pEGFP-N3-KLF2真核表达载体的构建及其对LPS诱导的THP-1中TF表达的影响

发布时间：2018-01-19 07:30

本文关键词： 组织因子脂多糖真核表达载体流式细胞术单核细胞　出处：《中南大学》2011年硕士论文　论文类型：学位论文

【摘要】：动脉血栓形成是急性心肌梗塞和脑梗死的主要诱因,也是发达国家最常见的死亡原因。组织因子(TF)是生理性止血和病理性形成血栓的启动因子,它在血栓形成中的起始作用越来越受到人们的重视。单核细胞作为动脉粥样硬化斑块中TF来源的细胞-泡沫细胞的前身,研究其TF的表达调控机制已经成为当前临床医学的热点之一。国内外大量研究表明,不稳定型心绞痛、急性冠状动脉综合症、高血压和高血脂等心脑血管疾病患者血液中单核细胞的TF都处在高表达的水平；同时发现,具有抗血栓作用以及血管内皮保护功能的KLF2基因在急性冠状动脉综合症病人的单核细胞中表达明显下调。因此,本研究旨在构建pEGFP-N3-KLF2高表达载体,并且观察LPS对KLF2高表达载体转染的THP-1细胞表达TF的影响,为丰富凝血理论提供新的实验依据。目的：构建pEGFP-N3-KLF2高表达载体,并且观察LPS对KLF2高表达载体转染的THP-1细胞表达TF的影响。方法：从NCBI获得klf2基因序列,运用premier5.0软件设计引物,运用PCR技术扩增klf2基因,电泳检测正确后,进行纯化回收,然后将纯化回收产物连pEGFP-N3载体进行转化,再挑单克隆以及摇菌,小提质粒,最后进行双酶切检测和PCR检测,当两项结果均正确时,送pEGFP-N3-KLF2新鲜菌液去测序。将单核细胞分为5组：①正常细胞组,②转染空质粒组,③转染高表达质粒组,④LPS组,⑤LPS+转染高表达质粒组。分别培养2 h、12 h、24 h、48 h和72 h后,用流式细胞术检测各组各时间点TF蛋白表达变化。结果：阳性克隆筛选和测序结果显示,pE-GFP-N3-KLF2高表达载体构建成功。在实验观察的2h至72h内,正常组细胞各时间点TF表达基本一致,各观察点间比较差异无统计学差异；与正常对照组相比,空载体组和高表达转染质粒组TF表达也无显著性差异(P0.05)。LPS组表达TF从12h起明显增加,至48h达到高峰,与对照组相比,差异具有统计学差异(p0.01),而LPS+高表达转染质粒组TF表达量明显降低,与LPS组同一时间点的TF表达量相比,差异具有显著性意义(P0.01)。结论：①利用具有标志绿色荧光蛋白的pEGFP-N3质粒载体构建了KLF2高表达质粒,并经测序鉴定后pEGFP-N3-KLF2高表达质粒载体构建成功。②LPS能显著地刺激THP-1表达TF,但这种诱导作用可被转染KLF2高表达的质粒所抑制。
[Abstract]:Arterial thrombosis is the main cause of acute myocardial infarction and cerebral infarction and the most common cause of death in developed countries. Mononuclear cells, the precursor of TF derived cells and foam cells in atherosclerotic plaques, have attracted more and more attention in the initiation of thrombosis. The study of TF expression regulation mechanism has become one of the hotspots in clinical medicine. A large number of studies at home and abroad show that, unstable angina pectoris, acute coronary syndrome, hypertension and hyperlipidemia in patients with cardiovascular and cerebrovascular diseases such as blood monocyte TF is in a high level of expression; It was also found that the expression of KLF2 gene with antithrombotic effect and vascular endothelial protection was significantly down-regulated in monocytes of patients with acute coronary syndrome. The purpose of this study was to construct a high expression vector of pEGFP-N3-KLF2 and to observe the effect of LPS on TF expression in THP-1 cells transfected with high expression vector of KLF2. It provides a new experimental basis for enriching clotting theory. Aim: to construct the high expression vector of pEGFP-N3-KLF2 and to observe the effect of LPS on TF expression in THP-1 cells transfected with high expression vector of KLF2. Methods: klf2 gene sequence was obtained from NCBI, primers were designed by premier5.0 software, and klf2 gene was amplified by PCR technique. Then the purified and recovered product was transformed into pEGFP-N3 vector, then the monoclonal and shaking bacteria were selected, and the plasmid was extracted. Finally, the double enzyme digestion and PCR detection were carried out, when the two results were correct. The mononuclear cells were divided into 5 groups: 1: 1 normal cell group: 1 / 2 transfected empty plasmid group and 3 transfected with high expression plasmid group (LPS-4). 5the high expression plasmid group was transfected with LPS. After cultured for 2 h for 12 h and 24 h for 48 h and 72 h, the changes of TF protein expression at each time point were detected by flow cytometry. Results: the results of positive clone screening and sequencing showed that the high expression vector pE-GFP-N3-KLF2 was successfully constructed and was observed within 2 to 72 hours. The TF expression of normal cells was basically the same at all time points, but there was no statistical difference among the observation points. There was no significant difference in TF expression between the empty vector group and the high expression plasmid group compared with the normal control group. The expression of TF in the P0.05 + LPS group was significantly increased from 12 h to the peak at 48 h. Compared with the control group, the difference was statistically significant (p0.01), while the TF expression in the high expression plasmid group of LPS was significantly lower than that in the LPS group at the same time point. The difference was significant (P 0.01). Conclusion the high expression plasmid of KLF2 was constructed by using pEGFP-N3 plasmid vector with green fluorescent protein marker. The recombinant plasmid vector with high expression of pEGFP-N3-KLF2 was successfully constructed by sequencing. 2 LPs could significantly stimulate the expression of TF in THP-1. However, this induction can be inhibited by the high expression plasmid transfected with KLF2.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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