【摘要】：目的:姜黃素是從姜黃根莖中提取的有效活性成分,是一種應(yīng)用前景廣泛的天然抗腫瘤藥物。但姜黃素水溶性差,生物利用度低,這就大大限制了姜黃素的臨床推廣。本研究制備了中空介孔二氧化硅納米粒子,負(fù)載姜黃素,得到中空介孔二氧化硅-姜黃素納米制劑,通過質(zhì)量評價和藥物釋放研究后,以肝癌SMMC-7721細(xì)胞和Hep G2細(xì)胞為研究對象,探索:1.中空介孔二氧化硅-姜黃素納米制劑對肝癌細(xì)胞增殖和凋亡的影響;2.中空介孔二氧化硅-姜黃素納米制劑是否激活肝癌細(xì)胞自噬。這不僅能解決姜黃素生物利用度低的問題,并且可明確中空介孔二氧化硅-姜黃素納米制劑抗肝癌的部分機理,為肝癌的臨床應(yīng)用提供理論基礎(chǔ)。方法:1.乳液聚合法制備陽離子聚苯乙烯模板(CPS),以CPS為模板,加入Na2Si O3·9H2O,制備二氧化硅納米粒子,經(jīng)高溫分層煅燒,得到中空介孔二氧化硅納米粒子(HMS)。掃描電鏡(SEM)對CPS和HMS進(jìn)行表征。2.HMS和姜黃素以一定比例混合,機械攪拌后得到中空介孔二氧化硅-姜黃素納米粒子(HMS-CUR),用透射電鏡(TEM)進(jìn)行表征,傅里葉紅外光譜(FT-IR)進(jìn)行質(zhì)量評價,紫外分光光度計進(jìn)行體外釋放研究。3.設(shè)立空白對照、單純HMS組、游離姜黃素組(濃度:5μM)、HMS-CUR組(實際姜黃素含量:3.5μM),分別處理肝癌細(xì)胞,采用CCK-8和平板克隆形成試驗檢測藥物對肝癌細(xì)胞的增殖抑制作用,并且用Western Blot半定量檢測藥物處理后細(xì)胞增殖相關(guān)蛋白的表達(dá)。4.Hoechst 33342染色定性觀察藥物處理后細(xì)胞凋亡情況,Annexin V/PI檢測細(xì)胞凋亡率,藥物處理后細(xì)胞凋亡相關(guān)蛋白的表達(dá)用Western Blot半定量檢測。5.Western Blot半定量檢測自噬相關(guān)蛋白表達(dá)。結(jié)果:1.SEM結(jié)果顯示CPS和HMS大小均一,粒徑在50-100nm之間。2.TEM和FT-IR證實,姜黃素負(fù)載在HMS上。UV-Vis分析HMS-CUR在乙醇中迅速釋放,在DMEM、10%FBS-DMEM中緩慢釋放。3.CCK-8和平板克隆形成試驗結(jié)果表明:HMS-CUR對肝癌細(xì)胞有增殖抑制作用(p0.05),且具緩釋效應(yīng)。HMS-CUR能下調(diào)增殖細(xì)胞核抗原(PCNA)的表達(dá)(p0.05)。4.Hoechst 33342染色和Annexin V/PI結(jié)果顯示,HMS-CUR能誘導(dǎo)肝癌細(xì)胞凋亡(p0.05),且可上調(diào)凋亡相關(guān)蛋白(Cleaved-PARP,Cleaved-Caspase3)的表達(dá)(p0.05)。5.HMS-CUR激活了肝癌細(xì)胞的自噬,Western Blot結(jié)果顯示HMS-CUR處理肝癌細(xì)胞后,LC3-I向LC3-II的轉(zhuǎn)化率增加,Beclin1表達(dá)上調(diào)(p0.05)。此外,在SMMC-7721細(xì)胞中,P62/SQSTM1表達(dá)下調(diào)(p0.05),但在Hep G2細(xì)胞中卻呈相反的上升趨勢(p0.05)。結(jié)論:HMS作為一種無毒,生物相容性良好的新型載藥體系,可有效解決姜黃素水溶性差的難題。與游離姜黃素相比,HMS-CUR能顯著抑制肝癌細(xì)胞增殖,誘導(dǎo)凋亡,激活自噬。且HMS-CUR對肝癌細(xì)胞的作用具有緩釋效應(yīng)。
[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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.7

【參考文獻(xiàn)】

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