發(fā)布時(shí)間：2018-06-04 19:11

  本文選題：絲氨酸蛋白酶抑制劑 + SPINK6��； 參考：《復(fù)旦大學(xué)》2012年碩士論文

【摘要】：目的：本實(shí)驗(yàn)室通過(guò)基因芯片分析技術(shù),分析了肝癌細(xì)胞及其細(xì)小病毒抗性株基因表達(dá)譜的差異,發(fā)現(xiàn)了在細(xì)小病毒抗性細(xì)胞株中Kazal型絲氨酸蛋白酶抑制劑6(SPINK6)基因表達(dá)明顯上調(diào)。但不清楚spink6基因在其他細(xì)胞系中的表達(dá)水平。本論文主要研究了spink6基因在不同腫瘤細(xì)胞和正常細(xì)胞系中的表達(dá)水平,并研究了SPINK6在細(xì)胞中的亞定位,對(duì)其作用機(jī)制可能有一定的揭示作用。方法：對(duì)多種腫瘤細(xì)胞和正常細(xì)胞抽取RNA,逆轉(zhuǎn)錄成cDNA后,通過(guò)半定量PCR和realtime-PCR的方法研究spink6在不同細(xì)胞系中的基因表達(dá)水平。根據(jù)realtime-PCR結(jié)果挑選了幾株高表達(dá)spink6基因的細(xì)胞系進(jìn)行免疫熒光實(shí)驗(yàn),檢測(cè)其在蛋白水平的表達(dá),以及天然狀態(tài)下SPINK6蛋白的定位。另外進(jìn)一步通過(guò)構(gòu)建pEGFP-N1-SPINK6載體,轉(zhuǎn)染細(xì)胞后,使用DAPI標(biāo)記細(xì)胞核,ER-Tracker Red標(biāo)記內(nèi)質(zhì)網(wǎng),Lyso-Tracker Red標(biāo)記溶酶體,Dil標(biāo)記細(xì)胞膜,共聚焦顯微鏡下觀察SPINK6的細(xì)胞器亞定位。結(jié)果：半定量PCR和realtime-PCR的結(jié)果顯示spink6在正常細(xì)胞中的表達(dá)水平高于腫瘤細(xì)胞中的表達(dá)水平。這一結(jié)果說(shuō)明SPINK6可能是與腫瘤的惡性程度相關(guān)。通過(guò)細(xì)胞器亞定位的研究,發(fā)現(xiàn)SPINK6在內(nèi)質(zhì)網(wǎng)和細(xì)胞膜上均有定位,揭示了SPINK6可能是通過(guò)出膜發(fā)揮作用的。
[Abstract]:Objective: to analyze the difference of gene expression profiles of hepatoma cells and parvovirus resistant strains by gene chip analysis. It was found that the gene expression of Kazal serine protease inhibitor 6 was up-regulated in parvovirus resistant cell lines. However, the expression level of spink6 gene in other cell lines was not clear. In this paper, we studied the expression level of spink6 gene in different tumor cells and normal cell lines, and studied the sublocalization of SPINK6 in the cells, which may reveal the mechanism of its action. Methods: RNAs were extracted from various tumor cells and normal cells and then transformed into cDNA. The gene expression of spink6 in different cell lines was studied by semi-quantitative PCR and realtime-PCR. According to the results of realtime-PCR, several cell lines with high expression of spink6 gene were selected for immunofluorescence assay to detect its expression at protein level and the localization of SPINK6 protein in natural state. In addition, the pEGFP-N1-SPINK6 vector was constructed and transfected into the cells. The cytoplasmic reticulum Lyso-Tracker Red was used to mark the cytoplasmic reticulum Lyso-Tracker Red to mark the cell membrane of lysosomal Dil. The sublocation of the organelle of SPINK6 was observed under confocal microscope. Results: the results of semi-quantitative PCR and realtime-PCR showed that the expression of spink6 in normal cells was higher than that in tumor cells. This result suggests that SPINK6 may be associated with the malignancy of the tumor. It was found that SPINK6 was localized in endoplasmic reticulum (ER) and cell membrane by sublocalization of organelle, which suggested that SPINK6 may play a role through membrane exudation.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R3416;Q78

【共引文獻(xiàn)】

相關(guān)期刊論文 前2條

1 錢(qián)雯瓊;葛葵葵;黃晉江;黃青山;;SPINK6蛋白在人乳腺癌細(xì)胞MCF-7中的表達(dá)與定位研究[J];復(fù)旦學(xué)報(bào)(自然科學(xué)版);2015年01期

2 戴薪傳;俞一鳴;葛葵葵;黃青山;;腺病毒介導(dǎo)的SPINK6表達(dá)在肝癌細(xì)胞QGY-7703中功能的初步研究[J];復(fù)旦學(xué)報(bào)(自然科學(xué)版);2015年01期

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本文編號(hào)：1978530