脂质体和PEI介导的pEGFP-N1-BmK CT对神经胶质瘤细胞迁移的影响

发布时间：2018-06-03 19:50

本文选题：神经胶质瘤细胞 + 东亚钳蝎氯离子通道毒素　；参考：《山西大学》2014年硕士论文

【摘要】：东亚钳蝎氯离子通道毒素(BmK CT)是一类短链蝎毒素,其蛋白质序列含有36个氨基酸、4对二硫键,与以色列蝎氯离子通道毒素有68%的同源性,推测它们有类似的生物学功能,可通过两条途径作用于神经胶质瘤细胞：其一是作用于神经胶质瘤细胞膜表面特异高水平表达的氯离子通道,抑制神经胶质瘤细胞的迁移和扩散。其二是结合并抑制神经胶质瘤细胞膜表面的基质金属蛋白酶-2的分泌和活性,从而抑制神经胶质瘤细胞的浸润。基质金属蛋白酶(MMPs)是一类能够降解细胞外基质的蛋白酶。肿瘤细胞侵袭到周围组织主要通过受体粘附到细胞外基质并穿过细胞外基质屏障,所以肿瘤的侵袭与细胞外基质的降解密切相关。本实验课题主要研究脂质体和PEI介导重组质粒pEGFP-N1-BmK CT的表达对大鼠神经胶质瘤C6细胞迁移的影响及作用机制。本实验将构建的重组质粒pEGFP-N1-BmK CT转染C6细胞后,激光共聚焦显微镜观察转染24 h后外源蛋白表达量达到最高。明胶酶谱实验表明转染pEGFP-N1-BmK CT后抑制了C6细胞pro-MMP-2, MMP-2的分泌水平,从而抑制肿瘤细胞的迁移能力。Western blot检测进一步证实转染pEGFP-N1-BmK CT后C6细胞降低了MMP-2的分泌水平。体外划痕擦伤实验结果表明,与pEGFP-N1转染组相比,pEGFP-N1-BmK CT转染组划痕两侧细胞向划痕中央生长的速度较慢,说明pEGFP-N1-BmK CT的表达抑制了MMP-2的分泌水平从而抑制了C6细胞的迁移。免疫荧光定位实验表明转染pEGFP-N1-BmKCT后,BmK CT与MMP-2在C6细胞的核膜周围有共定位。氯化锂是治疗躁狂症或抑郁症的常规药物。近年来研究发现,氯化锂可以有效的抑制神经胶质瘤细胞的迁移。本文尝试采用氯化锂和转染BmK CT联合作用C6细胞,以期辅助氯化锂在较低浓度下达到抑制C6细胞的增殖和迁移的作用。本实验研究发现转染pEGFP-N1-BmK CT后经氯化锂处理对C6细胞的增殖能力抑制效率高于仅转染pEGFP-N1-BmK CT处理。此外,pEGFP-N1-BmK CT转染组经氯化锂处理后C6细胞内pro-MMP-2分泌水平也更低,加强了对C6细胞的迁移能力的抑制作用。Western Blot实验结果表明,在pEGFP-N1和pEGFP-N1-BmK CT转染组中,经氯化锂作用后β-catenin在细胞核内的积累量均有所提高。开发肿瘤特异性复合纳米颗粒是当前研究的热点,已有研究发现部分纳米材料可以作为药物载体和造影剂。为了提高纳米材料的生物相容性、溶解度和特异性结合,需要对纳米颗粒表面改性,比如利用PEI进行修饰。PEI(聚乙烯亚胺)是一种常用的合成聚合物,能够作为基因载体且有较高的转染效率。本课题研究了PEI介导pEGFP-N1-BmK CT的表达对C6细胞迁移的影响。激光共聚焦显微镜观察到PEI介导重组质粒转染24 h后外源蛋白表达量达到60%以上。明胶酶谱实验结果表明pEGFP-N1-BmK CT的表达抑制了Pro-MMP-2的分泌水平。体外划痕擦伤实验表明与空对照和pEGFP-N1转染组比较,pEGFP-N1-BmK CT转染组抑制了神经胶质瘤细胞的迁移。随后本实验合成了四种纳米材料：ND-PEI、SiO2-PEI、MCM-41和SBA-15,并通过动态光散射检测了四种纳米材料在蒸馏水中的粒径分散。通过化学反应获得ND-PEI-pEGFP-N1-BmK CT,通过琼脂糖凝胶电泳对ND-PEI与DNA结合率分析,发现随着其比例的增加,结合DNA效率逐渐递增。这为后期开展纳米颗粒介导pEGFP-N1-BmK CT对C6细胞迁移抑制作用的效果研究奠定了基础。本实验旨在揭示脂质体和PEI及纳米颗粒介导的东亚钳蝎氯离子通道神经毒素基因BmK CT的抗肿瘤效应,为神经胶质瘤的治疗提供新的途径和方法。
[Abstract]:BmK CT is a class of short chain scorpion toxin. The protein sequence contains 36 amino acids, 4 pairs of two sulfur bonds and 68% homology of the Israeli scorpion chloride channel toxin. It is speculated that they have similar biological functions and can act on glioma cells through two pathways: one is the action of glial glia. The specific high level of chlorine ion channel expressed on the surface of the tumor cell membrane inhibits the migration and diffusion of glioma cells. The second is to inhibit the secretion and activity of matrix metalloproteinase -2, which combines and inhibits the surface of the glioma cell membrane, thus inhibiting the infiltration of glioma cells. The basic metalloproteinase (MMPs) is a class of degradation fine. The tumor cells invade the surrounding tissue mainly by adhesion to the extracellular matrix and through the extracellular matrix, so the invasion of the tumor is closely related to the degradation of the extracellular matrix. This experimental subject mainly studies the expression of the recombinant plasmid pEGFP-N1-BmK CT mediated by liposomes and PEI to the rat Neuroglia The effect and mechanism of tumor C6 cell migration. After transfection of recombinant plasmid pEGFP-N1-BmK CT to C6 cells, the expression of exogenous protein was highest after transfection of 24 h by laser confocal microscopy. The gelatinase assay showed that the transfection of pEGFP-N1-BmK CT inhibited the pro-MMP-2 of C6 cells and the secretion level of MMP-2, thus inhibiting swelling. .Western blot detection of tumor cell migration further confirmed that C6 cells reduced the secretion level of MMP-2 after transfection of