Objective To investigate the invasion ability of Panc-1 cells in vivo and in vitro af ter being t ransfected with tissue factor pathway inhibitor 2 gene ( TFPI-2) . Methods The expression vector pEGFP-C1-TFPI-2 was transfected into human pancreatic cancer line Panc-1 cells by using liposome. TFPI-2 mRNA and protein of transfected and nontransfected cells were detected by reverse t ranscription-polymerase chain reaction (RT-PCR) and Western blot respectively. The tumor cells invasive behavior of t ransfected ( Panc-1-TFPI-2) and nontransfected ( Panc-1-V and Panc-1-P) cells were assessed in vitro through Boyden Chamber method. The transfected and nontransfected cells were implanted into nude mice to observe it s growth and metastasis in vivo. Results Expressions of mRNA and protein of TFPI-2 were confirmed in transfected cells. Af ter TFPI-2 t ransfection , the number of Panc-1-TFPI-2 , Panc-1-V and Panc-1-P cells passing through membrane of Boyden Chamber were 24. 4 ±3. 5 ,61. 3 ±4. 1 and 60. 2 ±3. 9 , respectively. The number of TFPI-2-expressing cells to t raverse a Matrigel-coated membrane was obviously decreased compared with that of non-expressing cells , the invasion ability was lower than that before transfection in vitro. The subcutaneous tumor volume of the Panc-1-TFPI-2 group was (438. 0 ±69. 8) mm3 , the Panc-1-V group was (852. 0 ±102. 9) mm3 and the Panc-1-P group was (831. 0 ±78. 1) mm3 , P lt; 0. 05. The metastasis to liver and lung and muscular invasion occurred in the Panc-1-V group and the Panc-1-P group. There were no muscular invasion and metastatic lesions in the Panc-1-TFPI-2 group. Conclusion TFPI-2 gene expression may obviously inhibit the invasion ability of pancreatic cancer cells in vitro and in vivo , which provides an experimental basis for the treatment of human pancreatic cancer by gene therapy.
【Abstract】 Objective To reduce restenosis in vein grafts after coronary artery bypass grafting, to investigate theeffect of human tissue factor pathway inhibitor(TFPI) gene del ivery on neointima formation. Methods The eukaryotic expressed plasmid vector pCMV-(Kozak) TFPI was constructed. Forty-eight Japanese white rabbits were randomly divided into 3 groups with 16 rabbits in each group: TFPI group, empty plasmid control group and empty control group. Animal model of common carotid artery bypass grafting was constructed. Before anastomosis, vein endothel iocytes were transfected with cationic l iposome containing the plasmid pCMV- (Kozak) TFPI (400 μg) by pressurizing infusion (30 min) in TFPI group. In empty plasmid control group, vector pCMV- (Kozak) TFPI was replaced by empty plasmid pCMV (400 μg). In empty control group, those endothel iocytes were not interfered. After operation, vein grafts were harvested at 3 days for immunohistochemical, RTPCR and Western-blot analyses of exogenous gene expression and at 30 days for histopathology measurement of intimal areas, media areas and calculation of intimal/media areas ratio. Luminal diameter and vessel wall thickness were also measured byvessel Doppler ultrasonography and cellular category of neointima was analyzed by transmission electron microscope at 30 days after operation. Results Human TFPI mRNA and protein were detected in TFPI group. The mean luminal diameter of the TFPI group, empty plasmid control group and empty control group was (2.68 ± 0.32) mm, (2.41 ± 0.23) mm and (2.38 ± 0.21) mm respectively. There were statistically significant differences between TFPI group and control groups (P lt; 0.05). The vessel wall thickness of the TFPI group, empty plasmid control group and empty control group was (1.09 ± 0.11) mm, (1.28 ± 0.16) mm and (1.34 ± 0.14) mm respectively. There were statistically significant differences between TFPI group and other control groups (P lt; 0.01). The mean intimal areas, the ratio of the intimal/media areas of the TFPI group were (0.62 ± 0.05) mm2and 0.51 ± 0.08 respectively, which were reduced compared with those of the two control groups(P lt; 0.05). The mean media areas had no significant differences among three groups (P gt; 0.05). Through transmission electron microscope analyses, no smoothmuscle cells were seen in neointima of TFPI group in many visual fields, but smooth muscle cells were found in neointima of two control groups. Conclusion Human TFPI gene transfection reduced intimal thickness in vein grafts.
ObjectiveTo construct eukaryotic expression vector of pEGFP-N3-TFPI-2, and to provide the base of studying the function of TFPI-2 gene. MethodsExtraction of total RNA from placental tissue was extracted at first, and then reverse transcriptase synthesis of cDNA was carried out. The cDNA fragment of TFPI-2 gene which was obtained by real time PCR (RT-PCR) was inserted into eukaryotic expression vector of pEGFP-N3. After double digestion with XhoⅠand KpnⅠ, the recombinant vector of pEGFP-N3-TFPI-2 was identified in 1% agarose gel electrophoresis and was tested by the sequence analysis. Then, the recombinant vector of pEGFP-N3-TFPI-2 (transfection group) and vector of pEGFP-N3 (blank control group) were transfected into Top10 competent cells with LipofectamineTM 2000, but no transfection-related treatment was performed in cells of untransfection group. Western blot method was used to test the expression of TFPI-2 protein in cells of 3 groups. ResultsThe purity of total RNA which were analysis by agarose gel electrophoresis and spectrophotometry were fit for PCR. After coding of TFPI-2 gene fragment and eukaryotic expression vector of pEGFP-N3, the recombinant plasmid of pEGFP-N3-TFPI-2 were got double digestion with XhoⅠand KpnⅠ, and was identified in 1% agarose gel electrophoresis, of which showing that there were 2 specific amplification of strips at 708 bp and 4 700 bp. Result of sequence analysis confirmed that the size of recombinant vector was consistent with the theoretical value. Results of Western blot showed that the expression of TFPI-2 protein in transfection group (0.657 3±0.032 5) was higher than those of blank control group (0.301 7±0.028 7) and untransfection group (0.314 3±0.026 6), P < 0.01. ConclusionsThe eukaryotic expression vector of pEGFP-N3-TFPI-2 has been constructed successfully, which laiding the foundation for the analysis about function of TFPI-2 gene.