姜黄素在增殖性玻璃体视网膜病变中对表皮生长因子的作用
发布时间：2018-08-07 08:55
【摘要】：增殖性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)是1983年由美国视网膜专家协会提出用来描述孔源性视网膜脱离(rhegmatogenous retinal detachment,RRD)等疾病后玻璃体和/或视网膜前后面特异细胞增殖形成可以收缩的细胞性膜,进而引起视网膜牵拉、脱离与固定的一种病变,是一种常见的难治性致盲性眼病。据报道,5%~10%的RRD可继发PVR,而在复发性视网膜脱离(retinal detachment,RD)中,PVR的发生率增至75%。近年来,随着对PVR病理机制研究的逐渐深入,越来越多的研究发现表皮生长因子(epidermal growth factor,EGF)在RPE细胞的迁移、增生中起重要作用,是促进RPE细胞发生迁移进而发生PVR过程的关键因素之一。目前,临床上治疗PVR的主要方法就是玻璃体视网膜手术,但手术治疗效果并不理想,术后PVR的复发率较高。因此,随着对各种细胞和生长因子在PVR致病过程中作用机制了解的加深,针对PVR发展的不同阶段和相关因子采取不同药物来预防和治疗PVR成为目前进行研究的热点,并成为主流趋势。到现阶段为止,药物研究主要集中在西药领域,主要包括以下几类:皮质类固醇、维生素及其衍生物、抗代谢药、细胞外基质合成抑制剂和细胞信号转导抑制剂等。虽然应用西药来防治PVR的研究已有数年之久,但是由于其在眼内有较大的毒副作用、较单一的药理作用、较昂贵的价格等诸多的局限性致使迄今仍无一种特效的药物能够成功的在临床上得到广泛的应用。目前在西药上的研究难以取得突破性进展,那么应用我国传统中药来防治PVR的研究成为了充满希望的突破方向。中药具有药源丰富、作用广泛且毒副作用小等诸多优点,姜黄素是存在于姜科姜黄属植物根茎中的一种天然的中药单体成分,具有多种药理作用,例如抗炎症、抗细胞增生、抗微生物等。姜黄素的药理作用可以满足PVR防治药物的条件,并且安全性高、毒性低、药源广泛、价格低廉。我们的前期研究发现姜黄素具有抑制视网膜色素上皮细胞增殖的作用,那么姜黄素对在PVR形成过程中起重要作用的表皮生长因子是否有作用,本课题分别通过体外细胞实验和体内玻璃体腔注射姜黄素的动物实验研究姜黄素在pvr防治过程中对egf的作用和相关机制,为pvr的防治提供依据。第一部分表皮生长因子对体外培养的rpe细胞的作用及其在rpe细胞内的表达目的:研究egf对体外培养的兔rpe细胞的作用,探讨egf促进rpe细胞增殖的最佳质量浓度;观察egf在rpe细胞内的表达情况。方法:提取、培养青紫蓝兔rpe细胞,传至第三代并鉴定后,选取生长状态良好的第3代rpe细胞进行实验。将rpe细胞种植在载玻片上制备成细胞爬片,做免疫细胞化学染色进行rpe细胞鉴定和观察rpe细胞内egf的表达情况;将egf分为3、6、9、12ng/ml不同浓度组及空白对照组(10%fbs.dmem)4组;每组各设6个复孔,共接种3块培养板,加药后于24、48、72h随机抽取1块培养板采用四甲基偶氮唑盐(mtt)比色法检测不同浓度的egf在不同作用时间对rpe细胞增殖的影响。结果:rpe细胞培养早期生长活跃、胞核透明、胞浆含有丰富黑色素颗粒,免疫细胞化学染色提示角蛋白表达强阳性。egf在兔rpe细胞的细胞质中表达阳性,呈棕黄色。在相同时间点、不同浓度egf作用下,rpe细胞的吸光度(od值)随egf的浓度增加而增加,且与对照组比较差异均有统计学意义(p0.05)。egf浓度≥9ng/ml的相邻浓度实验组两组间比较差异无统计学意义(p0.05)。在相同浓度的egf作用下,rpe细胞吸光值(od值)随时间的增加而增加,且与相邻组比较差异均有统计学意义(p0.05)。结论:体外培养可以获得大量rpe细胞并可用于体外实验研究;egf在兔rpe细胞的细胞质中表达;egf对体外培养兔rpe细胞增生的调控存在一定的剂量-效应关系、时间-效应关系。egf对rpe细胞的促生长作用在9ng/ml以上逐渐达到饱和。研究结果提示:促体外培养的兔rpe细胞增殖的egf的最佳浓度为9ng/m1。第二部分姜黄素对体外培养的rpe细胞中egf表达的抑制作用目的:研究不同浓度的姜黄素对体外培养的rpe细胞中egf表达的影响,应用免疫细胞化学染色方法检测兔rpe细胞中egf的表达情况,寻找姜黄素抑制egf表达的最佳浓度;应用rt-pcr法、westernblot法检测姜黄素对兔rpe细胞中egfmrna和蛋白表达的影响,探讨姜黄素抑制egf的作用机制。方法:选取生长状态良好的兔第3代rpe细胞在盖玻片上制备成细胞爬片进行实验。分为空白对照组(含0.5‰dmso的10%fbs.dmem)和10、15、20ug/ml姜黄素4组;每组各设6个复孔,共接种3块培养板,加药后24、48、72h时随机抽取1块培养板行免疫组织化学染色,观察rpe细胞内egf的表达情况。rt-pcr、westernblot分别检测空白对照组(含0.5‰dmso的10%fbs.dmem)、姜黄素(15ug/ml)、egf(9ng/ml含0.5‰dmso)、egf(9ng/ml)+姜黄素(15ug/ml)作用24、48、72h后rpe细胞中egfmrna和蛋白表达的影响。结果:1不同浓度姜黄素对rpe内egf表达的抑制作用:时间依赖性:姜黄素各浓度组对rpe细胞内egf表达的抑制作用均随作用时间的延长而增强,各时间点之间差异均有统计学意义(p0.05),姜黄素对rpe细胞内egf表达的抑制作用具有时间依赖性。剂量依赖性:姜黄素各时间点对rpe细胞内egf表达的抑制均随药物质量浓度的增高而增强,除姜黄素15ug/ml和20ug/ml组之间差异无统计学意义(p0.05)外,其余各质量浓度之间差异均有统计学意义(p0.05)。2姜黄素对rpe细胞内egfmrna转录的影响:向培养液中加入姜黄素使其浓度为15ug/ml,在24h、48h、及72h检测egf的mrna含量,可见随时间延长细胞内egf的mrna表达量下降,和对照组相比较,差异有统计学意义(p0.05);不同时间点间做比较,相邻时间点组间相比差异均有统计学意义(p0.05)。姜黄素对egf作用下rpe细胞中egfmrna表达量在各时间点和对照组比较差异有统计学意义(p0.05),不同时间点间,相邻的时间组相比较差异均有统计学意义(p0.05)。3姜黄素对rpe细胞内egf蛋白表达的影响:向培养液中加入姜黄素使其浓度为15ug/ml,作用24h、48h、及72h,随着时间的延长细胞内egf蛋白表达量逐渐下降,与对照组相比较差异有统计学意义(p0.05);不同时间点间,相邻的时间组间相比较差异均有统计学意义(p0.05)。姜黄素对egf作用下rpe细胞中egf蛋白表达量在各时间点和对照组比较差异有统计学意义(p0.05),不同时间点间,相邻的时间组相比差异均有统计学意义(p0.05)。结论:姜黄素在体外抑制兔rpe细胞内egf的表达,最佳抑制浓度是15ug/ml,姜黄素在体外可显著抑制兔rpe细胞内egfmrna和蛋白的表达。第三部分姜黄素在兔眼增殖性玻璃体视网膜病变中对egf作用的实验研究目的:研究姜黄素在体内兔眼增殖性玻璃体视网膜病变形成过程中对egf的作用及对pvr的防治作用。方法:选择正常青紫蓝兔30只,所有兔在玻璃体注射前均抽出0.2ml玻璃体,随机选取一眼纳入对照组:共30眼,玻璃体腔注射0.1ml(2×106)培养的生长状态良好的第三代同种rpe细胞和含0.5‰dmso的生理盐水0.1ml;另一眼纳入实验组:共30眼,玻璃体腔注射0.1ml(2×106)培养的生长状态良好的同种第三代rpe细胞和质量浓度为1mg/ml的姜黄素0.1ml。注射后3、7、14、21、28天进行裂隙灯显微镜检查观察眼前节和前部玻璃体情况,间接眼底镜检查观察眼底情况,眼底彩色照相,眼部b超检查观察玻璃体浑浊情况和视网膜有无脱离及脱离的程度,检查完毕后,在每个时间点随机抽取6只兔,12只眼,对照组6只眼,实验组6只眼,分别抽取玻璃体,采用兔表皮生长因子elisa试剂盒检测玻璃体液中egf的含量。结果:1前房反应:玻璃体腔注射后第3天对照组和实验组兔眼前房内均可见少量浮游物,第7天时均消退。2玻璃体和视网膜情况:第3天两组玻璃体均有浑浊,实验组玻璃体混浊轻,第7天时玻璃体内有增殖膜形成,对照组增殖膜多且厚,实验组增殖膜局限且薄;第14天时对照组视网膜脱离发生率(11/18)61%,实验组玻璃体内增殖膜较薄,视网膜脱离发生率(2/18)11%;第21天时对照组视网膜脱离发生率(8/12)67%,增殖膜上可见新生血管,实验组视网膜脱离发生率(2/12)16%,视网膜脱离范围小,第28天对照组视网膜脱离渐加重,并有新生血管生长,视网膜脱离发生率(5/6)83%,实验组视网膜脱离局限,发生率(1/6)16%;视网膜脱离发生率实验组和对照组比较差异显著,有统计学意义(p0.05)。3玻璃体中egf含量测定(elisa结果):玻璃体液中egf含量对照组较高,实验组较少,各时间点实验组和对照组比较差异均具有统计学意义(P0.05)。结论:玻璃体腔内注射姜黄素可以有效抑制RPE细胞诱导的兔眼实验性PVR形成过程中表皮生长因子的水平,进而抑制PVR的发生和发展。
