【摘要】：外源性化合物是人類日常生活中接觸外界環(huán)境不可避免的一類化合物,這些化合物可能與機體接觸并進入機體,并且在機體內(nèi)產(chǎn)生一定的生物活性。有些化學物質(zhì)可能與機體組織發(fā)生生物化學作用,破壞正常生理功能,對人類健康產(chǎn)生負面影響,有些化學物質(zhì)也可能有益于人體健康。植物甾醇氧化物和膽固醇氧化物是存在人類日常飲食中一類化合物,大量的研究證明膽固醇氧化物對人體具有不良的生物效應(yīng),植物甾醇氧化物結(jié)構(gòu)與膽固醇相似,而關(guān)于植物甾醇氧化物的生物安全性尚未定論。隨著納米技術(shù)在生物醫(yī)學領(lǐng)域中的應(yīng)用,具有多功能的納米造影劑不斷問世,造影劑作為典型的臨床輔助診斷的外源性化合物,納米造影劑的產(chǎn)生為造影劑的發(fā)展和應(yīng)用帶來便利的同時,其生物安全性研究引起了國內(nèi)外許多學者的關(guān)注。目前納米造影劑的生物安全性研究尚處于起步階段,而且由于納米材料種類繁多,理化性質(zhì)各異,人們對于納米造影劑毒性效應(yīng)及安全評價的認識還相當局限。因此,探究這些外源性化合物對機體的生物學效應(yīng),明確其在機體內(nèi)的生物安全性具有重要意義。本研究主要以兩類(7-酮基和7β-羥基)植物甾醇氧化物和兩種不同結(jié)構(gòu)的納米核磁造影劑為研究對象,主要研究了這些外源性化合物對不同細胞的細胞毒性效應(yīng),獲得了如下主要結(jié)果:1.以膽固醇氧化物(7K-CH和7β-OH-CH)為陽性對照組,將兩類植物甾醇氧化物分別作用于HIC細胞后,在120μM作用濃度下,7-酮基植物甾醇氧化物對HIC細胞的毒性強度依次為7K-SI7K-CA7K-BR7K-ST7K-MIX,7β-羥基植物甾醇氧化物對HIC細胞的毒性強度依次為7β-OH-SI7β-OH-CA7β-OH-MIX7β-OH-ST≈7β-OH-BR。HIC細胞系可能對7-酮基系列氧化物要比對7β-羥基植物甾醇氧化物更敏感。所有被測氧化物都能明顯降低細胞活力,并具有濃度和時間依賴性,尤其是7K-SI和7K-CA活性最高,降低細胞活力最為明顯。此外,7K-SI和7K-CA不僅誘導早期凋亡細胞比例上升,而且還導致細胞周期S期含量上升,G1期含量下降,而7K-BR、7K-ST和7K-MIX沒有顯示出明顯的細胞毒性,與此同時,7K-SI、7K-CA和7K-BR通過激活caspase-3活性,調(diào)節(jié)Bcl-2蛋白誘導細胞凋亡,而7K-ST和7K-MIX不依賴于caspase-3和Bcl-2。2.兩類甾醇氧化物作用A549細胞后,被測氧化物均降低了A549的細胞活力且具有濃度和時間依賴關(guān)系。在120μM作用濃度下,7-酮基植物甾醇氧化物對A549細胞毒性強度依次為7K-SI7K-CA7K-BR≈7K-ST≈7K-MIX,7β-羥基-植物甾醇氧化物對A549細胞的毒性強度依次為7β-OH-SI≈7β-OH-CA7β-OH-MIX7β-OH-ST≈7β-OH-BR,這一結(jié)果表明A549細胞系可能對7β-羥基系列氧化物要比對7-酮基植物甾醇氧化物更敏感,這一結(jié)果正好與HIC細胞系結(jié)果相反。另外,7β-羥基系列氧化物不僅降低了細胞活力,還導致A549凋亡后期細胞比例上升,同時細胞周期實驗表明7β-羥基-CH、7β-羥基-SI、7β-羥基-CA和7K-CH導致sub-G1期的含量有所增加,但是被測氧化物對A549細胞周期的影響因化合物不同而效果不同,這也表明了甾醇氧化物對A549細胞周期與這些氧化物的化學結(jié)構(gòu)沒有直接的關(guān)聯(lián)性。此外,這些氧化物會導致A549細胞內(nèi)活性氧含量隨著時間延長而上升,同時通過激活caspase-3活性,調(diào)節(jié)Bcl-2蛋白誘導細胞凋亡。3.兩類甾醇氧化物作用于HepG2細胞后,被測的氧化物均降低了HepG2細胞活力并且具有濃度時間依賴關(guān)系。在120μM作用濃度下,7酮基-ST和7-酮基-MIX表現(xiàn)出強抑制細胞活性的特點,這一結(jié)果與HIC細胞和A549細胞實驗結(jié)果有很大差異。7β-羥基-CA和7酮基-CA不僅降低細胞活性,激發(fā)超氧化物歧化酶SOD和過氧化氫酶進而導致細胞凋亡,但是這兩種單體沒有導致脂質(zhì)過氧化物含量上升,而混合甾醇氧化物導致MDA含量略微上升,但是沒有影響細胞活力。實驗表明氧化應(yīng)激反應(yīng)與細胞死亡之間沒有必然的聯(lián)系,植物甾醇氧化物對HepG2細胞毒性與細胞對這些氧化物的攝取量有關(guān)系,因化合物不同而效果不同。4.本實驗中采用的納米材料Fe_3O_4@HSiO_2和負載了秋水仙堿的Fe_3O_4@HSiO_2-COLC是具抗腫瘤藥物載體和核磁造影劑功能的磁性納米材料體系。核磁共振成像實驗表明,納米材料Fe_3O_4@HSiO_2作為核磁造影劑沒有顯示出細胞毒性,同時具有很好的生物相容性和較高的核磁對比度,作為抗癌藥物載體(Fe_3O_4@HSiO_2 COLC)能在酸性環(huán)境中(pH3.0)有效釋放所負載的秋水仙堿(COLC),在磁場作用下能有效地靶向腫瘤細胞HepG2而不損傷正常細胞,且藥效要高于單純的COLC。5.由聚乙烯亞胺包裹葉酸和釓介孔2-50 nm的納米材料體系Gd-FA-Si,具有較好的生物相容性,兼具抗腫瘤藥物載體和核磁造影劑功能,同時受pH調(diào)控而釋放藥物。納米材料表面負載的葉酸能高效的靶向Hela和MDA-MB-231細胞上的葉酸受體,材料負載的阿霉素高效抑制細胞活性,比單純的阿霉素藥物給藥具有更高的藥效。
