钾通道对人支气管平滑肌细胞增殖与凋亡及其相关基因表达的影响

目的　探讨电压依赖性延迟整流钾通道 (KV)、钙激活钾通道 (KCa)和ATP敏感性钾通道 (KATP)对人支气管平滑肌细胞 (HBSMCs)增殖与凋亡及其相关基因表达的影响。方法　支气管组织取材于 5例肺癌切除术患者癌旁正常的肺组织 ,将培养的HBSMCs分为 4组 :(1)对照组 (用不含药物的无血清培养基处理 ) ;(2 ) 4 氨基吡啶 (4 AP)组 (含 4mmol/L的 4 AP) ;(3)四乙铵 (TEA)组 (含 1mmol/L的TEA) ;(4)格列本脲 (Glib)组 (含 0 1mmol/L的Glib)。采用流式细胞术观察细胞的周期 ;应用荧光光度法检测细胞内钙 ;四甲基偶氮唑盐 (MTT)微量比色分析法检测细胞的增殖 ;原位末端标记法 (TUNEL)检测细胞的凋亡 ;免疫细胞化学技术检测增殖细胞核抗原 (PCNA)及凋亡相关基因Fas和FasL的表达 ,以观察 3种钾通道阻断剂对培养的HBSMCs增殖及凋亡的影响。结果 (1)对照组HBSMCs吸光度 (A)值为 0 30± 0 0 8,4 AP组为 0 6 7± 0 14 ,两组比较差异有统计学意义 (P <0 0 1) ;对照组PCNA的阳性率为 (2 3± 5 ) % ,4 AP组为 (89± 7) % ,两组比较差异有统计学意义 (P <0 0 1) ;对照组细胞内Ca2 浓度为 (98± 7)nmol/L ,4 AP组为 (2 5 5± 17)nmol/L ,两组比较差异有统计学意义 (P <0 0 1) ;对照组S G2 M期细胞数为 (12 6±

Effect of potassium channel on the proliferation,apoptosis and related-gene expression in human bronchial smooth muscle cells

OBJECTIVE: To investigate the effects of three potassium channels, voltage-dependent K(+) channel (K(V)), calcium-activated K(+) channel (K(Ca)) and ATP-sensitive K(+) channel (K(ATP)), on the proliferation, apoptosis and related-gene expression in human bronchial smooth muscle cells (HBSMCs). METHODS: Normal human bronchial tissues were obtained from 5 patients undergoing lung partial resection for carcinomas. The cultured HBSMCs were divided into four groups: (1) control group; (2) 4-aminopyridine (4-AP) group: containing 4 mmol/L 4-AP; (3) TEA group: containing 1 mmol/L TEA; (4) Glib group: containing 0.1 mmol/L Glib. The cell cycles were observed by flow-cytometry; Ca(2+) concentration in HBSMCs were investigated by fluorescent quantification using fluorospectrophotometer. The proliferation and apoptosis were detected by MTT methods and TUNEL, respectively. Immunocytochemical staining was used to detect the effects of three potassium channel blockers on the expressions of PCNA and apoptosis related-gene Fas, FasL of HBSMCs. RESULTS: (1) The K(V) blocker 4-AP was shown to significantly increase the optical density value [the value of A of the 4-AP group was (0.67 +/- 0.14), compared to the control group (0.30 +/- 0.08), P< 0.01] and the expression of proliferating cell nucleus antigen [the positive percentage of PCNA of the 4-AP group was (89 +/- 7)%, compared to the control group (23 +/- 5)]%, P < 0.01). 4-AP significantly increased the Ca(2+) concentration [(255 +/- 17) nmol/L] in cultured HBSMCs, compared to the control group [(98 +/- 7) nmol/L, P < 0.01] and the numbers [(28.8 +/- 2.4)%] of S + G(2)M HBSMCs by flow-cytometry in cultured HBSMCs, compared to the control group [(12.6 +/- 4.8)%, P < 0.01]. The value of A of TEA group and Glib group were all (0.30 +/- 0.07). The positive percentage of PCNA of the two groups were (21 +/- 5)%, (20 +/- 4)%, respectively. Ca(2+) concentration of the two groups were (97 +/- 7) nmol/L, (99 +/- 6) nmol/L, respectively. And the numbers of S + G(2)M HBSMCs were (12.8 +/- 4.4)%, (11.0 +/- 4.4)%, respectively. But no significant differences were found between the two groups and the control group (all P > 0.05). (2) The level of TUNEL and the expressions of Fas and FasL of the control were (4.36 +/- 0.66)%, (2.92 +/- 0.25)%, (4.0 +/- 0.6)%, respectively. 4-AP significantly decrease the apoptosis rate and the expressions of Fas, FasL [(0.84 +/- 0.13)%, (0.92 +/- 0.16)]%, (1.4 +/- 0.6)%, respectively) (compared to control, all P < 0.01). The level of TUNEL of TEA group and Glib group were (4.47 +/- 0.93)%, (4.33 +/- 0.77)%, respectively. And the expressions of Fas, FasL of the two groups were (2.87 +/- 0.23)%, (4.2 +/- 0.8)%, (2.91 +/- 0.26)% and (4.2 +/- 0.9)%, respectively. There were no significant differences between the two groups and the control group (all P > 0.05). CONCLUSION: Inhibition of K(V) activity can increase the proliferation and intracellular [Ca(2+)]i, and decrease the apoptosis of HBSMCs, but inhibition of K(Ca) and K(ATP) showed no effects.