞产生氧化应激应答并严重干扰细胞内环境还原稳态。此外，早年有报道发现重金属铅、镉在眼底会发生沉着，而且在RPE层蓄积明显。因此，我们怀疑慢性铅暴露是否也是诱发RPE产生氧化应激并进一步导致其发生病变的潜在病因学因素。 本课题主要以本实验室建立的胎儿RPE细胞系fRPE-13及当下常用RPE系ARPE19作为主要的实验材料，RPE经不同浓度梯度铅染培养后，通过观察细胞形态学变化、鉴定氧化应激相关通路的分子表达差异以及对视网膜色素上皮生理学功能的测定与评价，来确认重金属铅是否也是诱发视网膜色素上皮氧化损伤的一个重要病理学因素。研究结果小结如下： 1、急性染铅培养72hr，用CCK-8法测定在各个铅剂量组RPE细胞活率。结果显示，无论是胎儿来源的fRPE-13还是成年来源的ARPE-19，都存在低剂量铅（≤20μM）兴奋性效应；而剂量高于50μM时，RPE细胞活率下降显著；此外，RPE对铅的可耐受性高于以往研究的其他类型细胞。我们并且由此确定了后续实验铅的考察剂量：0μM、10μM、50μM、250μM。 2、RPE细胞经间隔12hr连续的慢性染铅培养3天、5天、8天，细胞形态发生显著变化。铅染培养3天，0μM与10μM组细胞饱满生长旺盛，呈众小岛样，50μM、250μM组呈小岛样细胞相对较少，长势缓慢；染铅培养5天后，fRPE-13的10μM组培养皿外围细胞已开始皱缩；ARPE-19的50μM组细胞则成狭长弯钩状，而250μM组已经完全成皱缩样；当染铅培养8天后，fRPE-13中10μM组则大范围开始出现细胞皱缩，而ARPE-19的10μM组细胞也出现鹅卵石样上皮，但是细胞形态大小不一且不规整；50μM组与250μM组细胞则已经完全皱缩。 3、细胞满板后，划痕，并连续铅染培养3天，间隔一定时间在光镜下记录细胞迁移情况。采用计算空白区像素大小的方法进行统计学分析。结果表明，当铅浓度达到50μM，无论是fRPE-13还是ARPEA19，上皮的迁移能力几乎完全受到了抑制；而即使是低浓度铅（10μM）也会降低RPE细胞的划痕愈合能力，这种对上皮创伤愈合能力的抑制作用呈剂量依赖特征。 4、细胞经慢性铅染培养5天后，收集并重新铺入96孔板中连续培养7天，采用CCK-8法每隔24hr测定其细胞活率，并绘制细胞生长曲线。结果显示生长曲线右移，表明铅降低了RPE的增值活性。 5、依据RPE细胞生长曲线的特点，当染铅或无铅培养3.5天后达到对数生长期，收集细胞进行固定、PI染色并进行流式周期分析。结果表明，与不染铅组相比，低剂量（10μM）染铅组进入S与G2M期细胞明显增多，这进一步印证了低剂量铅的兴奋性效应；而随着铅浓度高于50μM，进入分裂相的细胞开始减少。 6、RPE细胞经慢性铅染培养5天，Trizol法提取总RNA，逆转录成cDNA后进行qPCR检测。结果表明铅能够诱导分子伴侣（如抗增值蛋白、HSP70）、抗氧化蛋白与酶均表达上调等一系列氧化应激应答成员的高表达。此外，氧化应激应答关键转录调控因子Nrf2的表达也发生显著上调。 7、RPE细胞经慢性铅染培养5天，收集贴壁或未贴壁细胞进行流式凋亡分析。结果显示细胞凋亡率呈铅剂量依赖性特征。 综上所述，从RPE细胞形态学、生理功能以及分子水平的研究结果表明，慢性铅暴露会诱发RPE产生氧化应激，对RPE造成损伤并进一步诱发凋亡或坏死。由此我们得到一个可以合理的推测：重金属铅也极有可能是诱发视网膜色素上皮氧化损伤的一个重要病理学因素，这为视网膜病变相关疾病的病因学机制提供了更多的参考、证据与支持。
[Abstract]:Retinal pigment epithelium (RPE) is a monolayer of cells located between the retinal and choroidal vascular layers and differentiated from the neuroectodermal layer. Normal RPE layers play an important role in the maintenance of visual cell structure, biological function and the smooth progress of the visual cycle. When RPE occurs, it will become diseased. It causes a series of retinal diseases. The pathogenesis of RPE is not fully elucidated, but these retinopathy diseases are usually accompanied by oxidative stress of RPE cells. This has become an important breakthrough in the study of the mechanism of retinopathy-related diseases. The main factors of stimulation are superoxide anion free radical, blue light radiation, toxic substance intake, smoking, obesity and so on. Some studies have found that heavy metal lead can induce some cells or tissues to produce free radicals in vivo after individual absorption. These components will further lead to oxidative stress and seriously interfere with the cellular environment. In addition, it was reported early that heavy metal lead and cadmium deposited in the fundus of the eye and accumulated in the RPE layer significantly. Therefore, we suspect that chronic lead exposure is also a potential etiological factor inducing oxidative stress in RPE and further contributing to the pathogenesis of RPE.
