叶酸—壳聚糖载药DAC影响SCC-9细胞p16基因甲基化状态的意义

发布时间：2018-03-26 00:33

本文选题：叶酸　切入点：壳聚糖　出处：《广西医科大学》2017年硕士论文

【摘要】：目的:研究叶酸偶联壳聚糖携带5′-氮杂-2′-脱氧胞苷(5-aza-2deoxycytidine,地西他滨,DAC)纳米粒对比5′-氮杂-2′-脱氧胞苷作用于体外培养的人鳞状上皮舌癌SCC-9细胞,了解叶酸偶联壳聚糖载药DAC对SCC-9细胞的p16基因和p16蛋白的表达情况,及对SCC-9细胞的抑制作用,从而探索叶酸偶联壳聚糖载药纳米粒携DAC对细胞的p16基因的高甲基化状态的还原能力的有效性,以及同时了解载药系统对细胞提高药效的能力。方法:合成叶酸壳聚糖载药DAC纳米粒,测其zeta电位证实其为稳定的纳米颗粒后,将SCC-9细胞分为0、1、2、3、4、5、6、7八组。2、3、4组选用三个梯度浓度1×l0-7mol/L、1×l0-6mol/L、1×l0-5mol/L的DAC进行处理细胞,5、6、7组采用同样三个浓度梯度1×l0-7mol/L、1×l0-6mol/L、1×l0-5mol/L的叶酸壳聚糖载药DAC纳米粒处理细胞,0组为空白生理盐水对照组,1组为空白叶酸壳聚糖处理组,0组内设3个浓度的组内对照a、b、c,1组内设3个组内对照,为e、f、c组。观察叶酸壳聚糖载药DAC纳米颗粒对比DAC作用细胞,用MTT法测各组药物处理细胞5天内的SCC-9细胞抑制率;药物各自作用48小时后,实时荧光定量PCR法检测细胞中p16基因m RNA的表达和应用免疫组化SP法检测细胞p16的蛋白表达。结果:MTT结果表明相同浓度的叶酸-壳聚糖载药DAC纳米颗粒组药物对细胞的抑制率高于同样浓度下的DAC组(P0.05),且药物对细胞的抑制率均与药物浓度呈正相关关系。实时荧光定量PCR结果显示2、3、4、5、6、7组的p16基因m RNA表达量显著高于0、1组(P0.05)。0组和1组无统计学意义(P0.05)。相同浓度的叶酸-壳聚糖载药DAC纳米颗粒组的p16基因的m RNA表达量高于同样浓度下的DAC组(P0.05);免疫组化结果显示,2、3、4、5、6、7组相较于0、1组呈强阳性表达,有统计学意义(P0.05),相同浓度的叶酸-壳聚糖载药DAC纳米颗粒组的p16蛋白的表达量高于同样浓度下的DAC组并具有统计学意义(P0.05),0组和1组没有统计学意义(P0.05)。结论:1.叶酸-壳聚糖载药纳米粒携带DAC和DAC均对SCC-9细胞有明显的去甲基化能力。2.叶酸-壳聚糖载药纳米粒携带DAC对比DAC具有更高效逆转SCC-9细胞p16基因甲基化状态,并具有促使p16基因m RNA和蛋白恢复表达的作用。3.叶酸-壳聚糖载药纳米粒携带DAC和DAC,去甲基化能力没有随着药物浓度升高而升高。4.叶酸-壳聚糖载药纳米粒携带DAC组对比DAC组,药物抑制率更明显。
[Abstract]:Aim: to study the effects of folic acid-coupled chitosan carrying 5-aza-2deoxycytidine (DAC-) nanoparticles on human squamous cell carcinoma (SCC-9) cells in vitro. To investigate the expression of p16 gene and p16 protein in SCC-9 cells and the inhibitory effect of folic acid-coupled chitosan loaded DAC on SCC-9 cells. To explore the effectiveness of folic acid-coupled chitosan loaded nanoparticles with DAC in reducing the hypermethylation of p16 gene. Methods: DAC nanoparticles loaded with chitosan folate were synthesized, and their zeta potential was determined to be stable nanoparticles. The SCC-9 cells were divided into 8 groups: 1 脳 l0-7 mol / L 1 脳 l0-6 mol / L 1 脳 1 脳 1 脳 L ~ (-1) mol / L ~ (1 脳 1 脳 10 ~ (-1) mol / L ~ (1)) l0-5mol/L. The cells were treated with the same concentration gradient of 1 脳 10 ~ (-7) mol / L ~ (-1) L ~ (-1) L _ (1 脳 10 ~ (-6) mol 路L ~ (-1)) DAC nanoparticles. Group 0 was treated as a blank normal saline control group (n = 1) with the same concentration gradient of 1 脳 10 ~ (-7) mol 路L ~ (-1) mol 路L ~ (-1) mol 路L ~ (-1) DAC. The control group was divided into 3 groups with 3 concentrations of folic acid chitosan treatment group, and 3 control groups with 3 concentrations in the control group (group 1), and the control group (group 1) was treated with folic acid chitosan. The DAC nanoparticles loaded with chitosan folate were observed to compare the effect of DAC on the cells. The inhibition rate of SCC-9 cells in each group was measured by MTT method within 5 days, and after 48 hours of treatment with each drug, the inhibition rate of SCC-9 cells was measured by MTT method. The expression of p16 gene m RNA was detected by real-time fluorescence quantitative PCR and the protein expression of p16 protein was detected by immunohistochemistry SP method. Results the results showed that the same concentration of folic acid-chitosan loaded DAC nanoparticles was used to detect the expression of p16 protein. The inhibition rate of cells was higher than that of DAC group at the same concentration, and the inhibitory rate of the drug on the cell was positively correlated with the drug concentration. The results of real-time quantitative PCR showed that the expression of p16 gene m RNA in group 2, 3, 3, 4, 5, 5, 5, 5, 7 was significantly higher than that in group 0, 0, 1, P0. 05 and 0. 0, respectively. The expression of m RNA of p16 gene in the same concentration of folic acid-chitosan loaded DAC nanoparticles was higher than that in the same concentration of DAC group P0.05.The immunohistochemical results showed that the expression of p16 gene was strongly positive in the same concentration of folate-chitosan loaded DAC group compared with the control group. The expression of p16 protein in the DAC nanoparticles with the same concentration of folic acid-chitosan was higher than that in the DAC group at the same concentration, and there was no significant difference in the expression of p16 protein between the two groups. Conclusion\\\%\\%\%\%\%\%\%? Both DAC and DAC carried by chitosan nanoparticles had obvious demethylation ability to SCC-9 cells. Compared with DAC, folic acid-chitosan nanoparticles carrying DAC could reverse the methylation of p16 gene in SCC-9 cells more efficiently. It can promote the expression of p16 gene m RNA and protein. 3. Folic acid-chitosan nanoparticles carry DAC and DAC, and the demethylation ability does not increase with the increase of drug concentration. 4. Folic acid-chitosan nanoparticles carrying DAC group compared with DAC group. The drug inhibition rate was more obvious.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.86

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