[Abstract]:Exogeneous compound is a kind of compound that comes into contact with the outside world in daily life. These compounds may come into contact with the organism and enter the organism, and produce certain biological activity in the organism. Some chemicals may have biochemical effects with body tissues, disrupt normal physiological functions, have negative effects on human health, and some chemicals may also be beneficial to human health. Phytochrome oxide and cholesterol oxide are a type of compound in the daily diet of humans, and a large number of studies have shown that cholesterol oxides have poor biological effects on the human body, and that the plant's oxide structure is similar to that of cholesterol. However, the biological safety of phytochrome oxide has not been decided yet. With the application of nanotechnology in the field of biomedicine, the multifunctional nano contrast agent is constantly invented, the contrast agent is taken as an exogenous compound for the typical clinical assistant diagnosis, and the production of the nano contrast agent brings convenience to the development and application of the contrast agent, Its biological safety research has attracted the attention of many scholars both at home and abroad. At present, the biological safety research of nano-contrast agent is still in the initial stage, and because of the wide variety of nano-materials and different physical and chemical properties, people's understanding of the toxicity effect and safety evaluation of the nano-contrast agent is quite limited. Therefore, it is important to explore the biological effect of these exogenous compounds on organism and to clarify its biological safety in organism. In this study, two kinds of nano-nuclear magnetic contrast agents (7-keto-and 7-hydroxy group) and two kinds of nano-nuclear magnetic contrast agents were studied. The cytotoxicity effect of these exogenous compounds on different cells was studied, and the following main results were obtained: 1. Using cholesterol oxide (7K-CH and 7H2O-OH-CH) as the positive control group, the toxicity intensity of the 7-keto-base plant oxide was 7K-SI7K-CA7K-BR7K-ST7K-MIX, respectively. The toxicity intensity of 7-keto-OH-SI7 CHA-OH-CA7 CHA-OH-MICACHE-OH-ST-7OH-OH-BR. 7721 cell line may be more sensitive to 7-keto-series oxides than for 7-keto-hydroxyphytochrome oxide. All the tested oxides can obviously decrease the activity of cells, and have the highest concentration and time dependence, especially the highest activity of 7K-SI and 7K-CA and the most obvious decrease of cell viability. In addition, 7K-SI and 7K-CA not only induce an increase in the proportion of early apoptotic cells but also lead to an increase in the S phase of the cell cycle and a decrease in G1 phase, while 7K-BR, 7K-ST and 7K-MIX show no significant cytotoxicity, while 7K-SI, 7K-CA and 7K-BR activate caspase-3 activity, Bcl-2 protein was