| The serine protease inhibitor, Kazal type3(SPINK3) is a trypsin inhibitor which is highly expressed inpancreatic cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer and other tumors, but theresearch on the effect of spink3on hepatic cancer cell strain has not been reported.Therefore, the expression vector (pEGFP-N1-spink3), interference vectors (pGenesil-1.0-spink3(123) and pGenesil-1.0-spink3(143)) and test vectors (pGenesil-1.0-spink3(123)-spink3, pGenesil-1.0-spink3(143)-spink3and pGenesil-1.0-HK-spink3) were established. The constructed test vectors wereadministrated using a hydrodynamics-based procedure in the tail vein of little rat to select the bestrecombinant interference plasmid using fluorescence microscopy technique to observe enhanced greenfluorescent protein (EGFP)-positive cell rate. Next, the expression vector pEGFP-N1-spink3and its controlpEGFP-N1, interference vector pGenesil-1.0-spink3(123) and its control pGenesil-1.0-HK were stablytransfected into hepatic cancer cell strain BEL-7402in vitro by lipofectamine and their stably transfectedcell strains were selected by G418. Then, the hepatic cancer cell strain BEL-7402and the four kinds ofstably transfected cell strains above were detected using the following methods, including cell morphologyobserved by HE staining, cell morphology and EGFP distribution observed under a fluorescencemicroscope, cell growth curve drew by cell counting and MTT assay, PCNA expression changes detectedby PCNA immuneocytochemistry, cell apoptosis detected by Hoechst33258staining, cell colony formationrate detected by Methyl cellulose semi-solid medium, and cell resistance to drugs detected by MTT assay.The results show that the SPINK3-EGFP fusion protein was evenly distributed in the cytoplasm, nofluorescence in the nucleus, more expression surrounding the outer nuclear membrane which is a typicalsecretory expression pattern, that spink3can inhibit hepatic cancer cell strain BEL-7402proliferation, butnot cause apoptosis, and that spink3can reduce the degree of malignancy of cells, but had no significanteffect on the resistance to drugs. |
