紫草素诱导自噬抑制胰腺癌细胞增殖的实验研究

Shikonin-induced Autophagy and Suppression of Pancreatic Cancer Cell Proliferation

目的:探讨紫草素是否通过诱导自噬来抑制胰腺癌细胞增殖。方法:采用噻唑蓝比色法(MTT法)研究紫草素对胰腺癌细胞Bxpc-3的增殖抑制作用。利用透射电镜观察紫草素作用胰腺癌细胞Bxpc-3后的细胞内具有双层膜结构的自噬体结构。利用荧光定量PCR(Real-time PCR)(荧光定量聚合酶链反应)和Western-blot法检测紫草素作用后胰腺癌Bxpc-3细胞自噬标记蛋白Beclin-1和LC3B的表达变化。结果:紫草素对胰腺癌细胞Bxpc-3具有明显的生长抑制作用,6μg/mL紫草素作用Bxpc-3细胞24h,其抑制率可达50%左右,且具有时间剂量依赖效应。透射电镜结果可见紫草素诱导后细胞内可见典型的双层膜结构的自噬体。Real-time PCR结果显示紫草素诱导后Bxpc-3细胞自噬相关标志物Beclin-1和LC3B表达增高。Western-blot结果显示与对照组相比,诱导后Beclin-1表达显著增高,LC3-Ⅰ表达变化不明显,但是LC3-Ⅱ/LC3-Ⅰ表达明显增高。结论:紫草素通过上调Beclin-1和LC3-Ⅱ/LC3-Ⅰ的表达来增加胰腺癌细胞Bxpc-3的自噬活性,从而抑制胰腺癌细胞的增殖作用。

Objective：To investigate whether Shikonin inhibits the proliferation of pancreatic cancer cells throught he autophagy pathway.Methods：After Shikonin effect,the proliferation of pancreatic cancer cell Bxpc-3 was meas ured by the MTT method,the morphologic changes of autophagy was observed with transmission electron micros cope.After treatment with Shikonin for 24h,the expression of autophagy related genes,such as Beclin-1 and LC3B,b oth in mRNA and protein level,were measured by using Real-time PCR and Western-blot methods.Results：Shik onin significantly inhibited the proliferation of Bxpc-3 cells in a dose and time dependent manner.When Bxp c-3 cells were treated with 6μg/ml Shikonin for 24h,the inhibition rate amounted to about 50%;the typicald ouble membrane structure of autophagosome was found under transmission electron microscope;the expression ofB eclin-1 and LC3B were up-regulated compared with negative control group.Western-blot analysis furthers howed that the Beclin-1 and LC3B（LC3-Ⅱ/LC3-Ⅰratio） were also significantly up-regulated after treated withS hikonin for 24h.Conclusions：Shikonin can inhibit the proliferation of pancreatic cancer cells with an autophag y activity in pancreatic cancer cell Bxpc-3 by increasing the expression of Beclin-1 and LC3-II/LC3-I ratio.