【摘要】：增殖性玻璃體視網(wǎng)膜病變(proliferative vitreoretinopathy,PVR)是1983年由美國視網(wǎng)膜專家協(xié)會提出用來描述孔源性視網(wǎng)膜脫離(rhegmatogenous retinal detachment,RRD)等疾病后玻璃體和/或視網(wǎng)膜前后面特異細(xì)胞增殖形成可以收縮的細(xì)胞性膜,進(jìn)而引起視網(wǎng)膜牽拉、脫離與固定的一種病變,是一種常見的難治性致盲性眼病。據(jù)報道,5%~10%的RRD可繼發(fā)PVR,而在復(fù)發(fā)性視網(wǎng)膜脫離(retinal detachment,RD)中,PVR的發(fā)生率增至75%。近年來,隨著對PVR病理機(jī)制研究的逐漸深入,越來越多的研究發(fā)現(xiàn)表皮生長因子(epidermal growth factor,EGF)在RPE細(xì)胞的遷移、增生中起重要作用,是促進(jìn)RPE細(xì)胞發(fā)生遷移進(jìn)而發(fā)生PVR過程的關(guān)鍵因素之一。目前,臨床上治療PVR的主要方法就是玻璃體視網(wǎng)膜手術(shù),但手術(shù)治療效果并不理想,術(shù)后PVR的復(fù)發(fā)率較高。因此,隨著對各種細(xì)胞和生長因子在PVR致病過程中作用機(jī)制了解的加深,針對PVR發(fā)展的不同階段和相關(guān)因子采取不同藥物來預(yù)防和治療PVR成為目前進(jìn)行研究的熱點(diǎn),并成為主流趨勢。到現(xiàn)階段為止,藥物研究主要集中在西藥領(lǐng)域,主要包括以下幾類:皮質(zhì)類固醇、維生素及其衍生物、抗代謝藥、細(xì)胞外基質(zhì)合成抑制劑和細(xì)胞信號轉(zhuǎn)導(dǎo)抑制劑等。雖然應(yīng)用西藥來防治PVR的研究已有數(shù)年之久,但是由于其在眼內(nèi)有較大的毒副作用、較單一的藥理作用、較昂貴的價格等諸多的局限性致使迄今仍無一種特效的藥物能夠成功的在臨床上得到廣泛的應(yīng)用。目前在西藥上的研究難以取得突破性進(jìn)展,那么應(yīng)用我國傳統(tǒng)中藥來防治PVR的研究成為了充滿希望的突破方向。中藥具有藥源豐富、作用廣泛且毒副作用小等諸多優(yōu)點(diǎn),姜黃素是存在于姜科姜黃屬植物根莖中的一種天然的中藥單體成分,具有多種藥理作用,例如抗炎癥、抗細(xì)胞增生、抗微生物等。姜黃素的藥理作用可以滿足PVR防治藥物的條件,并且安全性高、毒性低、藥源廣泛、價格低廉。我們的前期研究發(fā)現(xiàn)姜黃素具有抑制視網(wǎng)膜色素上皮細(xì)胞增殖的作用,那么姜黃素對在PVR形成過程中起重要作用的表皮生長因子是否有作用,本課題分別通過體外細(xì)胞實(shí)驗(yàn)和體內(nèi)玻璃體腔注射姜黃素的動物實(shí)驗(yàn)研究姜黃素在pvr防治過程中對egf的作用和相關(guān)機(jī)制,為pvr的防治提供依據(jù)。第一部分表皮生長因子對體外培養(yǎng)的rpe細(xì)胞的作用及其在rpe細(xì)胞內(nèi)的表達(dá)目的:研究egf對體外培養(yǎng)的兔rpe細(xì)胞的作用,探討egf促進(jìn)rpe細(xì)胞增殖的最佳質(zhì)量濃度;觀察egf在rpe細(xì)胞內(nèi)的表達(dá)情況。方法:提取、培養(yǎng)青紫藍(lán)兔rpe細(xì)胞,傳至第三代并鑒定后,選取生長狀態(tài)良好的第3代rpe細(xì)胞進(jìn)行實(shí)驗(yàn)。將rpe細(xì)胞種植在載玻片上制備成細(xì)胞爬片,做免疫細(xì)胞化學(xué)染色進(jìn)行rpe細(xì)胞鑒定和觀察rpe細(xì)胞內(nèi)egf的表達(dá)情況;將egf分為3、6、9、12ng/ml不同濃度組及空白對照組(10%fbs.dmem)4組;每組各設(shè)6個復(fù)孔,共接種3塊培養(yǎng)板,加藥后于24、48、72h隨機(jī)抽取1塊培養(yǎng)板采用四甲基偶氮唑鹽(mtt)比色法檢測不同濃度的egf在不同作用時間對rpe細(xì)胞增殖的影響。結(jié)果:rpe細(xì)胞培養(yǎng)早期生長活躍、胞核透明、胞漿含有豐富黑色素顆粒,免疫細(xì)胞化學(xué)染色提示角蛋白表達(dá)強(qiáng)陽性。