脑外伤后T型钙通道对神经干细胞增殖的诱导作用
T type calciumchannel activated by brain injury induces neural stem cell proliferation

摘要：目的 探讨脑外伤后T型钙通道对神经干细胞的诱导作用及其相关机制。方法 构建成年大鼠脑损伤模型，分为手术组和手术+米贝地尔组(米贝地尔组)，并另设正常对照组、假手术组。分离侧脑室壁的脑室下层(subventricular zone, SVZ)细胞，用神经干细胞悬浮培养法进行培养并鉴定其干细胞性质；在神经干细胞培养基中加入不同剂量的米贝地尔，通过计数神经干细胞球、噻唑蓝(MTT)法分析米贝地尔对神经干细胞增殖抑制作用，计算米贝地尔对神经干细胞的半抑制浓度(IC50)，Western blot法检测脑组织中Cav3.2以及神经干细胞中Cav3.2、Cyclin A和caspase-3的蛋白表达。结果 体内实验表明，手术组中神经干细胞的增殖能力显著高于正常对照组和假手术组，应用米贝地尔腹腔注射抑制脑组织中T型钙通道后，神经干细胞的增殖明显降低。体外细胞实验表明，米贝地尔能够明显抑制神经干细胞球的形成，MTT法显示米贝地尔浓度大于5μmol/L时，可显著抑制细胞增殖，5、10、20μmol/L组的吸光度(A值)与对照组比较，差异具有统计学意义(P<0.05)。IC50值为8.93μmol/L。Western blot检测结果显示，米贝地尔组的Cav3.2蛋白和Cyclin A蛋白的表达显著降低，caspase-3蛋白的表达显著升高。 结论 脑外伤后可激活脑组织中T型钙通道，上调神经干细胞的增殖能力。
ABSTRACT: Objective To investigate the mechanism and induction of T type calcium channel on neural stem cells after brain injury. Methods Adult mice brain injury model was established and divided into control, sham operation, surgery, and surgery+mebefradil groups. Neural stem cells were separated from the subventricular zone (SVZ) and identified. Moreover, we analyzed the results using MTT and neural stem cell sphere counting after adding different doses of mibefradil in culture medium, respectively. Then we calculated the half maximal inhibitory concentration (IC50) of mibefradil on neural stem cells. Finally, we analyzed the expression of Cav3.2 in SVZ and the protein expressions of Cav3.2, Cyclin A and caspase-3 in neural stem cells by Western blot. Results In vivo, neural stem cell proliferation was increased in surgery group compared with that in control and sham-operation groups. The proliferation of neural stem cells in surgery+mibefradil groups was significantly decreased compared with that in surgery group after mibefradil-induced inhibition of T type calcium channel protein. In vitro, the formation of neural stem cell sphere was significantly inhibited after adding mibefradil. The cell growth ratio was significantly decreased when the concentration of mibefradil was above 5μmol/L. A values in 5μmol/L, 10μmol/L and 20μmol/L groups were significantly lower than those in control group (P<0.05). IC50 was 8.93μmol/L. The protein expressions of Cyclin A and Cav3.2 were inhibited while that of caspase-3 was increased after mibefradil treatment. Conclusion Neural stem cell proliferation was enhanced by activating T type calcium channel after brain injury