pEGFP-N1-BmK CT. In vitro scratch scratch test results showed that compared with pEGFP-N1 transfected group, the growth rate of the scratched two sides cells in the pEGFP-N1-BmK CT transfected group was slower than that of the pEGFP-N1 transfected group, indicating that the expression of pEGFP-N1-BmK CT was inhibited. The secretory level of MMP-2 inhibits the migration of C6 cells. Immunofluorescence localization experiments show that BmK CT and MMP-2 are Co located around the nuclear membrane of C6 cells. Lithium chloride is a conventional drug for the treatment of mania or depression. In recent years, the study found that lithium chloride can effectively inhibit the migration of glioma cells. The effect of lithium chloride and transfection of BmK CT on C6 cells to inhibit the proliferation and migration of C6 cells under the lower concentration of lithium chloride was tried in this paper. The inhibitory effect of lithium chloride on the proliferation of C6 cells after pEGFP-N1-BmK CT transfection was higher than that of pEGFP-N1-BmK CT only after transfection of pEGFP-N1-BmK CT. The level of pro-MMP-2 secretion in C6 cells after lithium chloride treatment in pEGFP-N1-BmK CT transfected group was also lower, and the inhibitory effect of C6 cells on the migration ability of C6 cells was enhanced. The results of.Western Blot experiment showed that the accumulation of beta -catenin in the nucleus of the cell nucleus in the transfected group of pEGFP-N1 and pEGFP-N1-BmK CT increased. Specific composite nanoparticles are the hot spots of current research. Some research has been found that some nanomaterials can be used as drug carriers and contrast agents. In order to improve the biocompatibility of nanomaterials, the combination of solubility and specificity, the surface modification of nanoparticles, such as the use of PEI to modify.PEI (polyethylenimide), is a common combination. The effect of PEI mediated expression of pEGFP-N1-BmK CT on the migration of C6 cells was studied in this study. The expression of exogenous protein was more than 60% after PEI mediated recombinant plasmid transfection of 24 h. The results of gelatin zymogram showed the table of pEGFP-N1-BmK CT. The secretion level of Pro-MMP-2 was inhibited. In vitro scratch scratch test showed that the pEGFP-N1-BmK CT transfected group inhibited the migration of glioma cells compared with the empty control and pEGFP-N1 transfected groups. Then four nano materials were synthesized: ND-PEI, SiO2-PEI, MCM-41 and SBA-15, and four nanomaterials were detected by dynamic light scattering. The particle size of the distilled water was dispersed. ND-PEI-pEGFP-N1-BmK CT was obtained by chemical reaction. The binding rate of ND-PEI and DNA was analyzed by agarose gel electrophoresis. It was found that with the increase of its proportion, the efficiency of DNA combined with DNA gradually increased. This was the result of the study on the effect of nanoparticle mediated pEGFP-N1-BmK CT on the migration inhibition of C6 cells in the later period. This experiment aims to reveal the antitumor effect of liposomes and PEI and nanoparticles on the antitumor effect of the chlorion channel neurotoxin gene BmK CT of East Asia pincer scorpion, and to provide new ways and methods for the treatment of glioma.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.4

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