色素上皮衍生因子基因的克隆以及与肺癌细胞生长和肿瘤新生血管形成影响的研究  

Effects of pigment epithelial derived factor gene on growth of lung cancer cell and experiments with lung cancer cells and chick embryos

作　　者：陈晋峰[1] 赵威[2] 张建芝[1] Wen G Jiang 张力建[1] 

机构地区：[1]北京大学临床肿瘤学院,北京肿瘤医院暨北京市肿瘤防治研究所恶性肿瘤发病机制及转化研究教育部重点实验室,胸外科二病房,100142 [2]北京大学临床肿瘤学院,北京肿瘤医院暨北京市肿瘤防治研究所恶性肿瘤发病机制及转化研究教育部重点实验室,临床实验室,100142 [3]Metastasis & Angiogenesis Research Group, Wales College of Medicine, Cardiff University, Cardiff, United Kingdom

出　　处：《中华医学杂志》2009年第7期485-490,共6页National Medical Journal of China

基　　金：国家863课题基金资助项目（2007AA021010）;北京市科委基金资助项目（Z0005190043621）

摘　　要：目的克隆并在肺癌细胞SKMES1中表达色素上皮衍生因子（PEDF）蛋白；在体实验中观察PEDF蛋白过表达后对肺癌细胞生长和肿瘤相关性新生血管形成的影响。方法通过PCR技术扩增出可以表达PEDF蛋白的全长基因；利用基因重组技术与pEF6／v5-His质粒连接后构建出PEDF基因过表达载体PEDF^exp；将PEDF^exp载体转染SKMES1肺癌细胞，建立稳定过表达PEDF蛋白的肺癌细胞株；将过表达PEDF蛋白的肺癌细胞接种于鸡胚，在体实验中明确PEDF蛋白过表达后对肺癌细胞生长和肿瘤新生血管形成的影响。结果从人体组织的cDNA中成功扩增出PEDF全长序列，并正确构建了可以表达PEDF蛋白的载体，基因测序证实所构建的质粒载体中正确连接了没有核苷酸突变的目的片段；RT—PCR和Western印迹实验在RNA和蛋白水平证实，稳定转染PEDF^exp质粒的肺癌细胞与对照组肺癌细胞相比可以过表达PEDF蛋白。鸡胚实验证实PEDF基因过表达后，SKMES1^PEDFexp肺癌细胞形成病灶的体积明显小于对照组[（0．10±O．05）cm^3vs（0．17±0．07）cm^3]（P=0．016）；SKMES1^PEDFexp肺癌细胞形成病灶的质量也明显小于对照组细胞[（0．008±0．004）mgvs（0．024±0．009）mg]（P=0．006）。肿瘤相关新生血管计数结果显示，PEDF基因的过表达后，肿瘤病灶中一级血管的数量明显少于对照组细胞（15±3vs41±9）（P〈0．001）；二级血管的数量也明显少于对照组细胞（75±22vs175±39）（P=0．001）。结论成功构建出含有PEDF基因表达载体，为研究PEDF基因在肺癌中的功能奠定了基础。PEDF蛋白在肺癌生长以及肿瘤血管形成过程中的抑制作用说明PEDF蛋白是一种非常有前途的具有肺癌治疗作用的生物药物。Objective To investigate the effect of pigment epithelium derived factor （PEDF） on the growth of lung cancer cells and the cancer-related neovascularization. Methods The full length of human PEDF gene was amplified by polymerase chain reaction （PCR） . Human lung cancer cells of the line SKEMS1 were cultured and transfected with PEDF^exp, An eukaryotic expression vector constructed by recombinant DNA technology, so as to construct the SKMES1^PEDFexp cells over-expressing PEDF protein. RTPCR and Western blotting were used to confirm the mRNA and protein expression of PEDF in these ceils. Another SKMES1 lung cancer cells were transfected with blank plasmids （SKMES1^pEF/His cells）. The SKMES1 cells not transfected were called SKMES1^WT cells. The 3 kinds of SKMES1 cells were inoculated in the chorio-allntoic membrane （CAM） of chick embryo hatched for 7 days respectively. The size and weigh of the tumor were measured. The vessels density was examined. Results The tumor volume of the SKMES1^PEDFexp group was （0. 10 ± 0. 05 ） cm^3, significantly smaller than that of the control group [ （0. 17 ±0. 07 ） cm^3 ,P = 0. 016 ], and the mass of the SKME^SPEDFexp group was （0. 008 ± 0. 004 ） mg, significantly smaller than that of the control group too [ （0. 024 ± 0. 009） mg, P = 0. 006 ]. The amount of first class neovessels of the SKMES1^PEDFexp group was （ 15 ±3）, significantly fewer than that of the control group [ （41 ± 9） ,P 〈0. 0011. The amount of second class neo-vessels of the SKMES1^PEDFexp group was （75 ± 22）, also significantly fewer than ihat of the control group [ （ 175 ± 39 ）, P = 0. 001 ]. Conclusion Inhibiting the growth of lung cancer cells and neovascularization, PEDF protein may be used as a potential biological drug to treat lung cancer.

关 键 词：肺肿瘤 色素上皮 眼 新生血管化 病理性 

分 类 号：R686[医药卫生—骨科学]