In this study, the fetal RPE cell line fRPE-13 and ARPE19 were used as the main experimental materials. RPE was cultured in different concentration gradients of lead. The morphological changes of RPE cells were observed to identify the molecular expression differences of oxidative stress-related pathways and the physiological function of retinal pigment epithelium. The results are summarized as follows:1.
1. RPE cell viability was measured by CCK-8 method after 72 hours of acute lead exposure. The results showed that low dose lead (< 20 mu M) excitatory effect existed in both fetal fRPE-13 and adult ARPE-19, and RPE cell viability decreased significantly when the dose was higher than 50 mu M. Other types of cells studied. We also determined the dosage of lead for subsequent experiments: 0, 10, 50, 250.
2. RPE cells were cultured for 3 days, 5 days and 8 days after 12 hr interval of chronic lead staining. The cells in 0 and 10 mu M groups grew vigorously and showed islet-like morphology, while those in 50 and 250 mu M groups showed relatively small islet-like morphology and slow growth. After 5 days of lead staining, the peripheral cells in 10 Mu M group of fRPE-13 began to grow. The cells in the 50 mu M group of ARPE-19 contracted in a narrow hook-like shape, while those in the 250 mu M group had completely contracted. After 8 days of lead exposure, the cells in the 10 mu M group of fRPE-13 began to shrink in a large scale, while the cells in the 10 mu M group of ARPE-19 also showed pebble-like epithelium, but the cell morphology was different and irregular. It has completely shrunk.
3. The cells were scratched and cultured for 3 consecutive days with lead staining. The migration of the cells was recorded under light microscope at intervals of 3 days. Low concentration of lead (10 mu M) can also decrease the scratch healing ability of RPE cells, which is dose-dependent.
4. After 5 days of chronic lead staining, the cells were collected and re-planted in 96-well plate for 7 days. The cell viability was measured by CCK-8 method every 24 hours and the growth curve was drawn. The results showed that the growth curve shifted to the right, indicating that lead decreased the RPE activity.
5. According to the characteristics of RPE cell growth curve, when lead or lead-free culture reached logarithmic growth phase 3.5 days later, the cells were collected for fixation, PI staining and flow cytometry analysis. When the lead concentration is higher than 50 M, the cells entering the mitotic phase begin to decrease.
6. RPE cells were cultured for 5 days with chronic lead staining. Total RNA was extracted by Trizol method and then reverse transcribed into cDNA for qPCR detection. The results showed that lead could induce the overexpression of a series of oxidative stress response members, such as antiproliferative protein (HSP70), antioxidant protein and enzymes. The expression also increased significantly.
7. RPE cells were cultured for 5 days after chronic lead staining. Adherent or non-adherent cells were collected and analyzed by flow cytometry. The results showed that the apoptosis rate was dose-dependent.