egf在兔rpe細(xì)胞的細(xì)胞質(zhì)中表達(dá)陽性,呈棕黃色。在相同時間點(diǎn)、不同濃度egf作用下,rpe細(xì)胞的吸光度(od值)隨egf的濃度增加而增加,且與對照組比較差異均有統(tǒng)計學(xué)意義(p0.05)。egf濃度≥9ng/ml的相鄰濃度實(shí)驗(yàn)組兩組間比較差異無統(tǒng)計學(xué)意義(p0.05)。在相同濃度的egf作用下,rpe細(xì)胞吸光值(od值)隨時間的增加而增加,且與相鄰組比較差異均有統(tǒng)計學(xué)意義(p0.05)。結(jié)論:體外培養(yǎng)可以獲得大量rpe細(xì)胞并可用于體外實(shí)驗(yàn)研究;egf在兔rpe細(xì)胞的細(xì)胞質(zhì)中表達(dá);egf對體外培養(yǎng)兔rpe細(xì)胞增生的調(diào)控存在一定的劑量-效應(yīng)關(guān)系、時間-效應(yīng)關(guān)系。egf對rpe細(xì)胞的促生長作用在9ng/ml以上逐漸達(dá)到飽和。研究結(jié)果提示:促體外培養(yǎng)的兔rpe細(xì)胞增殖的egf的最佳濃度為9ng/m1。第二部分姜黃素對體外培養(yǎng)的rpe細(xì)胞中egf表達(dá)的抑制作用目的:研究不同濃度的姜黃素對體外培養(yǎng)的rpe細(xì)胞中egf表達(dá)的影響,應(yīng)用免疫細(xì)胞化學(xué)染色方法檢測兔rpe細(xì)胞中egf的表達(dá)情況,尋找姜黃素抑制egf表達(dá)的最佳濃度;應(yīng)用rt-pcr法、westernblot法檢測姜黃素對兔rpe細(xì)胞中egfmrna和蛋白表達(dá)的影響,探討姜黃素抑制egf的作用機(jī)制。方法:選取生長狀態(tài)良好的兔第3代rpe細(xì)胞在蓋玻片上制備成細(xì)胞爬片進(jìn)行實(shí)驗(yàn)。分為空白對照組(含0.5‰dmso的10%fbs.dmem)和10、15、20ug/ml姜黃素4組;每組各設(shè)6個復(fù)孔,共接種3塊培養(yǎng)板,加藥后24、48、72h時隨機(jī)抽取1塊培養(yǎng)板行免疫組織化學(xué)染色,觀察rpe細(xì)胞內(nèi)egf的表達(dá)情況。rt-pcr、westernblot分別檢測空白對照組(含0.5‰dmso的10%fbs.dmem)、姜黃素(15ug/ml)、egf(9ng/ml含0.5‰dmso)、egf(9ng/ml)+姜黃素(15ug/ml)作用24、48、72h后rpe細(xì)胞中egfmrna和蛋白表達(dá)的影響。結(jié)果:1不同濃度姜黃素對rpe內(nèi)egf表達(dá)的抑制作用:時間依賴性:姜黃素各濃度組對rpe細(xì)胞內(nèi)egf表達(dá)的抑制作用均隨作用時間的延長而增強(qiáng),各時間點(diǎn)之間差異均有統(tǒng)計學(xué)意義(p0.05),姜黃素對rpe細(xì)胞內(nèi)egf表達(dá)的抑制作用具有時間依賴性。劑量依賴性:姜黃素各時間點(diǎn)對rpe細(xì)胞內(nèi)egf表達(dá)的抑制均隨藥物質(zhì)量濃度的增高而增強(qiáng),除姜黃素15ug/ml和20ug/ml組之間差異無統(tǒng)計學(xué)意義(p0.05)外,其余各質(zhì)量濃度之間差異均有統(tǒng)計學(xué)意義(p0.05)。2姜黃素對rpe細(xì)胞內(nèi)egfmrna轉(zhuǎn)錄的影響:向培養(yǎng)液中加入姜黃素使其濃度為15ug/ml,在24h、48h、及72h檢測egf的mrna含量,可見隨時間延長細(xì)胞內(nèi)egf的mrna表達(dá)量下降,和對照組相比較,差異有統(tǒng)計學(xué)意義(p0.05);不同時間點(diǎn)間做比較,相鄰時間點(diǎn)組間相比差異均有統(tǒng)計學(xué)意義(p0.05)。姜黃素對egf作用下rpe細(xì)胞中egfmrna表達(dá)量在各時間點(diǎn)和對照組比較差異有統(tǒng)計學(xué)意義(p0.05),不同時間點(diǎn)間,相鄰的時間組相比較差異均有統(tǒng)計學(xué)意義(p0.05)。