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目的探讨脑源性神经营养因子(BDNF)对小鼠结肠平滑肌细胞(SMC)钙离子浓度及α-平滑肌激动蛋白(α-SMA)的影响及其相关调节机制。方法 Western blotting法检测BDNF基因敲除(BDNF~(+/-))小鼠与正常野生型(BDNF~(+/+))小鼠α-SMA表达水平的差异。培养原代小鼠结肠SMC,免疫荧光法检测SMC中酪氨酸激酶B(Trk B)受体的表达。同时以BDNF、TrkB受体阻滞剂(K252a)干预SMC,Western blotting法检测α-SMA和Trk BPLC-Ca~(2+)信号通路蛋白表达水平的变化,钙离子成像法检测SMC内钙离子浓度的变化。结果与BDNF+/+小鼠相比,BDNF~(+/-)小鼠结肠α-SMA表达水平明显降低。SMC表达Trk B受体,在BDNF作用下,SMC中α-SMA、Trk B-PLC-Ca~(2+)信号通路蛋白表达量和细胞内钙离子浓度增加,且加入K252a可阻断以上变化。结论 BDNF可能通过Trk B-PLC-Ca~(2+)信号通路作用于SMC,影响细胞内钙离子浓度及α-SMA的表达水平,进而影响肠道动力。
Objective To investigate the effect of brain derived neurotrophic factor (BDNF) on calcium concentration and α-smooth muscle actin (α-SMA) in mouse colon smooth muscle cells (SMC) and its related regulatory mechanism. Methods Western blotting was used to detect the difference of α-SMA expression between BDNF knockout (BDNF +/-) mice and normal wild type (BDNF ~ (+ / +)) mice. Primary mouse colon SMC was cultured and the expression of tyrosine kinase B (Trk B) receptor in SMC was detected by immunofluorescence. At the same time, SMC was intervened by BDNF and TrkB receptor blocker (K252a). The protein expression of α-SMA and Trk BPLC-Ca 2+ signal pathway was detected by Western blotting. The calcium ion in SMC Changes in concentration. Results Compared with BDNF + / + mice, the expression of α-SMA in the colon of BDNF +/- mice decreased significantly. SMC expressed Trk B receptor, and the expression of α-SMA and Trk B-PLC-Ca 2+ signal pathways and intracellular calcium increased in SMC treated with BDNF, and K252a could block the above changes . Conclusion BDNF may affect SMC via Trk B-PLC-Ca 2+ signal pathway, and affect the intracellular calcium concentration and the expression of α-SMA, which may affect the intestinal motility.