[Abstract]:Proliferative vitreoretinopathy (proliferative vitreoretinopathy, PVR) is a cell membrane that can constriction the specific cell proliferation of the vitreous and / or retina behind the retinal detachment (rhegmatogenous retinal detachment, RRD) by the American retina expert Association in 1983. Retinal distraction, detachment and fixation is a common refractory blindness. It is reported that 5%~10%'s RRD can be secondary to PVR, and the incidence of PVR in recurrent retinal detachment (retinal detachment, RD) has increased to 75%. in recent years. With the gradual deepening of the study of the pathogenesis of PVR disease, more and more studies have found the epidermis. Epidermal growth factor (EGF) plays an important role in the migration and proliferation of RPE cells. It is one of the key factors to promote the migration of RPE cells and the process of PVR. At present, the main method of treating PVR is vitreoretinal surgery, but the effect of operation is not ideal and the recurrence rate of PVR is high after operation. Therefore, with the understanding of the mechanism of various cells and growth factors in the pathogenesis of PVR, it is a hot topic to take different drugs to prevent and treat PVR according to the different stages and related factors of the development of PVR, and become the mainstream trend. The following are the following categories: corticosteroids, vitamins and their derivatives, anti metabolic drugs, extracellular matrix synthesis inhibitors and cellular signal transduction inhibitors. Although the use of Western medicine to prevent and control PVR has been for several years, it has large toxic and side effects in the eye, a single pharmacological effect, more expensive prices and many other bureaus. So far, no special drug can be successfully used in clinical practice. It is difficult to make a breakthrough in the research on Western medicine. So the application of traditional Chinese medicine to prevent and control PVR has become a promising breakthrough. Many advantages, curcumin is a natural Chinese medicine monomer in the rhizome of turmeric turmeric plant. It has a variety of pharmacological effects, such as anti inflammatory, anti cell proliferation, and antimicrobial resistance. The pharmacological effects of curcumin can meet the conditions of PVR control drugs, and have high safety, low toxicity, extensive drug source and low price. It is found that curcumin can inhibit the proliferation of retinal pigment epithelial cells. Then, curcumin plays an important role in the formation of PVR. The subject of this study is to study the effect of curcumin on the control of PVR in the process of in vitro cell experiments and intravitreal injection of curcumin in vivo. The role and related mechanisms of EGF provide the basis for the prevention and control of PVR. Part 1 the effect of epidermal growth factor on the cultured RPE cells and its expression in RPE cells: To study the effect of EGF on rabbit RPE cells cultured in vitro, to explore the optimal mass concentration of EGF to promote the proliferation of RPE cells, and to