摘    要    目的：现如今癌症仍是目前导致死亡的主要原因之一，肝癌是恶性肿瘤的一种，传统的手术治疗方法有肝切除和肝移植等，手术治疗后辅以化疗及放疗副作用明显，而中医中药联合治疗作为五大癌症防治手段之一越来越受到人们的关注，成为癌症治疗研究的热点。姜黄素中药姜黄的主要成分，它有抗氧化作用。有研究发现，姜黄素能通过调控细胞中Egr-1基因的表达来抑制癌细胞的增殖。本研究通过构建Egr-1基因外来过表达质粒，并将其转染至人肝癌HepG2细胞中，来检测分析转染细胞中Egr-1基因过表达对其增殖的影响。方法：用20 μmol/L的姜黄素处理HepG2细胞3小时后，Trizol法提取细胞总RNA，通过RT-PCR逆转录合成cDNA并进行扩增获取目的基因Egr-1片段。然后采用TA克隆技术将其构建到pMD18-Egr-1质粒，经测序正确后将pMD18-Egr-1重组质粒转化进大肠杆菌并小提质粒，随后用Xho I和Hind III双酶切出Egr-1片段并进一步连接到真核表达质粒pEGFP-C1上构建得到pEGFP-Egr-1重组表达质粒。随后将pEGFP-Egr-1重组质粒转染HepG2细胞，利用荧光显微镜和荧光定量PCR分析检测pEGFP-Egr-1重组质粒在HepG2细胞中的表达情况，最后采用WST-1细胞增殖检测法检测Egr-1基因外来过表达对HepG2细胞增殖的影响。结果：成功构建了能在HepG2细胞中表达的pEGFP-Egr-1重组质粒，并且发现转染了pEGFP-Egr-1的HepG2细胞与对照组（转染pEGFP-C1的HepG2细胞）相比，其增殖速率减慢，且随着转染质粒pEGFP-Egr-1浓度的提升，HepG2细胞其增殖速度明显减慢。结论：Egr-1基因外来过表达能抑制肝癌HepG2细胞的增殖，Egr-1基因在HepG2细胞增殖中起抑制作用。88834

Abstract Objective: Nowadays, cancer is still one of the leading causes of death, and liver cancer is a kind of malignant tumor, its traditional surgical treatment methods include liver resection and liver transplantation surgery。 Surgery combined with chemotherapy and radiotherapy has really clear side effect。 Chinese medicine treatment as one of the five major methods of cancer prevention has attracted more and more attention and become a hot topic study of cancer treatment。 Curcumin is a traditional Chinese medicine, and it has antioxidant effect。 Studies have found that curcumin can inhibit the proliferation of cancer cells by affecting the expression of Egr-1 gene in cells。 In this study, we detected the effect of Egr-1 gene overexpression on the proliferation of HepG2 cells through constructing the Egr-1 gene overexpression plasmid and transfecting it into human hepatocellular carcinoma HepG2 cells。 Methods: To treat HepG-2 cells with 20 mol/L curcumin for 3 hours, then extracted the total RNA by Trizol method, then reverse transcript cDNA by RT-PCR and amplify it to obtain the target gene fragment。 Using TA cloning technology construct PMD18-Egr-1 plasmid, after sequencing, the recombinant plasmid of pMD18-Egr-1 was transformed into E。 coli and metion the small plasmid。 Simultaneously enzyme digest to get Egr-1 gene by Xho I and Hind III for pEGFP-Egr-1 overexpression plasmid, sequencing the plasmid transfected into HepG-2 cells by lipofectin-mediated transfection and observe the expression by fluorescent microscope and fluorescence quantitative PCR。 Finally using the WST-1 method to detect the the influence of Egr-1 gene overexpression on hepatocarcinoma HepG2 cell proliferation。 Result: pEGFP-Egr-1 mammalian overexpression vector was constructed successfully, and compared with the control group (HepG2 transfected pEGFP-C1 cells), the proliferation rate of HepG2 cells transfected with pEGFP-Egr-1 decreased。 Also with the increase of the concentration of transfected pEGFP-Egr-1 plasmid, the proliferation rate of HepG2 cells was more and more slow。 Conclusion: Overexpression of Egr-1 gene inhibits proliferation of hepatocellular carcinoma HepG2 cells, and Egr-1 gene inhibits the proliferation of HepG2 cells。 Egr-1基因外来过表达对肝癌HepG2细胞增殖的影响研究:http://www.youerw.com/yixue/lunwen_168613.html