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目的将含入凝血酶调节蛋白(hTM)基因的真核表达载体质粒 pcDNA3.1/hTM 转染体外培养的脐静脉内皮细胞(HUVECs),观察外源凝血酶调节蛋白的表达及其所致的 HUVECs 抗凝功能的改变。方法由阳离子脂质体介导将 pcDNA3.1/hTM 质粒转入内皮细胞中,半定量 RT-PCR 测定各组 hTM mRNA 的表达强度;免疫组化法检测 hTM 在内皮细胞膜上的表达;测定各组内皮细胞对蛋白 C(PC)的激活;半自动凝血仪测定内皮细胞-PC 反应液对正常血浆活化部分凝血活酶时间(APTT)和凝血酶原时间(PT)的影响。结果 HUVECs pcDNA3.1/hTM 质粒转染率约为10%,TM mRNA 和 TM 蛋白的表达强度都有明显提高。重组质粒转染组、空载质粒组及未转染组 PC 反应液中活化蛋白 C(acti-vated protein C,APC)的含量分别是(2.80±0.43)μg/ml、(0.75±0.08)μg/ml、(0.85±0.11)μg/ml。APTT 值在重组质粒转染组、空载质粒组、未转染组、未激活 PC 组和正常对照组中分别为(51.68±2.73)s、(38.38±2.44)s、(39.65±2.39)s、(33.93±1.73)s 和(34.60±1.86)s。PT 值在各组中分别为(21.89±1.66)s、(20.56±1.74)s、(20.42±2.04)s、(19.57±1.36)s 和(20.16±1.35)s。结论pcDNA3.1/hTM 质粒能被导入内皮细胞中,表达的 TM 分子具有生物学活性,能明显提高对 PC 的激活。激活的 PC 能明显抑制血浆内源性凝血途径使 APTT 延长,也使外源性凝血途径受到轻度抑制。
OBJECTIVE: To transfect hUVECs cultured in vitro into eukaryotic expression vector pcDNA3.1 / hTM containing thrombin regulatory protein (hTM) gene to observe the expression of exogenous thrombin regulatory protein Changes of anticoagulant function in HUVECs. Methods The pcDNA3.1 / hTM plasmid was transfected into endothelial cells by cationic liposome. The expression of hTM mRNA in each group was detected by semi-quantitative RT-PCR. The expression of hTM in endothelial cell membrane was detected by immunohistochemistry. Group C was activated by proteinase C (PC). The semi-automatic coagulation analyzer was used to determine the effect of endothelial cell-PC reaction on APTT and PT. Results The transfection efficiency of HUVECs pcDNA3.1 / hTM plasmid was about 10%, and the expression intensity of TM mRNA and TM protein were significantly increased. The contents of acti-vated protein C (APC) in recombinant plasmid transfected group, empty plasmid group and untransfected group were (2.80 ± 0.43) μg / ml, (0.75 ± 0.08) μg / ml, (0.85 ± 0.1) μg / ml. APTT values were (51.68 ± 2.73) s, (38.38 ± 2.44) s, (39.65 ± 2.39) s in the recombinant plasmid transfected group, empty plasmid group, untransfected group, inactive PC group and normal control group , (33.93 ± 1.73) s and (34.60 ± 1.86) s, respectively. The PT values were (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 each group. Conclusion The pcDNA3.1 / hTM plasmid can be introduced into endothelial cells. The expressed TM molecules have biological activity and can significantly enhance the activation of PC. Activated PC can significantly inhibit the plasma endogenous coagulation pathway to APTT prolongation, but also the extrinsic coagulation pathway was mildly inhibited.