observe the expression of EGF in RPE cells. Methods: the RPE cells of blue and blue rabbit were extracted and cultured to third generations and identified to select the third generation RPE cells with good growth state. The RPE cells were planted on the slides to prepare the cell climbing tablets, and the immunocytochemical staining was used to identify the RPE cells and to observe the expression of EGF in the RPE cell, and EGF was divided into 3,6,9,12ng/ml. In the same concentration group and the blank control group (10%fbs.dmem) 4 groups, each group had 6 compound holes, 3 culture plates were inoculated, 1 culture plates were randomly selected, and four methyl azazolium salt (MTT) colorimetric method was used to detect the effects of different concentrations of EGF on the proliferation of RPE cells at different time. Results: the early growth of RPE cells was active, cell culture was active. Nuclear transparent, cytoplasm contains rich melanin particles. Immunocytochemical staining suggests that.Egf is positive in cytoplasm of rabbit RPE cells, and is brown in cytoplasm. At the same time point, the absorbance (OD value) of RPE cells increases with the increase of EGF concentration under the action of different concentrations of EGF, and there are different differences compared with those of the control group. There was no significant difference between the two groups of adjacent concentrations of.Egf concentration (P0.05) concentration (P0.05) in the experimental group (P0.05). In the same concentration of EGF, the absorbance value of RPE cells (OD value) increased with the time, and the difference was statistically significant (P0.05) compared with the adjacent groups. Conclusion: in vitro culture can obtain a large number of RPE cells and It can be used to study in vitro; EGF is expressed in the cytoplasm of rabbit RPE cells; EGF has a certain dose effect relationship on the regulation of proliferation of rabbit RPE cells in vitro. The time effect relationship of.Egf to RPE cells is gradually saturated with 9ng/ml above 9ng/ml. The results suggest that the proliferation of rabbit RPE cells in vitro is promoted. The best concentration of 9ng/m1. second part curcumin inhibits the expression of EGF in RPE cells cultured in vitro aim: To study the effect of curcumin on the expression of EGF in RPE cells cultured in vitro, and to detect the expression of EGF in rabbit RPE cells by immunocytochemical staining, and to find the best expression of curcumin to inhibit the expression of EGF. The effect of curcumin on the expression of egfmrna and protein in rabbit RPE cells was detected by RT-PCR and Westernblot, and the mechanism of curcumin on the inhibition of EGF was investigated. Methods: the third generation RPE cells with good growth state were selected to prepare the cell climbing tablets on the cover glass and divided into the blank control group (including 10%fbs.dmem of 0.5 per thousand DMSO) and 10 4 groups of 15,20ug/ml curcumin, each set of 6 compound holes, were inoculated with 3 pieces of culture plate, and 1 culture plates were randomly selected for immunohistochemistry after 24,48,72h, and the expression of EGF in RPE cells was observed.Rt-pcr, Westernblot was used to detect the blank control group (0.5% DMSO 10%fbs.dmem), curcumin (15ug/ml), EGF (9ng/ml 0.5 per thousand DMSO). The effect of EGF (9ng/ml) + curcumin (15ug/ml) on the expression of egfmrna and protein in RPE cells after 24,48,72h. Results: 1 the inhibitory effect of curcumin on the expression