蝙蝠葛酚性碱对BxPC-3裸鼠移植瘤Ptch1、Smo蛋白表达的影响

目的:通过观察蝙蝠葛酚性碱(Phenolic alkaloids from Menisphermum dauricum PAMD)对胰腺癌细胞株Bx PC-3裸鼠移植瘤的抑制情况,及其对裸鼠移植瘤Hedgehog信号通路中关键分子膜受体Patched 1(Ptch1)、偶联受体Smothened(Smo)基因、蛋白表达的影响,探讨其作用机制。方法:将30只裸鼠随机选择6只作为空白对照组,其余24只裸鼠接种人源性胰腺癌细胞株Bx PC-3细胞24小时后,随机分为4组:模型组、5-氟尿嘧啶组(5-FU)、PAMD高、低剂量组,每组6只。连续给药3周后,取出肿瘤组织进行抑瘤率计算,行免疫组化(IHC)、实时定量PCR和Western blot三种方法检测裸鼠移植瘤组织中Ptch1、Smo基因及蛋白的表达影响。结果:①抑瘤率:PAMD低、高剂量组和5-FU组与模型组比较,对胰腺癌裸鼠移植瘤均有不同程度的抑制作用,抑瘤率分别为36.14%、55.88%和30.88%,具有统计学意义(P0.05)。其中以PAMD高剂量组治疗效果最好,差异具有极显著统计学意义(P0.01)。②免疫组化:模型组与各治疗组比较,Ptch1和Smo蛋白均呈现高表达,而各治疗组Ptch1蛋白和Smo蛋白表达均出现不同程度的下调,差异具有统计学意义(P0.05);同组Ptch1和Smo蛋白相互之间具有相同的表达趋势,其中PAMD高剂量组Ptch1和Smo蛋白表达下调最明显,差异有极显著的统计学意义(P0.01)。③实时定量PCR:PAMD低、高剂量组与模型组比较,Ptch1蛋白相对表达量均有不同程度的降低,差异具有极显著统计学意义(P0.01);而Smo蛋白表达量降低但不明显,差异具有统计学意义(P0.05),其中以PAMD高剂量组表达效果最明显,差异具有极显著的统计学意义(P0.01)。④Western blot结果与上述趋势相一致。结论:PAMD可通过降低Hedgehog信号通路中Ptch1、Smo关键蛋白的含量,诱导肿瘤细胞发生凋亡,从而起到抑制BxPC-3裸鼠移植瘤的生长,延缓胰腺癌的发生发展。

Effects of Phenolic Alkaloids from Menisphermum Dauricumon on the Expression of Ptch1 and Smo Proteins in Pancreatic Cancer Cell Line BxPC-3 in Nude Mice

ABSTRACT Objective: To observe the inhibition of pancreatic cancer cell line BxPC-3 xenografts in nude mice by phenolic alkaloids from Menisphermum dauricum (PAMD), and to influence the key molecules of membrane receptor Ptch1, and coupled receptor Smo, and protein expression in hedgehog signaling pathway in nude mice. Besides, their mechanism of action was explored. Methods: Six nude mice were selected randomly as blank control group among thirty nude mice. The other 24 nude mice were inoculated with human pancreatic cancer cell line BxPC-3 for 24 hours. They were randomly divided into 4 groups: model group, 5-fluorouracil (5-FU) group, PAMD high and low dose groups, 6 in each group. After 3 weeks of continuous administration, the tumor tissues were taken out for tumor inhibition rate calculation. Immunohistochemistry (IHC), real-time quantitative PCR and Western blot were used to detect the expression of Ptch1 and Smo genes in nude mice. Results: 1. Tumor inhibition rate: PAMD low, high dose group and 5-FU group compared with the model group, had different degrees of inhibition on pancreatic cancer xenografts in nude mice, the tumor inhibition rates were 36.14%, 55.88% and 30.88 %, respectively, the difference was statistically significant (P<0.05). Among them, the high-dose PAMD group had the best effect, and the difference was statistically significant(P<0.01). 2. Immunohistochemistry: Compared with the treatment group, the expression of Ptch1 and Smo protein in the model group was high, while the expression of Ptch1 protein and Smo protein in each treatment group were down-regulated in different degrees, the difference was statistically significant (P<0.05); The Ptch1 and Smo proteins in the same group had the same expression trend. The expression of Ptch1 and Smo protein in the high dose group of PAMD was the most obvious, and the difference was statistically significant(P<0.01). 3. Real-time quantitative PCR: Compared with the model group, the relative expression of Ptch1 protein in PAMD low and high dose groups decreased to different degrees, the difference was statistically significant (P<0.01); while the expression of Smo protein decreased but not obvious. The difference was statistically significant (P<0.05). The expression of PAMD high dose group was the most obvious, and the difference was statistically significant (P<0.01). 4. Western blot results are consistent with the above trends. Conclusion: PAMD can induce the apoptosis of tumor cells by reducing the content of Ptch1 and Smo in the hedgehog signaling pathway, thereby inhibiting the growth of BxPC-3 xenografts in nude mice and delaying the development of pancreatic cancer.