蝎毒多肽干预CML K562细胞Hedgehog通路上游 活化因子的分子机制研究

摘要：目的 观察蝎毒多肽提取物（PESV）对K562细胞Hedgehog通路上游活化因子的影响并探讨分子调节机 制。方法 体外培养K562细胞，接种于24孔培养板，治疗组分别加入低、中及高浓度（10、20及40 mg/L）PESV 20 μL，阳性对照加入2 g/L的甲磺酸伊马替尼（GLEEVEC）20 μL（GLEEVEC组），阴性对照组加入生理盐水20 μL，共培 养48 h，应用Real-time PCR及Western blot法检测K562细胞BCR/ABL基因mRNA及融合蛋白P210bcr/abl表达的变化， 并检测Hedgehog通路上游活化因子Shh、Smo、Ptch mRNA及蛋白的表达。结果 与阴性对照组比较，PESV各剂量组 对K562细胞BCR/ABL融合基因mRNA及P210bcr/abl蛋白的表达均有不同程度的抑制作用（P＜0.05）；与阴性对照组比 较，中、高剂量组Shh、Smo、Ptch 基因mRNA 及蛋白在K562细胞中的表达水平均降低（P＜0.05）；与GLEEVEC 组比 较，中、高剂量组Shh、Smo、Ptch基因相对表达水平差异均无统计学意义，但低剂量组均升高（P＜0.05）。结论 PESV 可以抑制K562细胞BCR/ABL融合基因及P210bcr/abl蛋白的表达，并通过抑制Hedgehog通路上游活化因子的表达阻断 该通路的激活，进而抑制慢性粒细胞白血病的进展。

Study of PESV intervened upstream activating factors of Hedgehog pathway on CML K562 cells

Abstract: Objective To observe the effect of polypeptide extract from scorpion venom (PESV) on the upstream activating factors of Hedgehog pathway of K562 cells, and explore the molecular regulation mechanism. Methods K562 cells were cultured in vitro, and seeded in 24-well plates. Different concentrations of PESV (low dose 10 mg/L, middle dose 20 mg/L and high-dose 40 mg/L) were added to the experimental group, 20 μL was added to the control group, imatinib 2 g/L (20 μL) was given to the positive control group (GLEEVEC group) and saline 20 μL was given to the negative control group. Cells were cultured for 48 h. Real-time PCR and Western blot assay were used for detecting the expression of mRNA and fusion protein P210bcr/abl in BCR/ABL gene of K562 cells, and upstream activating factors and proteins Shh, Smo and Ptch of Hedgehog pathway. Results Compared with the negative control group, the expression of BCR / ABL gene mRNA and P210bcr/abl in K562 cells were inhibited in PESV groups (P＜0.05). Compared with the negative group, the expression levels of mRNA and protein in K562 cells were lower in the middle and high dose PESV groups (P＜0.05). There were no significant differences in the expression levels of Shh, Smo and Ptch between GLEEVEC group and the middle, high dose PESV groups， but which were increased in low dose PESV group (P＜0.05). Conclusion PESV can inhibit the expressions of BCRABL fusion gene and P210bcr/abl of K562 cells, which is related with the inhibiting the expression of the upstream activation factors of Hedgehog pathway, and thereby inhibiting the progression of chronic myeloid leukemia.