羟基喜树碱调控青光眼术区纤维化的实验研究

发布时间：2018-04-28 23:14

  本文选题：羟基喜树碱 + 丝裂霉素　； 参考：《南京医科大学》2011年硕士论文

【摘要】：目的:通过体外培养人眼Tenon’s囊成纤维细胞(Human Tenon’s capsule fibroblasts,HTFs),观察羟基喜树碱(HCPT)对HTFs增殖、移行及细胞周期的影响,探讨其可能的作用机制,并与丝裂霉素C(MMC)对比。 方法:1、HTFs的体外培养和鉴定:取正常供体新鲜的Tenon’s囊组织,采用组织块培养法,进行成纤维细胞的体外培养,并用光镜、免疫荧光法观察鉴定; 2、MTT法检测不同浓度的羟基喜树碱(0.031、0.062、0.125、0.25、0.5、1、2、4mg/l)、丝裂霉素(0.0031、0.0062、0.0125、0.025、0.05、0.1、0.2、0.4mg/l)作用24h、48h、72h后HTFs的A值变化,观察两种药物对HTFs的抑制作用,并进行对比; 3、流式细胞仪测定羟基喜树碱及丝裂霉素C对HTFs细胞周期的影响; 4、采用划痕法检测羟基喜树碱及丝裂霉素C对HTFs迁移的抑制能力; 5、通过台盼蓝染色法鉴定羟基喜树碱、丝裂霉素C对HTFs增殖、迁移的抑制作用是否由药物的细胞毒性引起; 6、琼脂糖凝胶电泳观察细胞凋亡结果; 7、Realtime PCR检测羟基喜树碱、丝裂霉素C作用24后HTFs的Smad7 mRNA基因表达的变化,探讨羟基喜树碱抑制HTFs增殖的可能机制。 结果:1、体外成功培养正常HTFs,细胞长梭形,胞浆丰富,胞核大,生长能力强,呈漩涡状或羽毛状生长;HTFs波形蛋白染色阳性、角蛋白染色阴性,证实所培养的细胞为成纤维细胞,而非上皮细胞; 2、MTT法显示不同浓度的HCPT组、MMC组作用24h、48h、72h后HTFs细胞的增值率均低于空白对照组,差异具有统计学意义;HCPT(0.031—0.125mg/l)、MMC(0.0031—0.0125mg/l)对HTFs的抑制率不显著增高,HCPT(0.25—4mg/l)、MMC(0.025—0.4mg/l)对HTFs的抑制率显著增高,呈现剂量依赖性和时间依赖性;HCPT作用24h、48h、72h的IC50分别为2.24mg/l、0.76mg/l、0.39mg/l,MMC作用24h、48h、72h的IC50分别为0.34mg/l、0.24mg/l、0.07mg/l,MMC对HTF增殖的抑制效应约要强于HCPT; 3、流式细胞仪检测HCPT(0、0.25、1、4mg/l)、MMC(0、0.025、0.1、0.4mg/l)对HTFs细胞周期的影响,结果显示HCPT主要影响HTFs的G2期和S期,MMC主要将HTFs阻滞于G1期; 4、划痕法检测不同浓度的HCPT、MMC作用24h、48h、72h后对HTFs迁移能力的影响,结果显示HCPT、MMC作用后HTFs的迁移能力受到抑制,这种抑制作用呈剂量依赖性而与时间无显著相关; 5、台盼蓝染色法检测HCPT(0、0.25、0.5、1、2、4mg/l)、MMC(0、0.0025、0.05、0.1、0.2、0.4)作用24h后HTFs的活细胞率,结果显示HCPT组、MMC组活细胞率和空白对照组比较均无统计学差异; 6、0.4mg/lMMC、4mg/lHCPT作用HTFs24h后,提取出的DNA经过1%琼脂糖凝胶电泳,呈现细胞凋亡后特异性梯状条带; 7、Realtime PCR检测0.4mg/lMMC、4mg/lHCPT作用HTFs24h后,Smad7 mRNA表达水平显著上调。 结论:1、羟基喜树碱和丝裂霉素C对体外培养HTFs的增殖、迁移有明显抑制作用,这种抑制作用呈剂量和时间依赖性。该两种药物对HTFs增殖、迁移的抑制效应:MMC约为HCPT的10倍左右,HCPT的安全性要高于MMC; 2、羟基喜树碱主要影响HTFs的G2和S期,丝裂霉素C主要将HTFs阻滞于G1期(静止期); 3、羟基喜树碱、丝裂霉素C抑制HTFs增殖、迁移的作用和药物的细胞毒性无关; 4、羟基喜树碱及丝裂霉素C均能诱导HTFs凋亡; 5、羟基喜树碱抑制HTFs增殖的机制可能是:通过上调Smad7 mRNA的表达阻断TGF—β信号通路从而抑制HTFs增殖和迁移。
[Abstract]:Objective: To observe the effect of Hydroxycamptothecin (HCPT) on the proliferation, migration and cell cycle of Tenon 's cysts (Human Tenon' s capsule fibroblasts, HTFs) in vitro, and to explore the possible mechanism of the action of HTFs, and compare with C (MMC) pairs of mitomycin.
Methods: 1, in vitro culture and identification of HTFs: fresh Tenon 's cyst tissue of normal donor, tissue culture method was used to culture the fibroblasts in vitro, and the identification was observed by light microscopy and immunofluorescence. 2, MTT method was used to detect the different concentrations of Hydroxycamptothecin (0.031,0.062,0.125,0.25,0.5,1,2,4mg/l) and Mitomycin (0.0031,0.0062,0.01). 25,0.025,0.05,0.1,0.2,0.4mg/l) change the A value of HTFs after 24h, 48h, 72h, observe the inhibitory effect of two drugs on HTFs, and compare them. 3, the effect of hydroxycamptothecin and mitomycin C on the HTFs cell cycle is measured by flow cytometry; 4, the inhibition ability of hydroxyl camptothecin and mitomycin C to HTFs migration is detected by the scratch method; 5 A trypan blue staining method was used to identify hydroxyl camptothecin and mitomycin C on HTFs proliferation and the inhibition of migration was caused by cytotoxicity of drugs; 6, agarose gel electrophoresis was used to observe the results of cell apoptosis; 7, Realtime PCR was used to detect the changes in the expression of Smad7 mRNA gene in HTFs after C action of mitomycin C, and to explore hydroxyl camptothecin The possible mechanism to inhibit the proliferation of HTFs.
