本文選題：絲氨酸蛋白酶抑制劑 + SPINK6　； 參考：《復旦大學》2012年碩士論文

【摘要】：目的：本實驗室通過基因芯片分析技術,分析了肝癌細胞及其細小病毒抗性株基因表達譜的差異,發(fā)現(xiàn)了在細小病毒抗性細胞株中Kazal型絲氨酸蛋白酶抑制劑6(SPINK6)基因表達明顯上調。但不清楚spink6基因在其他細胞系中的表達水平。本論文主要研究了spink6基因在不同腫瘤細胞和正常細胞系中的表達水平,并研究了SPINK6在細胞中的亞定位,對其作用機制可能有一定的揭示作用。方法：對多種腫瘤細胞和正常細胞抽取RNA,逆轉錄成cDNA后,通過半定量PCR和realtime-PCR的方法研究spink6在不同細胞系中的基因表達水平。根據realtime-PCR結果挑選了幾株高表達spink6基因的細胞系進行免疫熒光實驗,檢測其在蛋白水平的表達,以及天然狀態(tài)下SPINK6蛋白的定位。另外進一步通過構建pEGFP-N1-SPINK6載體,轉染細胞后,使用DAPI標記細胞核,ER-Tracker Red標記內質網,Lyso-Tracker Red標記溶酶體,Dil標記細胞膜,共聚焦顯微鏡下觀察SPINK6的細胞器亞定位。結果：半定量PCR和realtime-PCR的結果顯示spink6在正常細胞中的表達水平高于腫瘤細胞中的表達水平。這一結果說明SPINK6可能是與腫瘤的惡性程度相關。通過細胞器亞定位的研究,發(fā)現(xiàn)SPINK6在內質網和細胞膜上均有定位,揭示了SPINK6可能是通過出膜發(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.
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R3416;Q78

【共引文獻】

相關期刊論文 前2條

1 錢雯瓊;葛葵葵;黃晉江;黃青山;;SPINK6蛋白在人乳腺癌細胞MCF-7中的表達與定位研究[J];復旦學報(自然科學版);2015年01期

2 戴薪傳;俞一鳴;葛葵葵;黃青山;;腺病毒介導的SPINK6表達在肝癌細胞QGY-7703中功能的初步研究[J];復旦學報(自然科學版);2015年01期

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本文編號：1978530