人GFP-Sp1基因真核表达载体的构建及其对细胞周期调控作用研究  

Construction of a eukaryotic expression vector for the human GFP-Sp1 gene and its regulatory effect on cell cycle

作　　者：崔瀛书 孙园园 龙珊 徐媛媛[3] 李怡 胡佳[2] 李倩[2] 李晓松[2] CUI Yingshu;SUN Yuanyuan;LONG Shan;XU Yuanyuan;LI Yi;HU Jia;LI Qian;LI Xiaosong(Chinese PLA Medical School,Beijing 100853,China;Department of Oncology,the Seventh Medical Center,Chinese PLA General Hospital,Beijing 100700,China;Department of Laser,the First Medical Center,Chinese PLA General Hospital,Beijing 100853,China)

机构地区：[1]解放军医学院,北京100853 [2]解放军总医院第七医学中心肿瘤内科,北京100700 [3]解放军总医院第一医学中心激光科,北京100853

出　　处：《解放军医学院学报》2022年第8期867-872,共6页Academic Journal of Chinese PLA Medical School

基　　金：国家自然科学基金面上资助项目(61975239)。

摘　　要：背景乳腺癌目前已经成为女性发病率最高的恶性肿瘤,因此寻找有效治疗靶点进而控制肿瘤发生发展至关重要。目的构建带绿色荧光蛋白报告载体pEGFP-C1的Sp1真核表达载体,在获得pEGFP-Sp1融合表达蛋白后,对其功能进行验证。方法以人乳腺文库为模版,采用PCR技术扩增出Sp1的CDS区编码序列技术,将其插入到pEGFP-C1载体中,经酶切和测序验证成功后,转染至人胚肾293T细胞,蛋白质印迹法检测该融合蛋白的表达情况;分别转染pEGFP-C1、pEGFPSp1到人乳腺癌细胞ZR75-1细胞中,通过荧光显微镜观察Sp1蛋白在细胞中定位情况,通过逆转录PCR检测细胞周期基因转录水平。结果经双酶切及测序鉴定证明pEGFP-Sp1质粒构建成功,蛋白印迹检测蛋白表达成功;荧光显微镜结果显示Sp1主要定位于人乳腺癌细胞ZR75-1细胞的细胞核中,逆转录PCR结果显示过表达Sp1可改变细胞周期相关基因转录水平。结论通过构建真核表达载体pEGFP-Sp1,可确定Sp1蛋白在人乳腺癌细胞ZR75-1细胞中表达,且主要表达于细胞核中。同时Sp1可下调Cyclin D2与CDK6 mRNA水平,上调Cyclin G2和p18 mRNA水平。综上所述,我们的结果表明Sp1在细胞周期通路中发挥重要的功能,这为进一步深入研究Sp1转录因子的功能提供基础。Background Breast cancer has become the malignant tumor with the highest incidence rate in women,and therefore,it is of great importance to find effective therapeutic targets to control the development and progression of breast cancer.Objective To construct the eukaryotic expression vector of Sp1 with green fluorescent protein reporter(pEGFP-C1),and to validate the function of pEGFP-Sp1 protein.Methods With the human mammary library as the template,the CDS sequence of the Sp1 gene amplified by polymerase chain reaction was inserted into pEGFP-C1 vector;after enzyme digestion and sequence confirmation,the recombinant plasmid was transfected into human embryonic kidney 293T cells,and Western blotting was used to measure the expression level of pEGFP-Sp1 protein.After pEGFP-C1 and pEGFP-Sp1 were transfected into human breast cancer ZR75-1 cells,fluorescence microscopy was used to observe the localization of Sp1 protein in cells,and reverse transcription-polymerase chain reaction(RT-PCR)was used to measure the transcriptional levels of cell cycle genes.Results Double-enzyme digestion and sequencing confirmed that the pEGFP-Sp1 plasmid was successfully constructed,and Western blotting showed the successful expression of Sp1 protein.Fluorescence microscopy showed that Sp1 protein was mainly localized in the nucleus of human breast cancer ZR75-1 cells,and RT-PCR showed that Sp1 overexpression changed the transcriptional level of cell cycle-related genes.Conclusion The eukaryotic expression vector pEGFP-Sp1 is successfully constructed,and it is confirmed that Sp1 protein is expressed in human breast cancer ZR75-1 cells,mainly in the nucleus.In addition,Sp1 can downregulate the mRNA expression levels of Cyclin D2 and CDK6 and upregulate the mRNA expression levels of Cyclin G2 and p18.In summary,the results of this study show that Sp1 plays an important role in regulating the cell cycle pathway,which provides a basis for further in-depth research on the function of Sp1 transcription factor.

关 键 词：ZR75-1 转录因子SP1 pEGFP-Sp1 聚合酶链式反应 细胞周期 

分 类 号：R737.9[医药卫生—肿瘤] Q78[医药卫生—临床医学]