论文部分内容阅读
目的:观察非肌肉肌球蛋白重链ⅡB(nonmuscle myosin heavy chainⅡB,NMHC-ⅡB)对人脐静脉血管内皮细胞(Human umbilical vein endothelial cells,HUVECs)中肿瘤坏死因子受体1(TNF receptor 1,TN-FR1)转位的影响。方法:将成功构建的重组pEGFP-N1/TNFR1载体通过阳离子脂质体Lipofectin介导的方法转染入HUVECs,筛选获得稳定克隆株。设计并合成NMHC-ⅡB的小分子干扰RNA(small interference RNA,si RNA),用脂质体LipofectamineTM2000转染进入该稳定克隆株,差速离心加蔗糖密度梯度离心收获细胞质膜,Western印迹法观察TNFα诱导前和诱导后细胞质膜上TNFR1蛋白含量的变化。结果:与空白对照组比较,转染NMHC-ⅡB si RNA组进入HUVECs,可以明显抑制细胞内NMHC-ⅡB mRNA的表达[(0.670 4±0.024 2)∶(0.454 0±0.041 1),P<0.01],也可以明显减少TNFα诱导前[(0.430 6±0.028 9)∶(0.324 8±0.016 7),P<0.01]和诱导后[(0.660 9±0.026 5)∶(0.492 2±0.021 7),P<0.01]细胞质膜TNFR 1的含量。在转染NMHC-ⅡB si RNA的细胞,TNFα的诱导仍然可以提高细胞质膜TNFR 1的含量,诱导前、后比较[(0.324 8±0.016 7)∶(0.492 2±0.021 7),P<0.01]。结论:NMHC-ⅡB可能在TNFR1从反式高尔基体到细胞质膜的转位或运输中起正向作用,但是可能并不是惟一的或决定性的因素。
OBJECTIVE: To observe the effect of non-muscle myosin heavy chainⅡB (NMHC-ⅡB) on the expression of tumor necrosis factor receptor 1 (TNF) 1 in human umbilical vein endothelial cells (HUVECs) -FR1) translocation. Methods: The successfully constructed recombinant pEGFP-N1 / TNFR1 vector was transfected into HUVECs by Lipofectin-mediated cationic liposomes, and the stable clones were screened. The small interfering RNA (siRNA) of NMHC-ⅡB was designed and synthesized. The stable cloned strain was transfected with lipofectamineTM2000, and the cell plasma membrane was harvested by differential centrifugation and sucrose density gradient centrifugation. The expression of TNFα Changes of TNFR1 protein content on plasma membrane before and after induction. Results: Compared with the blank control group, the transfection of NMHC-ⅡB si RNA into HUVECs could significantly inhibit the expression of NMHC-ⅡB mRNA in the cells [(0.670 4 ± 0.024 2) :( 0.454 ± 0.041 1), P <0.01 ], But also significantly reduced the levels of [α (0.430 6 ± 0.028 9), (0.324 8 ± 0.016 7), P <0.01] and [0.660 9 ± 0.026 5] :( 0.492 2 ± 0.021 7) P <0.01] content of plasma membrane TNFR 1. In the transfected cells of NMHC-ⅡB si RNA, the induction of TNFα could still improve the content of TNFR 1 in the plasma membrane, compared with before and after induction [(0.324 8 ± 0.016 7) :( 0.492 2 ± 0.021 7, P <0.01] . CONCLUSIONS: NMHC-IIB may play a positive role in the translocation or transport of TNFRl from trans-Golgi to the plasma membrane, but may not be the only or decisive factor.