摘要
AIM: To construct a stable transfectant of human liver carcinoma cell line SMMC7721 that could secret human endostatin and to explore the effect of human endostatin expressed by the transfectant on endothelial cell proliferation. METHODS: Recombinant retroviral plasmid pLncx-Endo containing the cDNA for human endostatin gene together with rat albumin signal peptide was engineered and transferred into SMMC7721 cell by lipofectamine. After selection with G418, endostatin-transfected SMMC7721 cells were chosen and expanded. Immunohistochemical staining and Western blot were used to detect the expression of human endostatin in transfected SMMC7721 cells and its medium. The conditioned medium of endostatin-transfected and control SMMC7721 cells were collected to cultivate with human umbilical vein endothelial cells for 72 hours. The inhibitory effect of endostatin, expressed by transfected SMMC7721 cells, on endothelial proliferation in vitro was observed by using MTT assay. RESULTS: A 550 bp specific fragment of endostatin gene was detected from the PCR product of endostatin-transfected SMMC7721 cells. Immunohistochemistry and Western blot analysis confirmed the expression and secretion of foreign human endostatin protein by endostatin-transfected SMMC7721 cells. In vitro endothelial proliferation assay showed that 72 hours after cultivation with human umbilical vein endothelial cells, the optical density (OD) in group using the medium from endostatin-transfected SMMC7721 cells was 0.51 +/- 0.06, lower than that from RPMI 1640 group (0.98 +/- 0.09) or that from control plasmid pLncx-transfected SMMC7721 cells (0.88 +/- 0.11). The inhibitory rate for medium from endostatin-transfected SMMC7721 cells was 48%, significantly higher than that from empty plasmid pLncx-transfected SMMC7721 cells (10.2%, P【0.01). CONCLUSION: Human endostatin can be stably expressed by SMMC7721 cell transferred with human endostatin gene and its product can significantly inhibit the proliferation of human umbilical vein endothelial cell in
瞄准：为了构造人的肝癌房间的稳定的 transfectant，衬里 SMMC7721 能秘密人的 endostatin 并且在 endothelial 房间增长上由 transfectant 表示了探索人的 endostatin 的效果。方法：Recombinant 制动火箭和老鼠白朊信号肽为人的 endostatin 基因包含 cDNA 的病毒的原生质标志 pLncx-Endo 被设计并且由 lipofectamine 转了进 SMMC7721 房间。在有 G418 的选择以后， endostatin-transfected SMMC7721 房间被选择并且膨胀。Immunohistochemical 染色和西方的污点被用来在 transfected SMMC7721 房间和它的媒介检测人的 endostatin 的表示。endostatin-transfected 和控制 SMMC7721 房间的调节媒介是镇定的与人的脐的静脉 endothelial 房间栽培 72 个小时。endostatin 的禁止的效果，由 transfected SMMC7721 房间表示了，在 endothelial 上，在 vitro 的增长被使用 MTT 观察试金。结果：550 bp endostatin 基因的特定的碎片从 endostatin-transfected SMMC7721 房间的 PCR 产品被检测。Immunohistochemistry 和西方的污点分析由 endostatin-transfected SMMC7721 房间证实了外国人的 endostatin 蛋白质的表示和分泌物。在 vitro endothelial 增长，试金显示出那在有人的脐的静脉 endothelial 房间的耕作以后的 72 个小时，在用从 endostatin-transfected SMMC7721 房间的媒介的组的光密度( OD )是 0.51 +/- 0.06 ，从 RPMI 1640 组比那降低( 0.98 +/- 0.09 )或从控制原生质标志 pLncx-transfected SMMC7721 房间( 0.88 +/- 0.11 )。为从 endostatin-transfected SMMC7721 房间的媒介的禁止的率是 48% ，比那显著地高从空原生质标志 pLncx-transfected SMMC7721 房间(10.2% ， P<0.01 ) 。结论：人的 endostatin 能被与人的 endostatin 基因转移的 SMMC7721 房间稳定地表示，它的产品能显著地在 vitro 禁止人的脐的静脉 endothelial 房间的增长。
作者
Xuan Wang Fu-Kun Liu Xi Li Jai-Sou Li,Research Institute of General Surgery,Clinical School of Medicine,Nanjing University,Nanjing 210002,Jiangsu Province,China Gen-Xin Xu,Department of Molecular Biology,Nanjing Military Medical School,Nanjing 210002,Jiangsu Province,China
