發(fā)布時間：2018-06-03 19:50

  本文選題：神經(jīng)膠質(zhì)瘤細胞 + 東亞鉗蝎氯離子通道毒素��； 參考：《山西大學》2014年碩士論文

【摘要】：東亞鉗蝎氯離子通道毒素(BmK CT)是一類短鏈蝎毒素,其蛋白質(zhì)序列含有36個氨基酸、4對二硫鍵,與以色列蝎氯離子通道毒素有68%的同源性,推測它們有類似的生物學功能,可通過兩條途徑作用于神經(jīng)膠質(zhì)瘤細胞：其一是作用于神經(jīng)膠質(zhì)瘤細胞膜表面特異高水平表達的氯離子通道,抑制神經(jīng)膠質(zhì)瘤細胞的遷移和擴散。其二是結(jié)合并抑制神經(jīng)膠質(zhì)瘤細胞膜表面的基質(zhì)金屬蛋白酶-2的分泌和活性,從而抑制神經(jīng)膠質(zhì)瘤細胞的浸潤。基質(zhì)金屬蛋白酶(MMPs)是一類能夠降解細胞外基質(zhì)的蛋白酶。腫瘤細胞侵襲到周圍組織主要通過受體粘附到細胞外基質(zhì)并穿過細胞外基質(zhì)屏障,所以腫瘤的侵襲與細胞外基質(zhì)的降解密切相關(guān)。本實驗課題主要研究脂質(zhì)體和PEI介導(dǎo)重組質(zhì)粒pEGFP-N1-BmK CT的表達對大鼠神經(jīng)膠質(zhì)瘤C6細胞遷移的影響及作用機制。本實驗將構(gòu)建的重組質(zhì)粒pEGFP-N1-BmK CT轉(zhuǎn)染C6細胞后,激光共聚焦顯微鏡觀察轉(zhuǎn)染24 h后外源蛋白表達量達到最高。明膠酶譜實驗表明轉(zhuǎn)染pEGFP-N1-BmK CT后抑制了C6細胞pro-MMP-2, MMP-2的分泌水平,從而抑制腫瘤細胞的遷移能力。Western blot檢測進一步證實轉(zhuǎn)染pEGFP-N1-BmK CT后C6細胞降低了MMP-2的分泌水平。體外劃痕擦傷實驗結(jié)果表明,與pEGFP-N1轉(zhuǎn)染組相比,pEGFP-N1-BmK CT轉(zhuǎn)染組劃痕兩側(cè)細胞向劃痕中央生長的速度較慢,說明pEGFP-N1-BmK CT的表達抑制了MMP-2的分泌水平從而抑制了C6細胞的遷移。免疫熒光定位實驗表明轉(zhuǎn)染pEGFP-N1-BmKCT后,BmK CT與MMP-2在C6細胞的核膜周圍有共定位。氯化鋰是治療躁狂癥或抑郁癥的常規(guī)藥物。近年來研究發(fā)現(xiàn),氯化鋰可以有效的抑制神經(jīng)膠質(zhì)瘤細胞的遷移。本文嘗試采用氯化鋰和轉(zhuǎn)染BmK CT聯(lián)合作用C6細胞,以期輔助氯化鋰在較低濃度下達到抑制C6細胞的增殖和遷移的作用。本實驗研究發(fā)現(xiàn)轉(zhuǎn)染pEGFP-N1-BmK CT后經(jīng)氯化鋰處理對C6細胞的增殖能力抑制效率高于僅轉(zhuǎn)染pEGFP-N1-BmK CT處理。此外,pEGFP-N1-BmK CT轉(zhuǎn)染組經(jīng)氯化鋰處理后C6細胞內(nèi)pro-MMP-2分泌水平也更低,加強了對C6細胞的遷移能力的抑制作用。Western Blot實驗結(jié)果表明,在pEGFP-N1和pEGFP-N1-BmK CT轉(zhuǎn)染組中,經(jīng)氯化鋰作用后β-catenin在細胞核內(nèi)的積累量均有所提高。開發(fā)腫瘤特異性復(fù)合納米顆粒是當前研究的熱點,已有研究發(fā)現(xiàn)部分納米材料可以作為藥物載體和造影劑。為了提高納米材料的生物相容性、溶解度和特異性結(jié)合,需要對納米顆粒表面改性,比如利用PEI進行修飾。PEI(聚乙烯亞胺)是一種常用的合成聚合物,能夠作為基因載體且有較高的轉(zhuǎn)染效率。本課題研究了PEI介導(dǎo)pEGFP-N1-BmK CT的表達對C6細胞遷移的影響。激光共聚焦顯微鏡觀察到PEI介導(dǎo)重組質(zhì)粒轉(zhuǎn)染24 h后外源蛋白表達量達到60%以上。明膠酶譜實驗結(jié)果表明pEGFP-N1-BmK CT的表達抑制了Pro-MMP-2的分泌水平。體外劃痕擦傷實驗表明與空對照和pEGFP-N1轉(zhuǎn)染組比較,pEGFP-N1-BmK CT轉(zhuǎn)染組抑制了神經(jīng)膠質(zhì)瘤細胞的遷移。隨后本實驗合成了四種納米材料：ND-PEI、SiO2-PEI、MCM-41和SBA-15,并通過動態(tài)光散射檢測了四種納米材料在蒸餾水中的粒徑分散。通過化學反應(yīng)獲得ND-PEI-pEGFP-N1-BmK CT,通過瓊脂糖凝膠電泳對ND-PEI與DNA結(jié)合率分析,發(fā)現(xiàn)隨著其比例的增加,結(jié)合DNA效率逐漸遞增。這為后期開展納米顆粒介導(dǎo)pEGFP-N1-BmK CT對C6細胞遷移抑制作用的效果研究奠定了基礎(chǔ)。本實驗旨在揭示脂質(zhì)體和PEI及納米顆粒介導(dǎo)的東亞鉗蝎氯離子通道神經(jīng)毒素基因BmK CT的抗腫瘤效應(yīng),為神經(jīng)膠質(zhì)瘤的治療提供新的途徑和方法。
[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.
【學位授予單位】：山西大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R739.4

【參考文獻】

相關(guān)碩士學位論文 前1條

1 安娜;BmK CT活性位點分析及其抑制神經(jīng)膠質(zhì)瘤遷移的研究[D];山西大學;2012年

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本文編號：1973972