[Regulation of anticoagulation effect of human umbilical vein endothelial cells by thrombomodulin gene transfection].
OBJECTIVE
To transfect pcDNA3.1/hTM plasmids containing human thrombomodulin (hTM) gene into human umbilical vein endothelial cells (HUVECs), and investigate the expression of hTM and anticoagulating function of transfected HUVECs.


METHODS
HUVECs were transfected with pcDNA3.1/hTM by lipofectin. Expression of hTM mRNA was determined by semi-quantitative RT-PCR, hTM antigen on HUVECs membrane by immunohistochemistry, and activated protein C (PC) in HUVECs by chronometry. By using a semiautomatic coagulator, the effect of the reacting liquid from transfected HUVECs mixed with PC from normal peripheral blood was assayed.


RESULTS
About 10% HUVECs were transfected by pcDNA3.1/hTM with high-level hTM mRNA and protein expression. Activated PC produced by pcDNA3.1/hTM group, pcDNA3.1(+)/neo group and untransfected group was (2.80 +/- 0.43) microg/ml, (0.75 +/- 0.08) microg/ml and (0.85 +/- 0.11) microg/ml, respectively. APTT was (51.68 +/- 2.73) s, (38.38 +/- 2.44) s, (39.65 +/- 2.39) s, (33.93 +/- 1.73) s and (34.60 +/- 1.86) s and PT was (21.89 +/- 1.66) s, (20.56 +/- 1.74) s, (20.42 +/- 2.04) s, (19.57 +/- 1.36) s and (20.16 +/- 1.35) s in pcDNA3.1/hTM group, pcDNA3.1(+)/neo group, untransfected group, inactivating PC group and control, respectively.


CONCLUSIONS
The pcDNA3.1/hTM plasmid could be transfected into endothelial cells and expressed biologically functioning hTM protein on HUVECs membrane. Activated PC could inhibit intrinsic coagulation pathway obviously with slight effect on extrinsic pathway.
Dai
Yi
Y
Department of Vascular Surgery, The Affiliated Hospital, North Sichuan Medical College, Nanchong 637000, China.
Chen
Kai
K
Qiao
Zheng-rong
ZR
Zou
Lin
L
Zhang
Xue-mei
XM
Chen
Hui
H
Shi
De
D
D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology; D005786:Gene Expression Regulation; D041722:Genes, Transgenic, Suicide; D006801:Humans; D066298:In Vitro Techniques; D010314:Partial Thromboplastin Time; D010957:Plasmids; D011486:Protein C; D011517:Prothrombin Time; D018180:Thrombomodulin / Q000235:genetics*; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology
D011486:Protein C; D018180:Thrombomodulin