Tissue Factor Pathway Inhibitor-2Gene Cloning And Expression, And Its Effects On Gastrointestinal Stromal Tumor Cells

ObjectiveWe observe the expression of TFPI-2in gastrointestinal stromal tumor cellline GISTs cell，construct the eukaryotic expression vector pEGFP-N1-TFPI-2ofTFPI-2gene and then transfect it to GISTs cell.Afterwards，we detect andcompare the growth curve in TFPI-2expression experimental group, controlgroup and blank group cells by MTT，study the influence of TFPI-2expressionon growth inhibition of GISTs cell.Methods1、TFPI-2gene sequence was Retrieved in the GENBANK and Cloning ofcDNA synthesis.2、Construct eukaryotic expression vector pEGFP-N1-TFPI-2,after XhoI/KpnI endonuclease identification and gene sequencing to obtain recombinantadenovirus vector pEGFP-N1-TFPI-2.GISTs cell was transfected by means ofliposome Lipofectamine2000.3、Reverse transcriptase polymerase chain reaction: Amplification of TFPI-2gene and detection of TFPI-2mRNA after transfection.4、Western blot: Detection of TFPI-2protein before and after transfection.5、MTT method and absorbance were used to draw GISTs cell growth curve,and detect TFPI-2gene inhibition effect on proliferation of GISTs cell. Results1、The eukaryotic expression vector pEGFP-N1-TFPI-2of human TFPI-2gene was constructed successfully.2、After the pEGFP-N1-TFPI-2was transfected into GISTs cell, results ofRT-PCR and Western blot detection showed that: in GISTs-TFPI-2group,TFPI-2mRNA and protein expression were significantly higher than the othertwo groups with significant difference (P <0.05).3、After the pEGFP-N1-TFPI-2was transfected into GISTs cell,MTTmethod to draw growth curve, the results show that: the degree of cellproliferation decreased significantly after the experimental group,compared tothe former two groups, there was significant difference between them(P <0.05),and the blank group and the control group cells grew rapidly, thedifference between that two groups had no significant (P>0.05).Conclusions1、Exogenous TFPI-2gene can increase the expression of TFPI-2gene inGISTs cells by Liposome Lipofectamine2000mediated.2、TFPI-2can significantly inhibit the proliferation of GISTs cell.