| BackgroundGastric cancer is very common in tumor, ranks first in incidence and mortality rates of digestive cancer. Early diagnosis of gastric cancer is very important in reducing the morbidity of gastric cancer. As endoscopic technology is very important in diagnosing early stage gastric cancer, we developed advanced electronic staining endoscopic techniques. Current clinical application of a fluorescent probe material is Proflavine(a cell nucleus dye with absorption wavelength of 445 nm, emission wavelength of 515nm), with the defect of causing injury, limited penetration depth, limited accuracy and sensitivity. Upconversion nanoparticles(UCNPs) can make up for these shortcomings, demonstrat good prospects of bioinformatics, especially of the fluorescence probe. In 1999, the Tanke team carried out the research using UCNPs as biomolecular probes which reported the UCNPs as a marker applied to DNA chip fluorescence detection, the detection sensitivity is 3 times higher than the fluorescent dye Cy5. Subsequently, the researchers conducted researches about UCNPs in biological fluorescence detection. Compared with the traditional fluorescent labeling of biological materials such as organic dyes, quantum dots, UCNPs have advantages in light stability, large Stokes shift, low toxicity and chemical stability, narrow absorption and emission bands. Traditional UV excitation sources have the shortcomings such as easily impacted by nonspecific fluorescence, little penetration depth, prolonged exposure damage. Near-infrared light has the advantages of little damage to biological tissue, enough tissue penetration depth. While avoids biological samples autologous scattered light and fluorescence interference phenomena, reduces the detection backgroundand and improves the signal to noise ratio. In addition, UCNPs require only a low power density of near-infrared continuous laser(980nm),which is more universal, compared to the high power density generating a pulsed laser excitation of bis convert photon luminescence. All these advantages have confirmed that UNCPs have a bright prospect for biological applications. UCNPs become research focus, mainly concentrated in a few areas such as biomarker imaging, immunoassay analysis, and photodynamic therapy(PDT). But the traditional UCNPs have shortcomings such as poor water-soluble property, large diameter, little cell aggregation, weak fluorescence. This study aims to synthesis UCNPs to solve the problems and explore clinical application.Na YF4:20%Yb3+,3%Er3+ UCNPs was prepared using PVP as the surfactant by hydrothermal Synthesis, and identified its physical properties, optical properties. UCNPs imaging capability was identified by optical microscope imaging techniques, UCNPs cytotoxicity was identified through MTT assay, LDH escape assay.High concentration of UNCPs in cells showed significant cytotoxicity, the cytotoxicity of normal cells and the corresponding tumor cells indicated obvious differences, SGC-7901 cells were more sensitive to nanoparticles. ROS plays an important role in mediating cell death and sensitivity differences of tumor cells. Therefore nanoparticles trigger cell death was due to increaseing levels of ROS in the cytoplasm, and ROS induced apoptosis. The main ways of cell death include apoptosis and necrosis, apoptosis is primarily divided into exogenous apoptosis and endogenous apoptosis. Mitochondrial plays a key role in endogenous pathway. Decreasing ΔΨm represents early apoptosis signal, decreasing ΔΨm indicates that apoptosis entere irreversible stage. ΔΨm decreased with increasing cytoplasmic Ca2+, and Cyt C released as ΔΨm collapsed. Bax has a role in promoting Cyt C release, Bcl-2 and vice versa. Cyt C releasing and binding Apaf-1 activate the initial Caspase-9, activates Caspase-3, Caspase-3 causes DNA fragmentation, which ends in apoptosis. The path is closely related to the concentration of ROS. Tumor cells with high levels of ROS significantly apoptosis. High levels of ROS as a second messenger can induce Mitochondria apoptotic pathways. PI3K-Akt signaling pathway is important in regulating proliferation and apoptosis in cells, and this pathway is very important in malignant proliferation, angiogenesis, tumor metastasis and chemotherapy antagonism. Methods 1. Na YF4:20%Yb3+,3%Er3+ UCNPs was prepared using PVP as the surfactant. The nanometer particle diameter, size was identified by SEM. 2. The structure of the nanoparticle was indentified by XRD. 3. Optical properties of the UCNPs were identified by spectral characterization under 980 nm excitation. 