中空介孔二氧化硅—姜黄素纳米制剂的制备及其对肝癌细胞增殖、凋亡和自噬的影响
发布时间：2019-02-24 18:57
【摘要】：目的:姜黄素是从姜黄根茎中提取的有效活性成分,是一种应用前景广泛的天然抗肿瘤药物。但姜黄素水溶性差,生物利用度低,这就大大限制了姜黄素的临床推广。本研究制备了中空介孔二氧化硅纳米粒子,负载姜黄素,得到中空介孔二氧化硅-姜黄素纳米制剂,通过质量评价和药物释放研究后,以肝癌SMMC-7721细胞和Hep G2细胞为研究对象,探索:1.中空介孔二氧化硅-姜黄素纳米制剂对肝癌细胞增殖和凋亡的影响;2.中空介孔二氧化硅-姜黄素纳米制剂是否激活肝癌细胞自噬。这不仅能解决姜黄素生物利用度低的问题,并且可明确中空介孔二氧化硅-姜黄素纳米制剂抗肝癌的部分机理,为肝癌的临床应用提供理论基础。方法:1.乳液聚合法制备阳离子聚苯乙烯模板(CPS),以CPS为模板,加入Na2Si O3·9H2O,制备二氧化硅纳米粒子,经高温分层煅烧,得到中空介孔二氧化硅纳米粒子(HMS)。扫描电镜(SEM)对CPS和HMS进行表征。2.HMS和姜黄素以一定比例混合,机械搅拌后得到中空介孔二氧化硅-姜黄素纳米粒子(HMS-CUR),用透射电镜(TEM)进行表征,傅里叶红外光谱(FT-IR)进行质量评价,紫外分光光度计进行体外释放研究。3.设立空白对照、单纯HMS组、游离姜黄素组(浓度:5μM)、HMS-CUR组(实际姜黄素含量:3.5μM),分别处理肝癌细胞,采用CCK-8和平板克隆形成试验检测药物对肝癌细胞的增殖抑制作用,并且用Western Blot半定量检测药物处理后细胞增殖相关蛋白的表达。4.Hoechst 33342染色定性观察药物处理后细胞凋亡情况,Annexin V/PI检测细胞凋亡率,药物处理后细胞凋亡相关蛋白的表达用Western Blot半定量检测。5.Western Blot半定量检测自噬相关蛋白表达。结果:1.SEM结果显示CPS和HMS大小均一,粒径在50-100nm之间。2.TEM和FT-IR证实,姜黄素负载在HMS上。UV-Vis分析HMS-CUR在乙醇中迅速释放,在DMEM、10%FBS-DMEM中缓慢释放。3.CCK-8和平板克隆形成试验结果表明:HMS-CUR对肝癌细胞有增殖抑制作用(p0.05),且具缓释效应。HMS-CUR能下调增殖细胞核抗原(PCNA)的表达(p0.05)。4.Hoechst 33342染色和Annexin V/PI结果显示,HMS-CUR能诱导肝癌细胞凋亡(p0.05),且可上调凋亡相关蛋白(Cleaved-PARP,Cleaved-Caspase3)的表达(p0.05)。5.HMS-CUR激活了肝癌细胞的自噬,Western Blot结果显示HMS-CUR处理肝癌细胞后,LC3-I向LC3-II的转化率增加,Beclin1表达上调(p0.05)。此外,在SMMC-7721细胞中,P62/SQSTM1表达下调(p0.05),但在Hep G2细胞中却呈相反的上升趋势(p0.05)。结论:HMS作为一种无毒,生物相容性良好的新型载药体系,可有效解决姜黄素水溶性差的难题。与游离姜黄素相比,HMS-CUR能显著抑制肝癌细胞增殖,诱导凋亡,激活自噬。且HMS-CUR对肝癌细胞的作用具有缓释效应。
[Abstract]:Objective: Curcumin is an effective active component extracted from Rhizoma Curcumae, and is a kind of natural anti-tumor drug with wide application prospect. but the water solubility of the curcumin is poor and the bioavailability is low, which greatly limits the clinical popularization of the curcumin. In this study, the hollow mesoporous silica nanoparticles and the loaded curcumin were loaded to obtain the hollow mesoporous silica-curcumin nano-preparation. After the study of quality evaluation and drug release, the SMMC-7721 cells and the Hep G2 cells of the liver cancer were studied. The effect of hollow mesoporous silica-curcumin nano-preparation on the proliferation and apoptosis of hepatocellular carcinoma cells; Whether the hollow mesoporous silica-curcumin nano-preparation activates the autophagy of the liver cancer cells. The method not only can solve the problem of low bioavailability of the curcumin, and can clear the partial mechanism of the hollow mesoporous silica-curcumin nano-preparation to the liver cancer, and provides a theoretical basis for clinical application of the liver cancer. Method: 1. the cationic polystyrene template (CPS) is prepared by the emulsion polymerization method, the CPS is used as a template, and Na2SiO3 路 9H2O is added to prepare the silicon dioxide nano-particles, and the silicon dioxide nano-particles are prepared through high-temperature layering and sintering to obtain the hollow mesoporous silica nano-particles (HMS). Scanning electron microscope (SEM) was used to characterize the CPS and HMS. 2. HMS and curcumin were mixed in a certain proportion, and the hollow mesoporous silica-curcumin nano-particles (HMS-CUR) were obtained by mechanical stirring, and characterized by transmission electron microscopy (TEM), and the Fourier infrared spectrum (FT-IR) was used for quality evaluation. The in vitro release study was carried out by an ultraviolet spectrophotometer. A blank control group, a pure HMS group, a free curcumin group (concentration: 5. mu.M) and an HMS-CUR group (the actual curcumin content: 3.5. mu.M) were set up to respectively treat the liver cancer cells, and the inhibition of the proliferation of the liver cancer cells was detected by using the CCK-8 and the plate clone formation test. and the expression of the cell proliferation-related protein after the drug treatment was detected by Western Blot semi-quantitative method, and the apoptosis rate of the cells after the treatment of the drug after treatment with Hoechst 33342 was qualitatively observed, and the apoptosis rate of the Annexin V/ PI was detected. The expression of apoptosis-related protein in drug-treated cells was detected by Western Blot semi-quantitative method. Results: 1. SEM showed that the size of CPS and HMS was uniform and the particle size was between 50 and 100 nm. The rapid release of HMS-CUR in ethanol was analyzed by UV-Vis. The slow release of HMS-CUR in DMEM and 10% FBS-DMEM. HMS-CUR can downregulate the expression of proliferating cell nuclear antigen (PCNA) (p0.05). 4. Hoechst 33342 staining and Annexin V/ PI result show that HMS-CUR can induce apoptosis of liver cancer cells (p0.05), and the expression of apoptosis-related protein (Cleared-PARP, Cleared-Caspase3) (p0.05). The conversion of LC3-I to LC3-II was increased and the expression of Beclo1 was up-regulated (p0.05). In addition, in the SMMC-7721 cells, the expression of P62/ SQSTM1 was down-regulated (p0.05), but the contrary in the Hep G2 cells (p0.05). Conclusion: HMS, as a new drug-carrier system with no toxicity and good biocompatibility, can effectively solve the problem of water-solubility of curcumin. Compared with the free curcumin, the HMS-CUR can obviously inhibit the proliferation of the liver cancer cells, induce the apoptosis, and activate the autophagy. and the effect of the HMS-CUR on the liver cancer cells has a slow-release effect.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