of EGF in RPE: time dependence: the inhibitory effect of curcumin concentration groups on the expression of EGF in RPE cells is enhanced with the prolongation of action time, and the differences in time points are all The inhibitory effect of curcumin on the expression of EGF in RPE cells was time dependent. The inhibitory effect of curcumin on the EGF expression in RPE cells increased with the increase of drug mass concentration. There was no statistical significance (P0.05) except the difference between curcumin 15ug/ml and 20ug/ml group (P0.05). The difference in concentration was statistically significant (P0.05).2 curcumin's effect on egfmrna transcription in RPE cells: the concentration of curcumin was 15ug/ml, 24h, 48h, and 72h detected EGF mRNA content, and the EGF mRNA expression decreased with time, and the difference was statistically significant compared with the control group. Comparison between different time points was statistically significant (P0.05). The difference of egfmrna expression in RPE cells in RPE cells under the action of EGF was statistically significant (P0.05), and the difference of the time group was statistically significant (P0.05).3 curcumin between different time points. The effect on the expression of EGF protein in RPE cells: the concentration of curcumin was added to the culture medium to 15ug/ml, 24h, 48h, and 72h, and the expression of EGF protein decreased gradually in the cells with time. The difference was statistically significant (P0.05) compared with the control group (P0.05), and there were statistical differences between the adjacent time groups. Significance (P0.05). The expression of curcumin on the expression of EGF protein in RPE cells under the action of EGF was statistically significant (P0.05). The difference was statistically significant (P0.05) between the adjacent time groups at different time points (P0.05). Conclusion: the best inhibitory concentration of curcumin in rabbit RPE cells in vitro is 15ug/ml, Curcumin can inhibit the expression of egfmrna and protein in rabbit RPE cells in vitro. Third experimental study on the effect of curcumin on EGF in rabbit eye proliferative vitreoretinopathy: the effect of curcumin on EGF during the formation of vitreoretinopathy in rabbit eyes and the prevention and treatment of PVR in the process of rabbit eye proliferative vitreoretinopathy. 30 rabbits were selected from normal blue and blue rabbits. All rabbits were pumped out of 0.2ml vitreous body before vitreous injection. One eye was randomly selected as a control group: a total of 30 eyes were given a total of third generation RPE cells with good growth state of 0.1ml (2 x 106) and 0.1ml containing 0.5 per 1000 DMSO, and the other eyes were included in the experimental group: a total of 30 eyes, and glass cavity injection. 0.1ml (2 * 106) cultured RPE cells with good growth state and 1mg/ml - based curcumin 0.1ml. were injected into the anterior vitreous body at 3,7,14,21,28 days after 3,7,14,21,28 days. Indirect ophthalmoscopy was used to observe the fundus, fundus color photography, and eye B Ultrasound examination to observe the vitreous Hun After the examination, 6 rabbits were randomly selected, 12 eyes, 6 eyes of the control group, 6 eyes in the experimental group, and 6 eyes in the experimental group. The rabbit epidermal growth factor ELISA kit was used to detect the content of EGF in the glass body fluid. Results: 1 anterior chamber reaction: the third days after the glass cavity injection, the result was 