3姜黃素對rpe細(xì)胞內(nèi)egf蛋白表達(dá)的影響:向培養(yǎng)液中加入姜黃素使其濃度為15ug/ml,作用24h、48h、及72h,隨著時間的延長細(xì)胞內(nèi)egf蛋白表達(dá)量逐漸下降,與對照組相比較差異有統(tǒng)計學(xué)意義(p0.05);不同時間點(diǎn)間,相鄰的時間組間相比較差異均有統(tǒng)計學(xué)意義(p0.05)。姜黃素對egf作用下rpe細(xì)胞中egf蛋白表達(dá)量在各時間點(diǎn)和對照組比較差異有統(tǒng)計學(xué)意義(p0.05),不同時間點(diǎn)間,相鄰的時間組相比差異均有統(tǒng)計學(xué)意義(p0.05)。結(jié)論:姜黃素在體外抑制兔rpe細(xì)胞內(nèi)egf的表達(dá),最佳抑制濃度是15ug/ml,姜黃素在體外可顯著抑制兔rpe細(xì)胞內(nèi)egfmrna和蛋白的表達(dá)。第三部分姜黃素在兔眼增殖性玻璃體視網(wǎng)膜病變中對egf作用的實(shí)驗(yàn)研究目的:研究姜黃素在體內(nèi)兔眼增殖性玻璃體視網(wǎng)膜病變形成過程中對egf的作用及對pvr的防治作用。方法:選擇正常青紫藍(lán)兔30只,所有兔在玻璃體注射前均抽出0.2ml玻璃體,隨機(jī)選取一眼納入對照組:共30眼,玻璃體腔注射0.1ml(2×106)培養(yǎng)的生長狀態(tài)良好的第三代同種rpe細(xì)胞和含0.5‰dmso的生理鹽水0.1ml;另一眼納入實(shí)驗(yàn)組:共30眼,玻璃體腔注射0.1ml(2×106)培養(yǎng)的生長狀態(tài)良好的同種第三代rpe細(xì)胞和質(zhì)量濃度為1mg/ml的姜黃素0.1ml。注射后3、7、14、21、28天進(jìn)行裂隙燈顯微鏡檢查觀察眼前節(jié)和前部玻璃體情況,間接眼底鏡檢查觀察眼底情況,眼底彩色照相,眼部b超檢查觀察玻璃體渾濁情況和視網(wǎng)膜有無脫離及脫離的程度,檢查完畢后,在每個時間點(diǎn)隨機(jī)抽取6只兔,12只眼,對照組6只眼,實(shí)驗(yàn)組6只眼,分別抽取玻璃體,采用兔表皮生長因子elisa試劑盒檢測玻璃體液中egf的含量。結(jié)果:1前房反應(yīng):玻璃體腔注射后第3天對照組和實(shí)驗(yàn)組兔眼前房內(nèi)均可見少量浮游物,第7天時均消退。2玻璃體和視網(wǎng)膜情況:第3天兩組玻璃體均有渾濁,實(shí)驗(yàn)組玻璃體混濁輕,第7天時玻璃體內(nèi)有增殖膜形成,對照組增殖膜多且厚,實(shí)驗(yàn)組增殖膜局限且薄;第14天時對照組視網(wǎng)膜脫離發(fā)生率(11/18)61%,實(shí)驗(yàn)組玻璃體內(nèi)增殖膜較薄,視網(wǎng)膜脫離發(fā)生率(2/18)11%;第21天時對照組視網(wǎng)膜脫離發(fā)生率(8/12)67%,增殖膜上可見新生血管,實(shí)驗(yàn)組視網(wǎng)膜脫離發(fā)生率(2/12)16%,視網(wǎng)膜脫離范圍小,第28天對照組視網(wǎng)膜脫離漸加重,并有新生血管生長,視網(wǎng)膜脫離發(fā)生率(5/6)83%,實(shí)驗(yàn)組視網(wǎng)膜脫離局限,發(fā)生率(1/6)16%;視網(wǎng)膜脫離發(fā)生率實(shí)驗(yàn)組和對照組比較差異顯著,有統(tǒng)計學(xué)意義(p0.05)。3玻璃體中egf含量測定(elisa結(jié)果):玻璃體液中egf含量對照組較高,實(shí)驗(yàn)組較少,各時間點(diǎn)實(shí)驗(yàn)組和對照組比較差異均具有統(tǒng)計學(xué)意義(P0.05)。結(jié)論:玻璃體腔內(nèi)注射姜黃素可以有效抑制RPE細(xì)胞誘導(dǎo)的兔眼實(shí)驗(yàn)性PVR形成過程中表皮生長因子的水平,進(jìn)而抑制PVR的發(fā)生和發(fā)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R774.1

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