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AIM:To construct a stable transfectant of human livercarcinoma cell line SMVLC7721 that could secret humanendestatin and to explore the effect of human endostaUnexpressed by the transfectant on endothelial cell proliferation.METHODS:Recombinant retroviral plasmid pLncx-Endocontaining the cDNA for human endostain gene togetherwith rat albumin signal peptide was engineered andtransferred into SMMC7721 cell by lipofectamine.Afterselection with G418,endostatin-transfected SMMC7721 cellswere chosen and expanded.Immunohistochemical stainingand Western blot were used to detect the expression ofhuman endostaUn in transfected SMMC7721 cells and itsmedium.The conditioned medium of endostatin-transfectedand control SMMC7721 cells ware collected to cultivate withhuman umbilical vein endothelial cells for 72 hours.Theinhibitory effect of endostatin,expressed by transfectedSMMC7721 cells,on endothelial proliferation in vitro wasobserved by using MTT assay.RESULTS:A 550 bp specific fragment of endostaUn gene wasdetected from the PCR product of endostatin-transfectedSMMC7721 cells.Immunohistochemistry and Western blotanalysis confirmed the expression and secretion of foreighhuman endostatin protein by endostatin-transfectedSMMC7721 cells.In vitro endothelial proliferation assayshowed that 72 hours after cultivation with human umbilicalvein endothelial cells,the optical density(OD)in groupusing the medium from endostatin-transfected SMMC7721cells was 0.51±0.06,lower than that from RPMI 1640 group(0.98±0.09)or that from control plasmid pLncx-transfectedSMMC7721 cells(0.88±0.11).The inhibitory rate formedium 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 endostain gene and its product can significantly inhibit the proliferation of human umbilical vein endothelial cell in vitro.
AIM: To construct a stable transfectant of human liver carcinoma cell line SMVLC7721 that could secret humanendestatin and to explore the effect of human endostaUnexpressed by the transfectant on endothelial cell proliferation. METHODS: Recombinant retroviral plasmid pLncx-Endocontaining the cDNA for human endostain gene together with rat albumin signal peptide was engineered and transferred into SMMC7721 cells by lipofectamine. Airflection with G418, endostatin-transfected SMMC7721 cells were selected and expanded. Immunohistochemical staining and Western blot were used to detect the expression of human endostaUn in transfected SMMC7721 cells and its medium. The conditioned medium of endostatin-transfected and control SMMC7721 cells ware collected to cultivate with human umbilical vein endothelial cells for 72 hours. Inhibitory effect of endostatin, expressed by transfected SMMC7721 cells, on endothelial proliferation in vitro wasobserved by using MTT assay. RESULTS: A 550 bp specific fragment of endost aUn gene wasdetected from the PCR product of endostatin-transfected SMMC7721 cells. Immunohistochemistry and Western blot analysis confirmed the expression and secretion of foreighhuman endostatin protein by endostatin-transfected SMMC7721 cells. In vitro endothelial cell proliferation assays showed that 72 hours after cultivation with human umbilicalvein 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 from control plasmid pLncx-transfected SMMC7721 cells (0.88 ± 0.11). inhibitory rate for medium from Endostatin-transfected SMMC7721 cells was 48%, significantly higher than that from the empty plasmid pLncx-transfected SMMC7721 cells (10.2%, P <0.01) .CONCLUSION: Human endostatin can be stably expressed by SMMC7721 cells transferred with human endostain gene and its product can significantly inhibit the proliferation of human umbilical vein endothelial cell in vitro.