1. A small amount of floating objects were seen in the anterior chamber of the rabbits in the experimental group and the experimental group. The vitreous body and the retina of the.2 were eliminated at seventh days. The vitreous body in the two groups was cloudy on the third day, the vitreous opacity was light in the experimental group and the proliferation membrane in the glass body formed at seventh days. The proliferation membrane of the control group was thick and the proliferating membrane was limited and thin in the test group. At fourteenth days, the retina was the control group retina. In 11/18 61%, the proliferating membrane in the vitreous body of the experimental group was thinner and the retinal detachment incidence (2/18) was 11%, and the retinal detachment occurred at twenty-first days (8/12) 67%, the neovascularization was visible on the proliferation membrane, the retinal detachment rate (2/12) was 16%, the retinal detachment was small, and the retinal detachment gradually increased in the control group, and there were the twenty-eighth days in the control group. Neovascular growth, retinal detachment incidence (5/6) 83%, experimental group retinal detachment limited, the incidence of (1/6) 16%, retinal detachment incidence in the experimental group and the control group is significantly different, there is statistical significance (P0.05).3 vitreous EGF content determination (ELISA results): glass body fluid EGF content control group is higher, the experimental group is less, the time is less, the time is less. The difference between the experimental group and the control group was statistically significant (P0.05). Conclusion: Intravitreal injection of curcumin can effectively inhibit the level of epidermal growth factor in the formation of experimental PVR induced by RPE cells, and then inhibit the occurrence and development of PVR.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R774.1
本文编号：2169523
[Abstract]:Proliferative vitreoretinopathy (proliferative vitreoretinopathy, PVR) is a cell membrane that can constriction the specific cell proliferation of the vitreous and / or retina behind the retinal detachment (rhegmatogenous retinal detachment, RRD) by the American retina expert Association in 1983. Retinal distraction, detachment and fixation is a common refractory blindness. It is reported that 5%~10%'s RRD can be secondary to PVR, and the incidence of PVR in recurrent retinal detachment (retinal detachment, RD) has increased to 75%. in recent years. With the gradual deepening of the study of the pathogenesis of PVR disease, more and more studies have found the epidermis. Epidermal growth factor (EGF) plays an important role in the migration and proliferation of RPE cells. It is one of the key factors to promote the migration of RPE cells and the process of PVR. At present, the main method of treating PVR is vitreoretinal surgery, but the effect of operation is not ideal and the recurrence rate of PVR is high after operation. Therefore, with the understanding of the mechanism of various cells and growth factors in the pathogenesis of PVR, it is a hot topic to take different drugs to prevent and treat PVR according to the different stages and related factors of the development of PVR, and become the mainstream trend. The following are the following categories: corticosteroids, vitamins and their derivatives, anti metabolic drugs, extracellular matrix synthesis inhibitors and cellular signal transduction inhibitors. Although the use of Western