[Abstract]:Objective: Curcumin is an effective active component extracted from Rhizoma Curcumae, and is a kind of natural anti-tumor drug with wide application prospect. but the water solubility of the curcumin is poor and the bioavailability is low, which greatly limits the clinical popularization of the curcumin. In this study, the hollow mesoporous silica nanoparticles and the loaded curcumin were loaded to obtain the hollow mesoporous silica-curcumin nano-preparation. After the study of quality evaluation and drug release, the SMMC-7721 cells and the Hep G2 cells of the liver cancer were studied. The effect of hollow mesoporous silica-curcumin nano-preparation on the proliferation and apoptosis of hepatocellular carcinoma cells; Whether the hollow mesoporous silica-curcumin nano-preparation activates the autophagy of the liver cancer cells. The method not only can solve the problem of low bioavailability of the curcumin, and can clear the partial mechanism of the hollow mesoporous silica-curcumin nano-preparation to the liver cancer, and provides a theoretical basis for clinical application of the liver cancer. Method: 1. the cationic polystyrene template (CPS) is prepared by the emulsion polymerization method, the CPS is used as a template, and Na2SiO3 路 9H2O is added to prepare the silicon dioxide nano-particles, and the silicon dioxide nano-particles are prepared through high-temperature layering and sintering to obtain the hollow mesoporous silica nano-particles (HMS). Scanning electron microscope (SEM) was used to characterize the CPS and HMS. 2. HMS and curcumin were mixed in a certain proportion, and the hollow mesoporous silica-curcumin nano-particles (HMS-CUR) were obtained by mechanical stirring, and characterized by transmission electron microscopy (TEM), and the Fourier infrared spectrum (FT-IR) was used for quality evaluation. The in vitro release study was carried out by an ultraviolet spectrophotometer. A blank control group, a pure HMS group, a free curcumin group (concentration: 5. mu.M) and an HMS-CUR group (the actual curcumin content: 3.5. mu.M) were set up to respectively treat the liver cancer cells, and the inhibition of the proliferation of the liver cancer cells was detected by using the CCK-8 and the plate clone formation test. and the expression of the cell proliferation-related protein after the drug treatment was detected by Western Blot semi-quantitative method, and the apoptosis rate of the cells after the treatment of the drug after treatment with Hoechst 33342 was qualitatively observed, and the apoptosis rate of the Annexin V/ PI was detected. The expression of apoptosis-related protein in drug-treated cells was detected by Western Blot semi-quantitative method. Results: 1. SEM showed that the size of CPS and HMS was uniform and the particle size was between 50 and 100 nm. The rapid release of HMS-CUR in ethanol was analyzed by UV-Vis. The slow release of HMS-CUR in DMEM and 10% FBS-DMEM. HMS-CUR can downregulate the expression of proliferating cell nuclear antigen (PCNA) (p0.05). 4. Hoechst 33342 staining and Annexin V/ PI result show that HMS-CUR can induce apoptosis of liver cancer cells (p0.05), and the expression of apoptosis-related protein (Cleared-PARP, Cleared-Caspase3) (p0.05). The conversion of LC3-I to LC3-II was increased and the expression of Beclo1 was up-regulated (p0.05). In addition, in the SMMC-7721 cells, the expression of P62/ SQSTM1 was down-regulated (p0.05), but the contrary in the Hep G2 cells (p0.05). Conclusion: HMS, as a new drug-carrier system with no toxicity and good biocompatibility, can effectively solve the problem of water-solubility of curcumin. Compared with the free curcumin, the HMS-CUR can obviously inhibit the proliferation of the liver cancer cells, induce the apoptosis, and activate the autophagy. and the effect of the HMS-CUR on the liver cancer cells has a slow-release effect.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
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