[钾通道对人支气管平滑肌细胞增殖、凋亡及相关基因表达的影响]
[Effect of potassium channel on the proliferation, apoptosis and related-gene expression in human bronchial smooth muscle cells].
作者信息
Zhao Li-Min, Xu Yong-Jian, Zhang Zhen-Xiang, Ni Wang, Chen Shi-Xin
机构信息
Department of Respiratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
出版信息
Zhonghua Jie He He Hu Xi Za Zhi. 2004 Dec;27(12):841-6.
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.
目的
研究电压依赖性钾通道（K(V)）、钙激活钾通道（K(Ca)）和ATP敏感性钾通道（K(ATP)）三种钾通道对人支气管平滑肌细胞（HBSMCs）增殖、凋亡及相关基因表达的影响。
方法
从5例因肺癌行肺部分切除术的患者获取正常人支气管组织。将培养的HBSMCs分为四组：（1）对照组；（2）4-氨基吡啶（4-AP）组：含4 mmol/L 4-AP；（3）四乙铵（TEA）组：含1 mmol/L TEA；（4）格列本脲（Glib）组：含0.1 mmol/L Glib。采用流式细胞术观察细胞周期；用荧光分光光度计通过荧光定量法检测HBSMCs中的Ca(2+)浓度。分别采用MTT法和TUNEL法检测细胞增殖和凋亡情况。采用免疫细胞化学染色检测三种钾通道阻滞剂对HBSMCs中增殖细胞核抗原（PCNA）及凋亡相关基因Fas、FasL表达的影响。
结果
（1）K(V)阻滞剂4-AP可显著增加光密度值[4-AP组的A值为（0.67±0.14），对照组为（0.30±0.08），P<0.01]及增殖细胞核抗原的表达[4-AP组PCNA阳性率为（89±7）%，对照组为（23±5）%，P<0.01]。4-AP可显著增加培养的HBSMCs中的Ca(2+)浓度[（255±17）nmol/L]，与对照组[（98±7）nmol/L，P<0.01]相比，且通过流式细胞术检测培养的HBSMCs中S+G(2)M期细胞数量[（28.8±2.4）%]，与对照组[（12.6±4.8）%，P<0.01]相比。TEA组和Glib组的A值均为（0.30±0.07）。两组的PCNA阳性率分别为（21±5）%、（20±4）%。两组的Ca(2+)浓度分别为（97±7）nmol/L、（99±6）nmol/L。且两组S+G(2)M期HBSMCs数量分别为（12.8±4.4）%、（11.0±4.4）%。但两组与对照组之间均无显著差异（均P>0.05）。（2）对照组的TUNEL水平及Fas、FasL表达分别为（4.36±0.66）%、（2.92±0.25）%、（4.0±0.6）%。4-AP可显著降低凋亡率及Fas、FasL的表达[分别为（0.84±0.13）%、（0.92±0.16）%、（1.4±0.6）%]（与对照组相比，均P<0.01）。TEA组和Glib组的TUNEL水平分别为（4.47±0.93）%、（4.33±0.77）%。两组的Fas、FasL表达分别为（2.87±0.23）%、（4.2±0.8）%、（2.91±0.26）%和（4.2±0.9）%。两组与对照组之间均无显著差异（均P>0.05）。
结论
抑制K(V)活性可增加HBSMCs的增殖及细胞内[Ca(2+)]i，并降低其凋亡，但抑制K(Ca)和K(ATP)则无此作用。
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