盐酸高乌甲素通过pRb通路介导人宫颈癌Hela细胞周期阻滞

Lappaconitine Hydrochloride Mediates the Cell Cycle Arrest of Human Cervical Cancer Hela Cell Through pRb Pathway

关键词:  盐酸高乌甲素  Hela细胞  细胞增殖  细胞周期  细胞凋亡

Key words:  lappaconitine hydrochloride  Hela cells  cell proliferation  cell cycle  cell apoptosis

摘要:

目的 探讨盐酸高乌甲素对人宫颈癌Hela细胞体外增殖的抑制作用及其可能作用机制。方法 采用细胞计数试剂盒-8（cell counting kit-8，CCK-8）和5-乙炔基-2'-脱氧尿嘧啶核苷（5-ethynyl-2'-deoxyuridine，EdU）细胞增殖试剂盒法考察盐酸高乌甲素对Hela细胞的增殖抑制作用；4'，6-二脒基-2-苯基吲哚（4'，6-diamidino-2-phenylindole，DAPI）染色法研究其对Hela细胞核形态的影响；运用流式细胞技术，采用碘化丙啶（propidium iodide，PI）单标记法和膜联蛋白V-异硫氰酸荧光素/碘化丙啶（annexin V-fluorescein isothiocyanate/propidium iodide，Annexin V-FITC/PI）双标记法检测盐酸高乌甲素作用后Hela细胞的细胞周期分布情况及细胞凋亡情况；Western blotting检测盐酸高乌甲素对Hela细胞p53、p21、p16、Cyclin D1、Rb和p-Rb蛋白表达的影响。结果 盐酸高乌甲素对人宫颈癌Hela细胞的增殖具有抑制作用，且这种抑制作用具有明显的量效关系（r=0.999 3，P<0.01），其对Hela细胞的IC₅₀为0.772 6 mg·mL^-1。经盐酸高乌甲素作用后，Hela细胞的细胞核皱缩，并有变形和碎裂的现象，出现了明显的G₀/G₁期阻滞，且细胞凋亡率随盐酸高乌甲素浓度的增大而逐渐升高。盐酸高乌甲素能上调Hela细胞p53、p16和Rb的蛋白表达，下调Cyclin D1和p-Rb的蛋白表达，但对p21的表达无显著影响。结论 盐酸高乌甲素可能通过pRb通路介导Hela细胞发生G₀/G₁期阻滞，抑制Hela细胞的增殖，促进凋亡。

Abstract:

OBJECTIVE To explore the inhibitory effect of lappaconitine hydrochloride on proliferation of human cervical cancer Hela cells in vitro and its possible mechanism. METHODS Cell counting kit-8(CCK-8) and 5-ethynyl-2'-deoxyuridine(EdU) cell proliferation kit were used to detect the proliferation inhibition effect of lappaconitine hydrochloride on Hela cells. The effect of 4', 6-diamidino-2-phenylindole(DAPI) for nucleic acid staining on Hela nuclear morphology was studied. The technology of flow cytometry, propidium iodide(PI) single staining method and Annexin V-fluorescein isothiocyanate/propidium iodide(AnnexinV-FITC/PI) double staining method were employed to measure the cell cycle distribution and apoptosis of Hela cells after lappaconitine hydrochloride treatment. Western blotting was used to detect the effect of lappaconitine hydrochloride on the expression of p53, p21, p16, Cyclin D1, Rb and p-Rb. RESULTS Lappaconitine hydrochloride could inhibit the proliferation of human cervical cancer Hela cells in vitro, and the inhibition presented obvious dose-effect relationship(r=0.999 3, P<0.01). The IC₅₀ of lappaconitine hydrochloride on Hela cells was 0.772 6 mg·mL^-1. After dealing with lappaconitine hydrochloride, the nuclei of Hela cells were shrunken, deformed and fragmented. Hela cells showed obvious cell cycle arrest at G₀/G₁ phase, and the apoptotic ratio gradually increased with the increase of the concentration of lappaconitine hydrochloride. Lappaconitine hydrochloride could up-regulate the protein expression of p53, p16 and Rb in Hela cells, and down-regulate the protein expression of Cyclin D1 and p-Rb, but it had no significant effect on the expression of p21. CONCLUSION Lappaconitine hydrochloride might mediate G₀/G₁ phase arrest of Hela cells through pRb pathway, inhibit the proliferation of Hela cells and promote their apoptosis.