苦参碱对小鼠H22肝癌细胞凋亡作用的实验研究

Matrine induced apoptosis of murine H22 hepatocarcinoma cells

目的:研究中药苦参碱在体内外对小鼠H22肝癌细胞的诱导凋亡作用,探讨其可能的抗肿瘤作用机制。方法:An-nexinⅤ-FITC/PI双标记法检测苦参碱对H22细胞的早期促凋亡作用;免疫组织化学法检测苦参碱作用后H22细胞内Bc l-2、Bax两种凋亡相关蛋白的表达。建立H22肝癌移植瘤小鼠模型,观察苦参碱对荷瘤小鼠肿瘤生长情况的影响及肿瘤抑制率;电镜观察荷瘤小鼠肿瘤组织的超微结构改变;免疫组化方法检测小鼠肿瘤组织内Bc l-2、Bax的表达情况。结果:AnnexinⅤ法检测到1.0 mg/mL和1.5 mg/mL苦参碱作用48 h后H22细胞有早期凋亡改变,凋亡细胞百分率分别为11.71%和17.86%,与对照组相比差异有统计学意义(P<0.05)。苦参碱对荷瘤小鼠肿瘤的抑制率达60%以上;免疫组化显示,苦参碱作用后H22细胞内及小鼠肿瘤组织内Bc l-2蛋白的表达水平下降,Bax蛋白表达增强。电镜证实苦参碱治疗后荷瘤小鼠肿瘤组织内有凋亡细胞和凋亡小体的存在。结论:苦参碱在体内体外都对小鼠H22肝癌细胞表现出较强的抗肿瘤作用和诱导凋亡作用,促凋亡作用与其上调细胞内Bax表达,抑制Bc l-2表达有关。

Objective：To investigate the apoptosis-inducing effect of matrine on murine hepatocarcinoma cell line H22 in vivo and in vitro and explore the underlying mechanisms. Methods.The H22 cell apoptosis induced by matrine at the early stage was detected with Annexin V-FITC/PI double staining assay. The expressions of Bcl-2 and Bax proteins in H22 cells as well as in the BALB/c H22 xenograft tumor tissues were detected using immunohistochemical method. Transmission electron microscopy （ TEM ） was used to observe the ultramicro-structure alterations of H22 xenograft tumor cells in BALB/c mice. The effect of matrine on the kinetics of tumor growth after subcutaneous injection of H22 cells in BALB/c mice was observed and the tumor inhibition rate was calculated. Results： Annexin V staining detected early apoptosis of H22 cells after treatment with matrine 1.0 mg/mL and 1.5 mg/mL for 48 h. The apoptotic rates were 11.71% and 17.86%, respectively, both of which higher than that of control groups （ P 〈 0.05 ）. The tumor inhibition rate was above 60% after matrine treatment. Immunohistochemistry analysis revealed that matrine increased Bax protein expression and reduced Bcl-2 protein expression in both H22 cells in vitro and xenograft tumor tissues in vivo. TEM demonstrated the existence of apoptotic cells and apoptotic bodies in H22 xenograft tumor tissues after matrine treatment. Conclusion： Matrine significantly suppresses tumor growth and induced apoptosis both in vitro and in vivo. The apoptosis-inducing effects is related with up-regulation of Bax protein and down-regulation of Bcl-2 protein.