本文選題：漢防己甲素 + 5-氟尿嘧啶��； 參考：《山東中醫(yī)藥大學(xué)》2010年碩士論文

【摘要】：目的：1.研究Tet與5-Fu對(duì)兔眼體外RPE細(xì)胞增殖的抑制作用,并探討其抑制兔RPE細(xì)胞增殖的作用機(jī)制,以期初步評(píng)價(jià)藥物防治PVR的可行性問題。2.觀察Tet聯(lián)合5-Fu殼聚糖微球(Tet-5-FU-CS-MS)對(duì)實(shí)驗(yàn)性兔眼PVR模型的防治效果,研究藥物在眼內(nèi)的抗炎機(jī)制。 方法：1.提取培養(yǎng)兔RPE細(xì)胞,傳至第四代并鑒定后,選取生長(zhǎng)良好的第四代RPE細(xì)胞進(jìn)行試驗(yàn)。設(shè)Tet分為2、5、10、15μg/mL組及空白對(duì)照組(1O%FBS-DMEM,含0.5%oDMSO, DMSO終濃度≤1‰,對(duì)細(xì)胞無(wú)毒性作用)。5-Fu為5、10、15、20μg/mL組及空白對(duì)照組(同上)。噻唑藍(lán)比色(MTT)法檢測(cè)兩組藥物對(duì)RPE細(xì)胞增殖的抑制率,從而測(cè)定出增殖抑制率較高而藥物濃度相對(duì)較低的最佳有效濃度；流式細(xì)胞儀(FCM)檢測(cè)最佳有效濃度的兩組藥物及空白對(duì)照組作用72h后對(duì)RPE細(xì)胞周期的影響；流式細(xì)胞儀(FCM)檢測(cè)最佳有效濃度兩組藥物及空白對(duì)照組分別作用24h、48h、72h、96h后對(duì)RPE凋亡率、線粒體跨膜電位(ΔΨm)、細(xì)胞質(zhì)內(nèi)Ca2+濃度、Bax, Bcl-2蛋白表達(dá)的影響。2.健康成年青紫藍(lán)兔48只,隨機(jī)分為A、B、C三組,每組16只兔32眼,建立兔眼外傷性PVR模型,向玻璃體中后部注入藥物,A組注入含有Tet-5-FU-CS-MS的BSS懸浮液0.2ml,B組注入含有5-Fu聚乳酸微球的BSS懸浮液0.2ml,C組注入25mg無(wú)藥物殼聚糖微球的BSS懸浮液0.2ml,術(shù)后每天觀察眼底變化,必要時(shí)行眼科B超檢查直到注藥后第28天,藥效評(píng)價(jià)按Ryan分級(jí)法,觀察各組視網(wǎng)膜脫離的發(fā)生率。分別于術(shù)后7天、14天、28天等3個(gè)時(shí)間點(diǎn)抽取各術(shù)眼玻璃體液0.2ml,酶聯(lián)免疫吸附法(ELISA)檢測(cè)玻璃體液中TNF-α. IL-1β的含量。 成果：1.Tet與5-Fu明顯抑制兔體外RPE細(xì)胞的增殖,呈時(shí)間和劑量依賴性,除作用96h后,Tet1O、15μg/ml及5-Fu15,20μg/ml之間無(wú)統(tǒng)計(jì)學(xué)意義外,其余各質(zhì)量濃度及時(shí)間點(diǎn)之間的比較均有統(tǒng)計(jì)學(xué)意義(p0.05),分別選取RPE細(xì)胞的增殖抑制率較高而藥物濃度相對(duì)較低的Tet 10μg/ml)組和5-FU (15μg/ml)組進(jìn)行下面的實(shí)驗(yàn)。Tet與5-Fu兩組RPE細(xì)胞在72h時(shí)G0/G1(合成前期)細(xì)胞明顯增多,與相應(yīng)空白對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義(P0.05); Tet與5-Fu兩組各時(shí)間點(diǎn)RPE細(xì)胞凋亡率與相應(yīng)對(duì)照組相比明顯升高,差異均有統(tǒng)計(jì)學(xué)意義(p0.05)；隨時(shí)間延長(zhǎng)實(shí)驗(yàn)組RPE細(xì)胞線粒體ΔΨ逐漸降低,與相應(yīng)對(duì)照組相比差異均有統(tǒng)計(jì)學(xué)意義(P0.05)；隨時(shí)間延長(zhǎng)細(xì)胞質(zhì)內(nèi)Ca2+濃度逐漸升高,與相應(yīng)對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義(P0.05)。Tet(10μg/ml)及5-Fu(15μg/ml)實(shí)驗(yàn)組與相應(yīng)空白對(duì)照組相比,均出現(xiàn)Bax蛋白表達(dá)增加,Bcl2蛋白表達(dá)下降,Bcl-2/Bax比值下降,兩者比較差異有統(tǒng)計(jì)學(xué)意義(P0.05)。2Tet-5-FU-CS-MS組與5-Fu聚乳酸微球組及空白微球組相比,各時(shí)間點(diǎn)視網(wǎng)膜脫離的發(fā)生率明顯降低,其玻璃體液中TNF-α、IL-1B等炎性因子的含量也顯著低于其它兩組,方差分析p0.05,差異有統(tǒng)計(jì)學(xué)意義。 結(jié)論：1.Tet與5-Fu對(duì)體外兔RPE細(xì)胞的增生有明顯抑制作用,通過(guò)阻斷RPE細(xì)胞增生周期,干擾Bcl-2及Bax蛋白表達(dá)而誘導(dǎo)RPE細(xì)胞凋亡,兩藥聯(lián)用在防治PVR方面具有潛在價(jià)值。2Tet-5-FU-CS-MS顯著降低實(shí)驗(yàn)性兔眼PVR的發(fā)生率,有可能在PVR炎癥期發(fā)揮抗炎作用,抑制眼內(nèi)炎性因子的釋放,從而起到防治PVR發(fā)生的協(xié)同作用。
[Abstract]:Objective: 1. to study the inhibitory effect of Tet and 5-Fu on the proliferation of RPE cells in rabbit eye in vitro, and to explore the mechanism of its inhibitory effect on the proliferation of rabbit RPE cells in order to evaluate the feasibility of the drug control of PVR by.2. to observe the effect of Tet combined with 5-Fu chitosan microspheres (Tet-5-FU-CS-MS) on the experimental rabbit eye PVR model, and to study the resistance of drug in the eye. The mechanism of inflammation.
Methods: 1. the rabbit RPE cells were extracted and cultured to fourth generations and identified. The fourth generation of RPE cells with good growth were selected, and Tet was divided into 2,5,10,15 mu g/mL group and blank control group (1O%FBS-DMEM, 0.5%oDMSO, DMSO terminal concentration < 1 per 1000, non toxic effect on cells).5-Fu 5,10,15,20 u g/mL group and blank control group (same). Thiazole The inhibition rate of two groups of drugs on the proliferation of RPE cells was detected by blue colorimetric (MTT) method, and the best effective concentration of the proliferation inhibition rate and relatively low drug concentration was measured; the effect of two groups of drugs on the best effective concentration by flow cytometry (FCM) and the effect of 72h on the RPE cell cycle after the action of the blank control group; and the flow cytometry (FCM) detection The best effective concentration two groups of drugs and blank control group were treated with 24h, 