發(fā)布時(shí)間：2018-08-28 06:14

【摘要】：視網(wǎng)膜色素上皮細(xì)胞（retinal pigment epithelium, RPE）是位于神經(jīng)視網(wǎng)膜與脈絡(luò)膜血管層之間一單層細(xì)胞，由神經(jīng)外胚層分化而來。正常的RPE層對于視細(xì)胞結(jié)構(gòu)、生物學(xué)功能的維持以及視周期的順利進(jìn)行起著極為關(guān)鍵的作用。當(dāng)RPE發(fā)生病變，會引起一系列視網(wǎng)膜疾病。目前就RPE發(fā)生病變的機(jī)制并未完全闡明，，但這些視網(wǎng)膜病變疾病通常都伴隨有RPE細(xì)胞氧化應(yīng)激（oxidativestress）的發(fā)生。這成為了視網(wǎng)膜病變相關(guān)疾病機(jī)制研究的一個重要突破口。目前已經(jīng)知道誘發(fā)RPE產(chǎn)生氧化應(yīng)激的因素主要有超氧陰離子自由基、藍(lán)光輻射、致毒性物質(zhì)的攝入、吸煙、肥胖等等。有研究發(fā)現(xiàn)重金屬鉛經(jīng)個體吸收后在體內(nèi)會誘發(fā)某些細(xì)胞或組織產(chǎn)生自由基，這些成分會進(jìn)一步導(dǎo)致細(xì)胞產(chǎn)生氧化應(yīng)激應(yīng)答并嚴(yán)重干擾細(xì)胞內(nèi)環(huán)境還原穩(wěn)態(tài)。此外，早年有報(bào)道發(fā)現(xiàn)重金屬鉛、鎘在眼底會發(fā)生沉著，而且在RPE層蓄積明顯。因此，我們懷疑慢性鉛暴露是否也是誘發(fā)RPE產(chǎn)生氧化應(yīng)激并進(jìn)一步導(dǎo)致其發(fā)生病變的潛在病因?qū)W因素。 本課題主要以本實(shí)驗(yàn)室建立的胎兒RPE細(xì)胞系fRPE-13及當(dāng)下常用RPE系A(chǔ)RPE19作為主要的實(shí)驗(yàn)材料，RPE經(jīng)不同濃度梯度鉛染培養(yǎng)后，通過觀察細(xì)胞形態(tài)學(xué)變化、鑒定氧化應(yīng)激相關(guān)通路的分子表達(dá)差異以及對視網(wǎng)膜色素上皮生理學(xué)功能的測定與評價(jià)，來確認(rèn)重金屬鉛是否也是誘發(fā)視網(wǎng)膜色素上皮氧化損傷的一個重要病理學(xué)因素。研究結(jié)果小結(jié)如下： 1、急性染鉛培養(yǎng)72hr，用CCK-8法測定在各個鉛劑量組RPE細(xì)胞活率。結(jié)果顯示，無論是胎兒來源的fRPE-13還是成年來源的ARPE-19，都存在低劑量鉛（≤20μM）興奮性效應(yīng)；而劑量高于50μM時(shí)，RPE細(xì)胞活率下降顯著；此外，RPE對鉛的可耐受性高于以往研究的其他類型細(xì)胞。我們并且由此確定了后續(xù)實(shí)驗(yàn)鉛的考察劑量：0μM、10μM、50μM、250μM。 2、RPE細(xì)胞經(jīng)間隔12hr連續(xù)的慢性染鉛培養(yǎng)3天、5天、8天，細(xì)胞形態(tài)發(fā)生顯著變化。鉛染培養(yǎng)3天，0μM與10μM組細(xì)胞飽滿生長旺盛，呈眾小島樣，50μM、250μM組呈小島樣細(xì)胞相對較少，長勢緩慢；染鉛培養(yǎng)5天后，fRPE-13的10μM組培養(yǎng)皿外圍細(xì)胞已開始皺縮；ARPE-19的50μM組細(xì)胞則成狹長彎鉤狀，而250μM組已經(jīng)完全成皺縮樣；當(dāng)染鉛培養(yǎng)8天后，fRPE-13中10μM組則大范圍開始出現(xiàn)細(xì)胞皺縮，而ARPE-19的10μM組細(xì)胞也出現(xiàn)鵝卵石樣上皮，但是細(xì)胞形態(tài)大小不一且不規(guī)整；50μM組與250μM組細(xì)胞則已經(jīng)完全皺縮。 3、細(xì)胞滿板后，劃痕，并連續(xù)鉛染培養(yǎng)3天，間隔一定時(shí)間在光鏡下記錄細(xì)胞遷移情況。采用計(jì)算空白區(qū)像素大小的方法進(jìn)行統(tǒng)計(jì)學(xué)分析。結(jié)果表明，當(dāng)鉛濃度達(dá)到50μM，無論是fRPE-13還是ARPEA19，上皮的遷移能力幾乎完全受到了抑制；而即使是低濃度鉛（10μM）也會降低RPE細(xì)胞的劃痕愈合能力，這種對上皮創(chuàng)傷愈合能力的抑制作用呈劑量依賴特征。 4、細(xì)胞經(jīng)慢性鉛染培養(yǎng)5天后，收集并重新鋪入96孔板中連續(xù)培養(yǎng)7天，采用CCK-8法每隔24hr測定其細(xì)胞活率，并繪制細(xì)胞生長曲線。結(jié)果顯示生長曲線右移，表明鉛降低了RPE的增值活性。 5、依據(jù)RPE細(xì)胞生長曲線的特點(diǎn)，當(dāng)染鉛或無鉛培養(yǎng)3.5天后達(dá)到對數(shù)生長期，收集細(xì)胞進(jìn)行固定、PI染色并進(jìn)行流式周期分析。結(jié)果表明，與不染鉛組相比，低劑量（10μM）染鉛組進(jìn)入S與G2M期細(xì)胞明顯增多，這進(jìn)一步印證了低劑量鉛的興奮性效應(yīng)；而隨著鉛濃度高于50μM，進(jìn)入分裂相的細(xì)胞開始減少。 6、RPE細(xì)胞經(jīng)慢性鉛染培養(yǎng)5天，Trizol法提取總RNA，逆轉(zhuǎn)錄成cDNA后進(jìn)行qPCR檢測。結(jié)果表明鉛能夠誘導(dǎo)分子伴侶（如抗增值蛋白、HSP70）、抗氧化蛋白與酶均表達(dá)上調(diào)等一系列氧化應(yīng)激應(yīng)答成員的高表達(dá)。此外，氧化應(yīng)激應(yīng)答關(guān)鍵轉(zhuǎn)錄調(diào)控因子Nrf2的表達(dá)也發(fā)生顯著上調(diào)。 7、RPE細(xì)胞經(jīng)慢性鉛染培養(yǎng)5天，收集貼壁或未貼壁細(xì)胞進(jìn)行流式凋亡分析。結(jié)果顯示細(xì)胞凋亡率呈鉛劑量依賴性特征。 綜上所述，從RPE細(xì)胞形態(tài)學(xué)、生理功能以及分子水平的研究結(jié)果表明，慢性鉛暴露會誘發(fā)RPE產(chǎn)生氧化應(yīng)激，對RPE造成損傷并進(jìn)一步誘發(fā)凋亡或壞死。由此我們得到一個可以合理的推測：重金屬鉛也極有可能是誘發(fā)視網(wǎng)膜色素上皮氧化損傷的一個重要病理學(xué)因素，這為視網(wǎng)膜病變相關(guān)疾病的病因?qū)W機(jī)制提供了更多的參考、證據(jù)與支持。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R774.1

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