medicine to prevent and control PVR has been for several years, it has large toxic and side effects in the eye, a single pharmacological effect, more expensive prices and many other bureaus. So far, no special drug can be successfully used in clinical practice. It is difficult to make a breakthrough in the research on Western medicine. So the application of traditional Chinese medicine to prevent and control PVR has become a promising breakthrough. Many advantages, curcumin is a natural Chinese medicine monomer in the rhizome of turmeric turmeric plant. It has a variety of pharmacological effects, such as anti inflammatory, anti cell proliferation, and antimicrobial resistance. The pharmacological effects of curcumin can meet the conditions of PVR control drugs, and have high safety, low toxicity, extensive drug source and low price. It is found that curcumin can inhibit the proliferation of retinal pigment epithelial cells. Then, curcumin plays an important role in the formation of PVR. The subject of this study is to study the effect of curcumin on the control of PVR in the process of in vitro cell experiments and intravitreal injection of curcumin in vivo. The role and related mechanisms of EGF provide the basis for the prevention and control of PVR. Part 1 the effect of epidermal growth factor on the cultured RPE cells and its expression in RPE cells: To study the effect of EGF on rabbit RPE cells cultured in vitro, to explore the optimal mass concentration of EGF to promote the proliferation of RPE cells, and to observe the expression of EGF in RPE cells. Methods: the RPE cells of blue and blue rabbit were extracted and cultured to third generations and identified to select the third generation RPE cells with good growth state. The RPE cells were planted on the slides to prepare the cell climbing tablets, and the immunocytochemical staining was used to identify the RPE cells and to observe the expression of EGF in the RPE cell, and EGF was divided into 3,6,9,12ng/ml. In the same concentration group and the blank control group (10%fbs.dmem) 4 groups, each group had 6 compound holes, 3 culture plates were inoculated, 1 culture plates were randomly selected, and four methyl azazolium salt (MTT) colorimetric method was used to detect the effects of different concentrations of EGF on the proliferation of RPE cells at different time. Results: the early growth of RPE cells was active, cell culture was active. Nuclear transparent, cytoplasm contains rich melanin particles. Immunocytochemical staining suggests that.Egf is positive in cytoplasm of rabbit RPE cells, and is brown in cytoplasm. At the same time point, the absorbance (OD value) of RPE cells increases with the increase of EGF concentration under the action of different concentrations of EGF, and there are different differences compared with those of the control group. There was no significant difference between the two groups of adjacent concentrations of.Egf concentration (P0.05) concentration (P0.05) in the experimental group (P0.05). In the same concentration of EGF, the absorbance value of RPE cells (OD value) increased with the time, and the difference was statistically significant (P0.05) compared with the adjacent groups. Conclusion: in vitro culture can obtain a large number of RPE cells and It can be used to study in vitro; EGF is expressed in the cytoplasm of rabbit RPE cells; EGF has a certain dose effect relationship on the regulation of proliferation of rabbit RPE cells in vitro. The time effect