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目的研究静水压作用下人膀胱平滑肌细胞(human bladder smooth muscle cells,hb-SMCs)内游离钙离子浓度(intracellular free calcium concentration,[Ca2+]i)的变化及香草型瞬时受体电位(transient receptor potential vanilloid,TRPV)基因的表达情况,初步探讨机械应力在介导hb-SMCs增殖中的分子机制。方法取第6～7代细胞进行实验,采用新型Ca2+荧光染色剂Fluo-3/AM负载hb-SMCs,应用倒置激光扫描共聚焦显微镜分别测定静水压加压0 cm H2O(1 cm H2O=0.098 kPa)(A组)、200 cm H2O作用30 min(B组)及撤销200 cm H2O静水压(C组)后hb-SMCs内[Ca2+]i。采用RT-PCR技术测定200 cm H2O静水压作用0、2、6、12、24 h时TRPV1、TRPV2及TRPV4基因mRNA的表达量。结果 A、B、C组hb-SMCs内[Ca2+]i分别为(100.808±1.724)、(122.008±1.575)、(99.918±0.887)U,B组[Ca2+]i明显高于A、C组(P<0.001),C组撤销200 cm H2O静水压力后[Ca2+]i下降至A组基线水平(t=0.919,P=0.394)。RT-PCR检测示,200 cm H2O静水压加压0 h组、2 h组、6 h组、12 h组及24 h组TRPV1、TRPV2及TRPV4在hb-SMCs中均有表达,随时间推移表达无明显变化;各组间TRPV1、TRPV2、TRPV4 mRNA表达量比较差异均无统计学意义(P>0.05)。结论高水平静水压作用下hb-SMCs内[Ca2+]i明显升高。作为可能表达为机械敏感性阳离子通道的TRPV1、TRPV2及TRPV4基因在hb-SMCs中均有表达,机械应力可能通过调节其开放量而非上调其表达量使hb-SMCs内[Ca2+]i升高。
Objective To study the changes of intracellular free calcium concentration ([Ca2 +] i) in human bladder smooth muscle cells (hb-SMCs) under hydrostatic pressure and the changes of vanilloid type transient receptor potential potential vanilloid (TRPV) gene expression and to investigate the molecular mechanism of mechanical stress in mediating the proliferation of hb-SMCs. Methods Sixth to seventh passage cells were harvested, and hb-SMCs were loaded with Fluo-3 / AM, a new fluorescent dye Ca2 +. Hydrostatic pressure 0 cm H2O (1 cm H2O = 0.098 (group A), [Ca2 +] i in hb-SMCs after 200 cm H2O for 30 min (group B) and withdrawal of 200 cm H2O hydrostatic pressure (group C). The expression of TRPV1, TRPV2 and TRPV4 mRNA was detected by RT-PCR at 0, 2, 6, 12 and 24 h after hydrostatic pressure of 200 cm H2O. Results The [Ca2 +] i in hb-SMCs of groups A, B and C were (100.808 ± 1.724), (122.008 ± 1.575) and (99.918 ± 0.887) U, respectively. (P <0.001). After withdrawal of hydrostatic pressure of 200 cm H2O in group C, [Ca2 +] i decreased to baseline in group A (t = 0.919, P = 0.394). RT-PCR results showed that TRPV1, TRPV2 and TRPV4 were expressed in hb-SMCs at 200 h H2O hydrostatic pressure for 0 h, 2 h, 6 h, 12 h and 24 h, respectively, There was no significant difference in the expression of TRPV1, TRPV2 and TRPV4 between the groups (P> 0.05). Conclusions [Ca2 +] i in hb-SMCs significantly increased under high hydrostatic pressure. The expression of TRPV1, TRPV2 and TRPV4 genes in hb-SMCs, which may be expressed as mechanosensitive cation channels, may increase [Ca2 +] i in hb-SMCs by regulating its open volume instead of increasing its expression level .