48h, 72h, 96h, the apoptosis rate of RPE, the mitochondrial transmembrane potential (delta m), the concentration of Ca2+ in the cytoplasm, Bax, and the expression of Bcl-2 protein in 48 rabbits of.2. healthy adult blue and blue rabbits, randomly divided into A, B, three groups, 16 rabbits in each group, 32 eyes, to establish the rabbit ocular traumatic model and to vitreous body. The drug was injected into the middle and posterior parts, the BSS suspension containing Tet-5-FU-CS-MS was injected into the A group 0.2ml, the B group injected the BSS suspension containing 5-Fu polylactic acid microspheres 0.2ml, the C group injected the BSS suspensions of 25mg without the drug chitosan microspheres, and observed the changes of the fundus daily. The incidence of retinal detachment was observed in each group. The vitreous fluid 0.2ml was extracted from all eyes at 7 days, 14 days and 28 days, respectively, and enzyme linked immunosorbent assay (ELISA) was used to detect the content of TNF- alpha. IL-1 beta in the vitreous fluid.
Results: 1.Tet and 5-Fu obviously inhibited the proliferation of RPE cells in rabbit in vitro, which was time and dose dependent. Except for 96h, there was no statistical significance between Tet1O, 15, g/ml and 5-Fu15,20 mu g/ml, the rest of the mass concentration and time points were statistically significant (P0.05), and the proliferation inhibition rate of RPE cells was higher and the drug concentration was stronger respectively. The lower Tet 10 mu g/ml) group and the 5-FU (15 g/ml) group carried out the experimental.Tet and 5-Fu two groups of RPE cells in the G0/G1 (Synthesis prophase) cells significantly increased, compared with the corresponding blank control group, the difference was statistically significant (P0.05), Tet and 5-Fu two at the time point of apoptosis rate compared with the corresponding control group was significantly higher than the corresponding control group. The differences were statistically significant (P0.05). The mitochondrial delta of RPE cells in the experimental group decreased gradually with time, and the difference was statistically significant compared with the corresponding control group (P0.05). The concentration of Ca2+ in the cytoplasm increased gradually with the time, and the difference was statistically significant (P0.05).Tet (10 mu g/ml) and 5-Fu (15 u g/ml) experimental group compared with the corresponding control group. Compared with the corresponding blank control group, the expression of Bax protein increased, the expression of Bcl2 protein decreased and the Bcl-2/Bax ratio decreased. The difference was statistically significant (P0.05) compared with the 5-Fu polylactic acid microspheres group and the blank microsphere group, the incidence of retinal detachment at each time point decreased significantly, and the TNF- alpha and IL- in the glass body fluid were TNF-, IL-. 1B and other inflammatory factors were also significantly lower than those of the other two groups. The variance analysis P0.05 showed a significant difference.
Conclusion: 1.Tet and 5-Fu have obvious inhibitory effect on the proliferation of rabbit RPE cells in vitro. By blocking the proliferation cycle of RPE cells and interfering with the expression of Bcl-2 and Bax protein, the apoptosis of RPE cells can be induced. The potential value of two drugs in the prevention and control of PVR has a significant decrease in the incidence of PVR in the experimental rabbit eye, and it may play a possible role in the PVR inflammatory period. Anti inflammatory effect can inhibit the release of endophthalmitis factor, thereby playing a synergistic role in the prevention and treatment of PVR.

【學(xué)位授予單位】：山東中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R774.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 龔守良,劉淑春,呂U，

本文編號(hào)：1840212