relationship of.Egf to RPE cells is gradually saturated with 9ng/ml above 9ng/ml. The results suggest that the proliferation of rabbit RPE cells in vitro is promoted. The best concentration of 9ng/m1. second part curcumin inhibits the expression of EGF in RPE cells cultured in vitro aim: To study the effect of curcumin on the expression of EGF in RPE cells cultured in vitro, and to detect the expression of EGF in rabbit RPE cells by immunocytochemical staining, and to find the best expression of curcumin to inhibit the expression of EGF. The effect of curcumin on the expression of egfmrna and protein in rabbit RPE cells was detected by RT-PCR and Westernblot, and the mechanism of curcumin on the inhibition of EGF was investigated. Methods: the third generation RPE cells with good growth state were selected to prepare the cell climbing tablets on the cover glass and divided into the blank control group (including 10%fbs.dmem of 0.5 per thousand DMSO) and 10 4 groups of 15,20ug/ml curcumin, each set of 6 compound holes, were inoculated with 3 pieces of culture plate, and 1 culture plates were randomly selected for immunohistochemistry after 24,48,72h, and the expression of EGF in RPE cells was observed.Rt-pcr, Westernblot was used to detect the blank control group (0.5% DMSO 10%fbs.dmem), curcumin (15ug/ml), EGF (9ng/ml 0.5 per thousand DMSO). The effect of EGF (9ng/ml) + curcumin (15ug/ml) on the expression of egfmrna and protein in RPE cells after 24,48,72h. Results: 1 the inhibitory effect of curcumin on the expression of EGF in RPE: time dependence: the inhibitory effect of curcumin concentration groups on the expression of EGF in RPE cells is enhanced with the prolongation of action time, and the differences in time points are all The inhibitory effect of curcumin on the expression of EGF in RPE cells was time dependent. The inhibitory effect of curcumin on the EGF expression in RPE cells increased with the increase of drug mass concentration. There was no statistical significance (P0.05) except the difference between curcumin 15ug/ml and 20ug/ml group (P0.05). The difference in concentration was statistically significant (P0.05).2 curcumin's effect on egfmrna transcription in RPE cells: the concentration of curcumin was 15ug/ml, 24h, 48h, and 72h detected EGF mRNA content, and the EGF mRNA expression decreased with time, and the difference was statistically significant compared with the control group. Comparison between different time points was statistically significant (P0.05). The difference of egfmrna expression in RPE cells in RPE cells under the action of EGF was statistically significant (P0.05), and the difference of the time group was statistically significant (P0.05).3 curcumin between different time points. The effect on the expression of EGF protein in RPE cells: the concentration of curcumin was added to the culture medium to 15ug/ml, 24h, 48h, and 72h, and the expression of EGF protein decreased gradually in the cells with time. The difference was statistically significant (P0.05) compared with the control group (P0.05), and there were statistical differences between the adjacent time groups. Significance (P0.05). The expression of curcumin on the expression of EGF protein in RPE cells under the action of EGF was statistically significant (P0.05). The difference was statistically significant (P0.05) between the adjacent time groups at different time points (P0.05). Conclusion: the best inhibitory concentration of curcumin in rabbit RPE cells in vitro is 15ug/ml, Curcumin can inhibit the expression of egfmrna and protein in rabbit RPE cells in vitro. Third experimental study on the effect of curcumin on EGF in rabbit eye proliferative vitreoretinopathy: the effect of curcumin on EGF during the formation of vitreoretinopathy in rabbit eyes and the prevention and treatment of PVR in the process of rabbit eye proliferative vitreoretinopathy. 