Results: 1, the normal HTFs was successfully cultured in vitro, the cells grew spindle shaped, the cytoplasm was rich, the nucleus was large, the growth ability was strong, the cells were whirlpool or featherlike growth, and HTFs vimentin was positive, and the keratin staining was negative, which proved that the cultured cells were fibroblasts, not epithelial cells.
2, MTT method showed different concentrations of HCPT group. The increment rate of HTFs cells in group MMC was lower than that of blank control group after 24h, 48h and 72h. The difference was statistically significant. HCPT (0.031 0.125mg/l), MMC (0.0031 - 0.0125mg/l) did not significantly increase the inhibition rate of HTFs. (0.25 - 0.025), the inhibition rate increased significantly. The effects of HCPT on 24h, 48h, and 72h are 2.24mg/l, 0.76mg/l, 0.39mg/l, MMC, 24h, 48h, respectively.
3, the effect of HCPT (0,0.25,1,4mg/l) and MMC (0,0.025,0.1,0.4mg/l) on the cycle of HTFs cells was detected by flow cytometry. The results showed that HCPT mainly affected the G2 phase and S phase of HTFs, and MMC mainly blocked the HTFs in G1 stage.
4, the effect of different concentrations of HCPT and MMC on the migration of HTFs after 24h, 48h and 72h was detected. The results showed that the migration ability of HTFs was inhibited after the action of HCPT and MMC, and this inhibitory effect was dose-dependent but had no significant correlation with time.
5, trypan blue staining was used to detect the living cell rate of HCPT (0,0.25,0.5,1,2,4mg/l) and MMC (0,0.0025,0.05,0.1,0.2,0.4) after 24h, and the results showed that there was no significant difference in the rate of living cells between the HCPT group and the MMC group compared with the blank control group.
After 6,0.4mg/lMMC and 4mg/lHCPT were applied to HTFs24h, the extracted DNA was shown by 1% agarose gel electrophoresis, showing a specific ladder like band after apoptosis.
7, Realtime PCR detected 0.4mg/lMMC, 4mg/lHCPT after HTFs24h, Smad7 mRNA expression level was significantly up-regulated.
Conclusion: 1, hydroxycamptothecin and mitomycin C have obvious inhibitory effect on the proliferation and migration of HTFs in vitro. This inhibitory effect is dosed and time dependent. The inhibitory effects of the two drugs on HTFs proliferation and migration are about 10 times as much as HCPT, and the safety of HCPT is higher than that of MMC.
2, HCPT mainly affected the G2 and S phases of HTFs. Mitomycin C mainly blocked HTFs in G1 phase (quiescent stage).
3, hydroxycamptothecin and mitomycin C inhibited HTFs proliferation.
4, hydroxycamptothecin and mitomycin C both induce HTFs apoptosis.
5, the mechanism of HCPT inhibiting the proliferation of HTFs is probably to inhibit the proliferation and migration of HTFs by up regulating the expression of Smad7 mRNA and blocking the TGF - beta signaling pathway.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R779.6

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