To sum up, the results of RPE cell morphology, physiological function and molecular level studies show that chronic lead exposure can induce oxidative stress in RPE, damage RPE and further induce apoptosis or necrosis. An important pathological factor of injury provides more evidence and support for the etiological mechanism of retinopathy-related diseases.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R774.1
本文编号：2208502
[Abstract]:Retinal pigment epithelium (RPE) is a monolayer of cells located between the retinal and choroidal vascular layers and differentiated from the neuroectodermal layer. Normal RPE layers play an important role in the maintenance of visual cell structure, biological function and the smooth progress of the visual cycle. When RPE occurs, it will become diseased. It causes a series of retinal diseases. The pathogenesis of RPE is not fully elucidated, but these retinopathy diseases are usually accompanied by oxidative stress of RPE cells. This has become an important breakthrough in the study of the mechanism of retinopathy-related diseases. The main factors of stimulation are superoxide anion free radical, blue light radiation, toxic substance intake, smoking, obesity and so on. Some studies have found that heavy metal lead can induce some cells or tissues to produce free radicals in vivo after individual absorption. These components will further lead to oxidative stress and seriously interfere with the cellular environment. In addition, it was reported early that heavy metal lead and cadmium deposited in the fundus of the eye and accumulated in the RPE layer significantly. Therefore, we suspect that chronic lead exposure is also a potential etiological factor inducing oxidative stress in RPE and further contributing to the pathogenesis of RPE.
In this study, the fetal RPE cell line fRPE-13 and ARPE19 were used as the main experimental materials. RPE was cultured in different concentration gradients of lead. The morphological changes of RPE cells were observed to identify the molecular expression differences of oxidative stress-related pathways and the physiological function of retinal pigment epithelium. The results are summarized as follows:1.
1. RPE cell viability was measured by CCK-8 method after 72 hours of acute lead exposure. The results showed that low dose lead (< 20 mu M) excitatory effect existed in both fetal fRPE-13 and adult ARPE-19, and RPE cell viability decreased significantly when the dose was higher than 50 mu M. Other types of cells studied. We also determined the dosage of lead for subsequent experiments: 0, 10, 50, 250.
2. RPE cells were cultured for 3 days, 5 days and 8 days after 12 hr interval of chronic lead staining. The cells in 0 and 10 mu M groups grew vigorously and showed islet-like morphology, while those in 50 and 250 mu M groups showed relatively small islet-like morphology and slow growth. After 5 days of lead staining, the peripheral cells in 10 Mu M group of fRPE-13 began to grow. The cells in the 50 mu M group of ARPE-19 contracted in a narrow hook-like shape, while those in the 250 mu M group had completely contracted. After 8 days of lead exposure, the cells in the 10 mu M group of fRPE-13 began to shrink in a large scale, while the cells in the 10 mu M group of ARPE-19 also showed pebble-like epithelium, but the cell morphology was different and irregular. It has completely shrunk.
3. The cells were scratched and cultured for 3 consecutive days with lead staining. The migration of the cells was recorded under light microscope at intervals of 3 days. Low concentration of lead (10 mu M) can also decrease the scratch healing ability of RPE cells, which is dose-dependent.
4. After 5 days of chronic lead staining, the cells were collected and re-planted in 96-well plate for 7 days. The cell viability was measured by CCK-8 method every 24 hours and the growth curve was drawn. The results showed that the growth curve shifted to the right, indicating that lead decreased the RPE activity.
5. According to the characteristics of RPE cell growth curve, when lead or lead-free culture reached logarithmic growth phase 3.5 days later, the cells were collected for fixation, PI staining and flow cytometry analysis. When the lead concentration is higher than 50 M, the cells entering the mitotic phase begin to decrease.
6. RPE cells were cultured for 5 days with chronic lead staining. Total RNA was extracted by Trizol method and then reverse transcribed into cDNA for qPCR detection. The results showed that lead could induce the overexpression of a series of oxidative stress response members, such as antiproliferative protein (HSP70), antioxidant protein and enzymes. The expression also increased significantly.
7. RPE cells were cultured for 5 days after chronic lead staining. Adherent or non-adherent cells were collected and analyzed by flow cytometry. The results showed that the apoptosis rate was dose-dependent.
To sum up, the results of RPE cell morphology, physiological function and molecular level studies show that chronic lead exposure can induce oxidative stress in RPE, damage RPE and further induce apoptosis or necrosis. An important pathological factor of injury provides more evidence and support for the etiological mechanism of retinopathy-related diseases.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R774.1
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2 郝长付;郝卫东;;MAPK信号转导通路在CdCl_2、HgCl_2诱导RAW264.7细胞低剂量兴奋效应中的作用[J];毒理学杂志;2011年03期
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本文编号：2208502
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