OHAP-1对大鼠C6胶质瘤细胞bcl-2/bax基因表达和氧化应激的影响

目的：探讨冲绳巴布蛇毒蛋白-1（OHAP-1）对大鼠C6胶质瘤细胞的增殖抑制作用及其机制。方法：MTT法检测2.5、5.0和10.0 mg?L^-1 OHAP-1对C6胶质瘤细胞的增殖抑制作用；RT-PCR法检测OHAP-1对C6胶质瘤细胞bcl-2和bax基因表达的影响；分光光度法检测胶质瘤细胞中丙二醛 (MDA）的含量和超氧化物歧化酶(SOD）的活性。结果：2.5、5.0和10.0 mg?L^-1OHAP-1对C6胶质瘤细胞生长抑制率（49.77％、67.65％和76.42％）高于对照组（P<0.001）。随着OHAP-1剂量的增加，bcl-2 mRNA的表达逐渐下降，bax mRNA的表达逐渐升高，bcl-2/bax亦逐渐减小。2.5、5.0和10.0 mg?L^-1组MDA含量高于对照组(P<0.01)，且SOD活性低于对照组(P<0.01)；5.0和10.0 mg?L^-1组MDA含量高于2.5 mg?L-1 组和对照组(P<0.001)，10.0 mg?L^-1组SOD活性低于2.5 mg?L^-1 组和对照组(P<0.001)；10.0 mg?L^-1组SOD活性低于5.0、2.5 mg?L^-1组和对照组(P<0.01)。结论：OHAP-1通过氧化应激使bcl-2/bax mRNA表达降低可能是抑制C6胶质瘤细胞增殖的重要原因之一。

Effects of OHAP-1 on expression of bcl-2/bax mRNA and oxidative stress in rat C6 glioma cells

Objective To observe the effects of Okinawa Habu apoxin protein-1 (OHAP-1) on the proliferation inhibition of rat C6 glioma cells and its mechanisms. Methods MTT colorimetric analysis was used to measure the inhibitory effect of OHAP-1 with different doses(2.5,5.0,and 10.0 mg·L-1) on C6 glioma cells .RT-PCR was used to evaluate the mRNA expressions of bcl-2 and bax genes.The activity of superoxide dismutase (SOD) and the level of maleicdialdehyde (MDA) in the C6 glioma cells were also examined. Results The proliferation of C6 glioma cells was significantly inhibited by different doses of OHAP-1(2.5,5.0,and 10.0 mg·L-1).The inhibitory rate were 49.77%,67.65%,and 76.42%,respectively.The inhibitory rate in 2.5,5.0, and 10.0 mg·L-1 groups were higher than that in control group(P<0.001).With the increasing of the dose of OHAP-1,bax mRNA expression increased,and bcl-2 mRNA decreased markedly,the ratio of bcl-2/bax mRNA also decreased.In 2.5,5.0, and 10.0 mg·L-1 OHAP-1 groups,MDA concentrations increased significantly compared with control group(P<0.01),and SOD decreased (P<0.01); In 5.0 and 10.0 mg·L-1 OHAP-1 groups,MDA concentrations were higher than those in 2.5 mg·L-1 OHAP-1 and control groups (P<0.001);in 10.0 mg·L-1 OHAP-1 group, SOD activity was lower than those in 2.5 mg·L-1 and control groups (P<0.001); in 10.0 mg·L-1 OHAP-1 group, SOD activity was lower than those in 5.0 ,2.5 mg·L-1OHAP-1 and control groups (P<0.001).Conclusion The proliferation inhibition of C6 glioma cell induced by OHAP-1 may be related to the expression of bcl-2/bax mRNA decreasing resulted from oxidative stress.