TPA基因体外转染对血管平滑肌细胞增殖和迁移的影响

The Effects of Transfected tPA Gene on Vascular Smooth Muscle Cells Proliferation and Migration in Vitro

目的探讨组织型纤溶酶原激活因子(tPA)基因体外转染对鼠血管平滑肌细胞(VSMC)黏附、增殖和迁移的影响,为tPA基因在预防移植血管再狭窄中的应用奠定理论基础。方法通过阳离子质体介导,将tPA基因转染体外培养鼠血管平滑肌细胞,RT-PCR、Western-Blot及ELSIA检测转基因平滑肌细胞基因表达,底物发色法检测转基因VSMC纤溶活性变化,MTT、细胞黏附、细胞迁移实验等方法,检测体外转染tPA基因对血管平滑肌细胞黏附、增殖和迁移的影响。结果RT-PCR和Western-Blot检测到转基因血管平滑肌细胞tPA基因转录及蛋白表达,转基因组、载体组和空白对照组tPA含量分别为(518.38±125.32)ng/106cells/24h、(12.53±4.27)ng/106cells/24h和(13.21±5.02)ng/106cells/24h,转基因组明显高于各对照组;转基因组tPA活性为(26.6±7.02)IU/106cells/24h,而载体组和空白对照组没有检测出tPA活性。转基因平滑肌细胞和对照组相比,其细胞的增殖、黏附和迁移能力没有明显变化。结论转tPA基因血管平滑肌细胞纤溶活性增高,但对血管平滑肌细胞的黏附、增殖和迁移能力没有影响。

Objective To investigate the effects of transfected tPA gene on vascular smooth muscle cell proliferation and migration in vitro and to provide a basis for further study on preventing vein graft restenosis. Methods Recombinant plasmid pcDNA 3. 1-Myc-His B（-）/tPA was transfected into mice vascular smooth muscle cells mediated by lipofectamine2000. The transcription and expres- sion of tPA gene were investigated by RT-PCR and Western-Bloting respectively. The levels of human tPA antigen in culture media was measured suing enzymelinked inmmnosorbent assay. The tPA activity was measured by chromogenic substrate easy. The cell attachment assay, cell proliferation assay and cell migration assay were carried out to detect the cell attachment, proliferation and migration re- spectively. Results RTPCR and Western-Blot confirmed the expression of tPAmRNA and the presence of tPA protein in the transfected tPA gene group, and not in the control groups. The tPA activity detected in the transfected tPA gene group was （26. 6±7. 02） IU/106cells/24h, and could not he detected in the control groups. The tPA contains in the transfected tPA gene group, vector group and blank group were （518. 38±125. 32）ng/106cells/24h, （12. 53±4.27）ng/lO6cells/24h and（13. 21± 5. 02）ng/106cells/24h respectively. The tPA contained was significantly higher than that in the control group. There were no difference observed in cell attachment, proliferation and migration among the three groups. Conclusion The expression of high levels of tissue plasminogen activator does not specifically affect VSMC attachment, proliferation and migration.