摘要目的：现今癌症仍是最主要的致死原因，但一直以来治疗癌症都是以手术治疗、放射治疗和化学治疗为主。近年来，中医中药治疗逐渐成为热点，而姜黄素就是其中一种能够抑制肿瘤细胞增殖的中药，然而其具体机制却不甚了解。有研究发现，在姜黄素抑制肿瘤细胞的过程中，多种基因的表达量发生了变化，而Egr-1就是其中一种。本研究旨在构建pEGFP-Egr-1 真核过表达质粒，并将其导入到肝癌细胞HepG-2中，观察转染效果，从而为进一步探讨Egr-1基因在姜黄素抑制肿瘤细胞中的作用。方法：用20 μmol/L的姜黄素处理HepG-2细胞3小时，然后提取其总RNA，逆转录成cDNA后经RT-PCR得到Egr-1目的基因，运用TA克隆技术构建pMD18-Egr-1重组载体，转化大肠杆菌，经鉴定正确后小提质粒，接着用Xho I 和Hind III同时双酶切pMD18-Egr-1重组载体和pEGFP-C1空载体，得到pEGFP-Egr-1哺乳动物过表达重组质粒，同样转化大肠杆菌，利用菌落PCR鉴定，鉴定正确后大提质粒，最后测序正确后再转染HepG-2细胞，并检验最终的转染效率。结果： pEGFP-Egr-1哺乳动物过表达载体构建成功，并能在HepG-2中正常表达，而且负载比（质粒DNA和转染试剂的比值）不同会导致转染效果有所不同。结论：pEGFP-Egr-1的成功构建为之后姜黄素抑制肿瘤细胞的内在机制以及Egr-1作为一种新的肿瘤抑制基因的研究打下了一定的实验基础。71956

Abstract Objective: Today, cancer is still the most important cause of death, but the treatment is always based on surgery, radiotherapy and chemotherapy。 In recent years, Chinese medicine has gradually become a hot topic, and curcumin is one of the traditional Chinese medicine can inhibit the proliferation of tumor cells, however its mechanism has yet to be elucidated。 Some studies have found that in the process of inhibiting the tumor cells, the expression of many genes has changed, and Egr-1 is one of them。 The purpose of this study was to construct the eukaryotic overexpression plasmid of pEGFP-Egr-1, and transfected it into HepG-2 of liver cancer cells, to observe the effect of transfection, so as to further investigate the role of Egr-1 in the inhibition of tumor cells by curcumin。 Methods: To treat HepG-2 cells with 20 mol/L curcumin for 3 hours, then extracted the total RNA, and after reverse transcription as well as RT-PCR, we can obtain cDNA, which is the Egr-1。 Using TA cloning to construct the pMD18-Egr-1 recombinant vector, and then transformed it into E。coli。 After the recombinant plasmid was confirmed by digestion, used the Plasmid Mini Kit to acquire plasmid vector。 Simultaneously enzyme digest pMD18-Egr-1 and pEGFP-C1 by Xho I and Hind III for pEGFP-Egr-1 overexpression plasmid, and then transformed it into E。coli。 Only after confirming by digestion, it can be extracted by Plasmid Maxi Kit。 Finally, sequencing the plasmid transfected into HepG-2 cells by lipofectin-mediated transfection and observe the expression by fluorescent microscope。 Result: pEGFP-Egr-1 mammalian overexpression vector was constructed successfully, and can be expressed normally in HepG-2。 Moreover, the load ratio (ratio of plasmid DNA and transfection reagent) can result in different transfection efficiency。 Conclusion: The successful construction of pEGFP-Egr-1 provided a foundation for the research of the intrinsic mechanism of curcumin in inhibiting the tumor cells and Egr-1 as a new tumor suppressor gene。

毕业论文关键词：Egr-1基因；pEGFP-Egr-1重组质粒；HepG-2细胞；基因表达；姜黄素

Keyword: Egr-1 gene; pEGFP-Egr-1 recombinant plasmid; HepG-2 cells; gene expression;  curcumin

目    录

引言 5

1 材料和方法 6

1。1 材料与仪器 6