Fibrocystin对常染色体隐性遗传多囊肾病囊肿细胞增殖的影响

目的 研究fibrocystin对表皮生长因子（EGF）诱导的细胞过度增殖的影响并探讨其机制。 方法 通过RNA干扰技术建立PKHD1-沉默细胞株，分别检测EGF诱导的细胞增殖率、ERK1/2磷酸化水平和细胞内Ca2+浓度。通过降低HEK 293细胞和增高PKHD1-沉默细胞内Ca2+浓度，研究Ca2+在其中的可能作用。 结果 与HEK 293细胞比较，PKHD1-沉默 HEK 293细胞对EGF作用高度敏感，增殖率为231.5%，显著高于HEK 293细胞的152.8%(P ＜ 0.01)，细胞内Ca2+浓度也显著降低（P ＜ 0.01）。同时PKHD1沉默可显著增高EGF诱导的ERK1/2信号通路活化。运用Ca2+通道阻断剂维拉帕米降低HEK 293细胞内Ca2+浓度后，与未经维拉帕米处理的细胞比较，细胞增殖率为202.2%，显著增高。维拉帕米处理的HEK 293细胞的ERK1/2磷酸化水平明显高于未处理的细胞。而运用Ca2+通道激活剂Bay K8644增高PKHD1-沉默 HEK 293细胞内Ca2+浓度后，显著降低了EGF诱导的PKHD1-沉默HEK 293细胞的增殖率（135.5%）， Bay K8644处理的PKHD1-沉默HEK 293细胞的ERK1/2磷酸化水平明显低于未处理的细胞。 结论 PKHD1基因的突变导致fibrocystin功能缺失，引起细胞内Ca2+浓度降低，导致EGF诱导的ERK1/2信号通路过度活化，可能是常染色体隐性遗传多囊肾疾病（ARPKD）患者肾囊肿衬里上皮细胞异常增殖导致ARPKD肾囊肿发生的机制之一。

Effect of fibrocystin on the proliferation of kidney cyst epithelial cells in autosomal recessive polycystic kidney disease

Objective To explore whether the inhibited expression of fibrocystin by RNA interference can increase epidermal growth factor (EGF)-induced cell proliferation and its possible mechanism . Methods A stable PKHD1-silenced HEK 293 cell line was established . Cell proliferation rate, intracellular Ca2+ concentration and extracellular signal-reguhted kinase 1/2(ERK1/2) activity were assessed after treatment with EGF, verapamil and Bay K8644 . Results The proliferation rate of PKHD1-silenced HEK-293 cells was found to be significantly higher after EGF stimulation compared to the control HEK 293 cell (231 .5% vs 152 .8%, P＜0 .01) . PKHD1-silencing lowered the intracellular Ca2+ concentration and caused EGF-induced ERK1/2 overactivation in the cells(P＜0 .01 ) . When cells were treated with verapamil for 4 hours to lower the intracellular Ca2+ concentration, the cell proliferation rate was significantly increased after 20 ng EGF for 24 hours . The verapamil treatment increased the level of activated ERK1/2 in EGF-treated cells . An increase of intracellular Ca2 + in PKHD1-silenced ceils repressed the EGF-dependent ERK1/2 activation and the hyperproliferative response to EGF stimulation . Conclusions Inhibition of fibrocystin can cause EGF-induced excessive proliferation through decreasing intracellular Ca2+ resulting in EGF-induced ERK1/2 activation . The loss of fibrocystin may lead to abnormal proliferation in kidney epithelial cells and cyst formation in ARPKD through modulation of intracellular Ca2+ concentration .