汉防己甲素联合5-氟尿嘧啶壳聚糖微球防治实验性PVR的实验研究

发布时间：2018-05-03 21:36

本文选题：汉防己甲素 + 5-氟尿嘧啶　；参考：《山东中医药大学》2010年硕士论文

【摘要】：目的：1.研究Tet与5-Fu对兔眼体外RPE细胞增殖的抑制作用,并探讨其抑制兔RPE细胞增殖的作用机制,以期初步评价药物防治PVR的可行性问题。2.观察Tet联合5-Fu壳聚糖微球(Tet-5-FU-CS-MS)对实验性兔眼PVR模型的防治效果,研究药物在眼内的抗炎机制。方法：1.提取培养兔RPE细胞,传至第四代并鉴定后,选取生长良好的第四代RPE细胞进行试验。设Tet分为2、5、10、15μg/mL组及空白对照组(1O%FBS-DMEM,含0.5%oDMSO, DMSO终浓度≤1‰,对细胞无毒性作用)。5-Fu为5、10、15、20μg/mL组及空白对照组(同上)。噻唑蓝比色(MTT)法检测两组药物对RPE细胞增殖的抑制率,从而测定出增殖抑制率较高而药物浓度相对较低的最佳有效浓度；流式细胞仪(FCM)检测最佳有效浓度的两组药物及空白对照组作用72h后对RPE细胞周期的影响；流式细胞仪(FCM)检测最佳有效浓度两组药物及空白对照组分别作用24h、48h、72h、96h后对RPE凋亡率、线粒体跨膜电位(ΔΨm)、细胞质内Ca2+浓度、Bax, Bcl-2蛋白表达的影响。2.健康成年青紫蓝兔48只,随机分为A、B、C三组,每组16只兔32眼,建立兔眼外伤性PVR模型,向玻璃体中后部注入药物,A组注入含有Tet-5-FU-CS-MS的BSS悬浮液0.2ml,B组注入含有5-Fu聚乳酸微球的BSS悬浮液0.2ml,C组注入25mg无药物壳聚糖微球的BSS悬浮液0.2ml,术后每天观察眼底变化,必要时行眼科B超检查直到注药后第28天,药效评价按Ryan分级法,观察各组视网膜脱离的发生率。分别于术后7天、14天、28天等3个时间点抽取各术眼玻璃体液0.2ml,酶联免疫吸附法(ELISA)检测玻璃体液中TNF-α. IL-1β的含量。成果：1.Tet与5-Fu明显抑制兔体外RPE细胞的增殖,呈时间和剂量依赖性,除作用96h后,Tet1O、15μg/ml及5-Fu15,20μg/ml之间无统计学意义外,其余各质量浓度及时间点之间的比较均有统计学意义(p0.05),分别选取RPE细胞的增殖抑制率较高而药物浓度相对较低的Tet 10μg/ml)组和5-FU (15μg/ml)组进行下面的实验。Tet与5-Fu两组RPE细胞在72h时G0/G1(合成前期)细胞明显增多,与相应空白对照组相比差异有统计学意义(P0.05); Tet与5-Fu两组各时间点RPE细胞凋亡率与相应对照组相比明显升高,差异均有统计学意义(p0.05)；随时间延长实验组RPE细胞线粒体ΔΨ逐渐降低,与相应对照组相比差异均有统计学意义(P0.05)；随时间延长细胞质内Ca2+浓度逐渐升高,与相应对照组相比差异有统计学意义(P0.05)。Tet(10μg/ml)及5-Fu(15μg/ml)实验组与相应空白对照组相比,均出现Bax蛋白表达增加,Bcl2蛋白表达下降,Bcl-2/Bax比值下降,两者比较差异有统计学意义(P0.05)。2Tet-5-FU-CS-MS组与5-Fu聚乳酸微球组及空白微球组相比,各时间点视网膜脱离的发生率明显降低,其玻璃体液中TNF-α、IL-1B等炎性因子的含量也显著低于其它两组,方差分析p0.05,差异有统计学意义。结论：1.Tet与5-Fu对体外兔RPE细胞的增生有明显抑制作用,通过阻断RPE细胞增生周期,干扰Bcl-2及Bax蛋白表达而诱导RPE细胞凋亡,两药联用在防治PVR方面具有潜在价值。2Tet-5-FU-CS-MS显著降低实验性兔眼PVR的发生率,有可能在PVR炎症期发挥抗炎作用,抑制眼内炎性因子的释放,从而起到防治PVR发生的协同作用。
[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.

【学位授予单位】：山东中医药大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R774.1

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