4. The study of fluorescence imaging of SGC-7901 in vitro was completed by laser confocal imaging technology. 5. The cytotoxicity of nanoparticles was evaluated by MTT assay, LDH escape assay. 6. Annexin V-FITC apoptosis detection and cell cycle flow cytometry showed that 400μg/m L nanoparticles inhibited cell proliferation. 7. DCFH-DA was used to detect the ROS level in SGC-7901 coexisted with nanoparticles. 8. Aimed to ascertain the mechanism and influence factors that ROS triggered apoptosis, examined the ΔΨm, cytoplasmic Ca2+, Caspase-9, Caspase-3, Cyt C, Bax, Bcl-2, Akt, p Akt(Ser 473) expression. Results 1. The XRD pattern of the UNCPs indicated that diffraction peaks match the data of cubic-phase Na YF4 nanocrystals, and there no other peaks of impurities were detected. 2. It indicated that the UNCPs was single-phase pure cubic Na YF4 crystals and demonstrated the highly crystalline nature of the nanocrystals. 3. In the SEM image, the UCNPs had small size. Spectrum of UCNPs indicated that the strongest peak was centered in 650 nm and 540 nm, and therefore the UCNPs dispersed aqueous solution exhibited bright yellow luminescence under the excitation of 980 nm infrared light. 4. Imaging effect of nanoparticles could be seen in the bright field detecting image, the cell morphology and number showed no obvious changes, instructed a range of concentration of nanoparticle(200μg/m L) and cells can coexist. 5. MTT indicated that the proliferation rate of the cells decreased while the concentrations of nanoparticles increased. When the concentration of nanoparticles increased to 400μg/m L, after 24 h, cell activity was to a minimum value, Hela cells fell 22.13%, GES-1 declined 30.08%, SGC-7901 declined 36.13%. SGC-7901 was the most sensitive to nanoparticles, when the concentration up to 400μg/m L, reaction time up to 72 h, cell proliferation rate fell 65.72%. LDH release assay showed SGC-7901 cells was correlated with nanoparticle concentration and reaction time.When the concentration of 400μg/m L, reaction time 24 h, SGC-7901 LDH release rate increased 36.21%. 6. Main ways that inhibit cell proliferation were apoptosis and cell cycle arrest by Annexin V-FITC apoptosis detection(decline 17.4% in normal cells, necrotic cells increased by 10.1%, an increase of early apoptotic cells was 6%) and cell cycle detection(arrested in G1). 7. ROS levels were significantly increased by DCFH-DA assay(71.43%). 8. ROS triggered elevating cytosolic Ca2+(91.59%) and decreasing ΔΨm(152.38%), which in turn accelerated Cyt C releasing(40.35%). Bax was up-regulated(130.21%), while Bcl-2 was down-regulated(48.75%), Caspase-9(170.35%), Caspase-3(50.15%) releasing triggered apoptosis. The process simultaneously triggered p Akt(Ser473) reducing(46.17%). Conclusions 1. UCNPs are nanopaticles with 30 nm measurerment, stable property, easily uptaked by SGC-7901 cells. 2. Infrared laser confocal fluorescence microscopy showed strong fluorescence in SGC-7901 cells, indicated the nanoparticle(200μg/m L) have good penetrability of the cell membrane and stability of fluorescence imaging capacity. 3. A preliminary evaluation of cytotoxicity showed a certain concentration range nanomaterials have biological safety. 4. 400μg/m L UCNPs inhibited SGC-7901 cells proliferation rate by cell cycle arrest and apoptosis, the main incentive was that UCNPs triggered ROS increasing and mitochondrial apoptotic pathway activating, which was also mediated by PI3K-Akt pathway signaling pathways. |