30 rabbits were selected from normal blue and blue rabbits. All rabbits were pumped out of 0.2ml vitreous body before vitreous injection. One eye was randomly selected as a control group: a total of 30 eyes were given a total of third generation RPE cells with good growth state of 0.1ml (2 x 106) and 0.1ml containing 0.5 per 1000 DMSO, and the other eyes were included in the experimental group: a total of 30 eyes, and glass cavity injection. 0.1ml (2 * 106) cultured RPE cells with good growth state and 1mg/ml - based curcumin 0.1ml. were injected into the anterior vitreous body at 3,7,14,21,28 days after 3,7,14,21,28 days. Indirect ophthalmoscopy was used to observe the fundus, fundus color photography, and eye B Ultrasound examination to observe the vitreous Hun After the examination, 6 rabbits were randomly selected, 12 eyes, 6 eyes of the control group, 6 eyes in the experimental group, and 6 eyes in the experimental group. The rabbit epidermal growth factor ELISA kit was used to detect the content of EGF in the glass body fluid. Results: 1 anterior chamber reaction: the third days after the glass cavity injection, the result was 1. A small amount of floating objects were seen in the anterior chamber of the rabbits in the experimental group and the experimental group. The vitreous body and the retina of the.2 were eliminated at seventh days. The vitreous body in the two groups was cloudy on the third day, the vitreous opacity was light in the experimental group and the proliferation membrane in the glass body formed at seventh days. The proliferation membrane of the control group was thick and the proliferating membrane was limited and thin in the test group. At fourteenth days, the retina was the control group retina. In 11/18 61%, the proliferating membrane in the vitreous body of the experimental group was thinner and the retinal detachment incidence (2/18) was 11%, and the retinal detachment occurred at twenty-first days (8/12) 67%, the neovascularization was visible on the proliferation membrane, the retinal detachment rate (2/12) was 16%, the retinal detachment was small, and the retinal detachment gradually increased in the control group, and there were the twenty-eighth days in the control group. Neovascular growth, retinal detachment incidence (5/6) 83%, experimental group retinal detachment limited, the incidence of (1/6) 16%, retinal detachment incidence in the experimental group and the control group is significantly different, there is statistical significance (P0.05).3 vitreous EGF content determination (ELISA results): glass body fluid EGF content control group is higher, the experimental group is less, the time is less, the time is less. The difference between the experimental group and the control group was statistically significant (P0.05). Conclusion: Intravitreal injection of curcumin can effectively inhibit the level of epidermal growth factor in the formation of experimental PVR induced by RPE cells, and then inhibit the occurrence and development of PVR.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R774.1
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