色素上皮衍生因子的分段克隆表达对人皮肤鳞状细胞癌细胞增殖的影响  

Segmented cloning and expression of the pigment epithelium derived factor and its effect on the proliferation of human SCL-1 cells

作　　者：张志彬 彭胜男[2] 刘藕根[1] 张颖鹏[1] 李春明[1] 彭亚婷 张淑兰[1] ZHANG Zhi-bin;PENG Sheng-nan;LIU Ou-gen;ZHANG Ying-peng;LI Chun-ming;PENG Ya-ting;ZHANG Shu-lan(Department of Dermatology,the Second Affiliated Hospital of Nanchang University,Nanchang 330006,Jiangxi,China;Science and Technology College of Jiangxi Traditional Chinese Medicine University,Nanchang330004,Jiangxi,China)

机构地区：[1]南昌大学第二附属医院皮肤科,南昌330006 [2]江西中医药大学科技学院,南昌330004

出　　处：《医学研究生学报》2019年第10期1014-1018,共5页Journal of Medical Postgraduates

基　　金：国家自然科学基金(81960569);江西省教育厅科学技术研究项目(GJJ170090)

摘　　要：目的有关色素上皮衍生因子(PEDF)短肽对人皮肤鳞状细胞癌细胞增殖的研究鲜有报道。文中旨在获得PEDF蛋白的分段克隆表达,并观察对人皮肤鳞状细胞癌细胞系(SCL-1)细胞增殖的影响。方法采用PCR扩增PEDF1、PEDF2和PEDF3目的基因,将回收的片段用NheI/HindⅢ酶切,插入至pET28a(+)载体。连接产物转化入大肠埃希菌BL21并诱导表达,并把融合蛋白的进行分离、纯化。采用CCK-8方法检测24 h、48 h及72 h,不同浓度100、400、800和1000 nmol/L PEDF1、PEDF2和PEDF3肽段对SCL-1细胞增殖影响。结果成功构建PEDF1、PEDF2和PEDF3的原核表达载体,PEDF1、PEDF2和PEDF3融合蛋白,相对分子质量约为18 000、17 000和13 000。24 h时,800 nmol/L、1000 nmol/L浓度PEDF3 SCL-1细胞增殖[(0.61±0.03)、(0.78±0.07)]较0 nmol/L PEDF3(1.00±0.00)明显降低(P<0.05);48 h时,400、800、1000nmol/L浓度PEDF3SCL-1细胞增殖呈现显著抑制,并呈剂量依赖性(P<0.05);72 h时,800、1000 nmol/L浓度PEDF3 SCL-1细胞增殖[(0.53±0.05)、(0.51±0.05)]较400、0 nmol/L[(0.60±0.05)、(1.00±0.00)]显著降低(P<0.05)。结论成功分段克隆表达PEDF融合蛋白,PEDF3抑制了SCL-1细胞增殖,为进一步筛选PEDF活性功能短肽提供了基础。Objective Studies are rarely reported on the effect of short peptides of the pigment epithelium derived factor(PEDF)on the proliferation of human cutaneous squamous(SCL-1)cells.The purpose of this study is to investigate segmented cloning and expression of the PEDF protein and observe its effect on the proliferation of human SCL-1 cells.MethodsThe target genesof PEDF1,PEDF2 and PEDF3 were amplified by PCR and the recovered fragments subjected to double digestion of NheⅠand HindⅢand inserted into the pET28 a(+)plasmid.The product was transformed into human E coli BL21 and induced to express,followed by isolation and purification of the fusion protein.CCK-8 assay was used to detect the proliferation of the SCL-1 cells with PEDF1,PEDF2 and PEDF3 at 100,400,800 and 1000 nmol/L at 24,48 and 72 hours.ResultsThe prokaryotic expression vectors of PEDF1,PEDF2 and PEDF3 were successfully constructed,and their fusion proteins prepared,with the molecular weight of 18 000,17 000 and 13 000,respectively.The proliferation of the SCL-1 cells was significantly decreased in the 800 and 1000 nmol/L PEDF3 groups compared with that in the 0 nmol/L PEDF3 group at 24 hours(0.16±0.03 and 0.78±0.07 vs 1.00±0.00,P<0.05),inhibited in a concentration-dependent manner in the 400,800 and 1000 nmol/L PEDF3 groups at 48 hours(P<0.05),markedly lower in the 800 and 1000 nmol/L PEDF3 groups at 72 hours(0.53±0.05 and 0.51±0.05)than in the in the 400 and 0 nmol/L PEDF3 groups(0.60±0.05 and 1.00±0.00)(P<0.05).ConclusionThe PEDF fusion proteins were successfully segmentally cloned and expressed and PEDF3 inhibited the proliferation of SCL-1 cells,which has paved the ground for further screening of active functional short peptides of PEDF.

关 键 词：色素上皮衍生因子 人皮肤鳞状细胞癌 分段克隆 细胞增殖 

分 类 号：R739.5[医药卫生—肿瘤]