Influence of RNA interference-induced silencing Smad4 gene on proliferation and migration of ECV304 cell line / 肿瘤

Objective: To investigate the effect of silencing Smad4 gene expression by using small interfering RNA (siRNA) on proliferation and migration of ECV304 cell line and explore the role of Smad4 in angiogenesis. Methods: Two siRNA sequences targeting Smad4 gene including siRNA1 and siRNA2 were designed and then synthetized. The two sequences of siRNA were transfected into ECV304 cells via lipofectamine mediation. The alteration of Smad4 mRNA and protein expression was examined by real-time RT-PCR and Western blot, respectively. The proliferation capacity of ECV304 cells was measured by cell cycle analysis and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) flow cytometry. The monostratal wound healing test was used to determine the migration capacity of ECV304 cells. Results: siRNA2 was successfully screened out of the two sequences. Transfection of siRNA2 significantly knocked down the Smad4 mRNA and protein expressions. The proliferation and migration capacity of ECV304 cells are significantly enhanced after transfection with siRNA2. Conclusions: Highly effective and specific siRNA targeting Smad4 gene can be successfully screened by RNA interference technique. Selective silencing of Smad 4 gene greatly promotes the proliferation and migration of ECV304 cells.

Entery of calcium into ECV304 endothelial cell via CaCa~(2+) activated non-selective cation channel / 中国药理学通报

AIM To investigate the pathways of Ca 2+ entry into ECV304 endothelial cell and the effect of angiotensin Ⅱ(AⅡ) on calcium activated non setective cation channel(CAN). METHODS The cell attachment and whole cell configurations of patch clamp technique were used to record channel activity. RESULTS (1) The single channel conductance is ? o=(12 90?2 11) pS( n =4) for Ca 2+ passing through CAN of ECV304 cell in condition of pipette solution without K + and Na + but composed 120 mmol?L -1 CaCl 2. The channel current amplitude and open time can be enhanced by 1?10 -7 mol?L -1 AⅡ. The enhanced conductance in CAN is ? 1=(22 18?2 29) pS( n =4). The results of whole cell recording are identified with single channel recording. (2) The whole cell configuration was carried out for recording voltage dependent Ca 2+ channel in ECV304 cell. The peak current amplitude was (29 32?3 56) pA( n =4). This current was inhibited to (6 00?3 94) pA( n =4) by nifedipine and activated by BayK8644. CONCLUSIONS (1)Ca 2+ enters ECV304 cell via Ca 2+ activated non selective cation channel and voltage dependent L type calcium channel. (2) AⅡ can significantly enhance the calcium entry via CAN in ECV304 cell.