regulated to induce apoptosis, while 7K-ST and 7K-MIX were independent of caspase-3 and Bcl-2. After A549 cells were treated with two kinds of exogenous oxides, the measured oxides reduced the viability of A549 cells and had a concentration and time-dependent relationship. The toxic strength of the 7-keto-phytochrome oxide on A549 cells was 7K-SI7K-CA7K-BR-7K-ST-7K-MIX in order of 120. m This result shows that the A549 cell line may be more sensitive to the 7-keto-group oxide than to the 7-keto-based plant, and this result is just contrary to the results of the cell line. in addition, that 7-hydroxy-hydroxy-series oxide not only reduce the activity of the cell, but also lead to the increase of the cell proportion in the late stage of apoptosis of the A549 cell, and meanwhile, the cell cycle experiment shows that the content of the sub-G1 phase is increased due to the 7-hydroxy-CH, 7OH-hydroxy-SI, 7OH-hydroxy-CA and 7K-CH, However, the effect of oxide on the cell cycle of A549 is different because of the different compounds, which also indicates that the cell cycle of the A549 cells is not directly related to the chemical structure of these oxides. In addition, these oxides lead to increased active oxygen content in A549 cells over time, while modulating Bcl-2 protein-induced apoptosis by activating caspase-3 activity. Both of the two types of oxygenic oxide have reduced the activity of HepG2 cells and have a concentration-time dependent relationship after HepG2 cells. At the concentration of 120. m u.M, the 7-keto-ST and 7-keto-MIX exhibit strong inhibitory cellular activity. The result is very different from the experimental results of Jurkat cells and A549 cells. The 7-keto-hydroxy-CA and 7-keto-CA not only decrease cell activity, Activation of superoxide dismutase (SOD) and catalase in turn led to apoptosis, but the two monomers did not cause the lipid peroxide content to rise, but the mixed oxide resulted in a slight increase in MDA content, but did not affect the viability of the cells. The results showed that there was no definite relationship between oxidative stress response and cell death. The toxicity of phytochrome oxide to HepG2 cells was related to the uptake of these oxides. Fe _ 3O _ 4 @ HSiO _ 2 and Fe _ 3O _ 4 @ HSiO _ 2-COLC, which are used in this experiment, are magnetic nano-materials with anti-tumor drug carrier and nuclear magnetic contrast agent. The nuclear magnetic resonance imaging experiments show that the nano-material Fe _ 3O _ 4 @ HSiO _ 2 has not shown cytotoxicity as a nuclear magnetic contrast agent, but also has good biocompatibility and high nuclear magnetic contrast. 浣滀負鎶楃檶鑽墿杞戒綋(Fe_3O_4@HSiO_2 COLC)鑳藉湪閰告，

本文編號：2270977