SPINK6在不同细胞系中的表达和定位

发布时间：2018-06-04 19:11

本文选题：丝氨酸蛋白酶抑制剂 + SPINK6　；参考：《复旦大学》2012年硕士论文

【摘要】：目的：本实验室通过基因芯片分析技术,分析了肝癌细胞及其细小病毒抗性株基因表达谱的差异,发现了在细小病毒抗性细胞株中Kazal型丝氨酸蛋白酶抑制剂6(SPINK6)基因表达明显上调。但不清楚spink6基因在其他细胞系中的表达水平。本论文主要研究了spink6基因在不同肿瘤细胞和正常细胞系中的表达水平,并研究了SPINK6在细胞中的亚定位,对其作用机制可能有一定的揭示作用。方法：对多种肿瘤细胞和正常细胞抽取RNA,逆转录成cDNA后,通过半定量PCR和realtime-PCR的方法研究spink6在不同细胞系中的基因表达水平。根据realtime-PCR结果挑选了几株高表达spink6基因的细胞系进行免疫荧光实验,检测其在蛋白水平的表达,以及天然状态下SPINK6蛋白的定位。另外进一步通过构建pEGFP-N1-SPINK6载体,转染细胞后,使用DAPI标记细胞核,ER-Tracker Red标记内质网,Lyso-Tracker Red标记溶酶体,Dil标记细胞膜,共聚焦显微镜下观察SPINK6的细胞器亚定位。结果：半定量PCR和realtime-PCR的结果显示spink6在正常细胞中的表达水平高于肿瘤细胞中的表达水平。这一结果说明SPINK6可能是与肿瘤的恶性程度相关。通过细胞器亚定位的研究,发现SPINK6在内质网和细胞膜上均有定位,揭示了SPINK6可能是通过出膜发挥作用的。
[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.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R3416;Q78

【共引文献】