二甲双胍抑制下咽癌裸鼠移植瘤生长的实验研究

Metformin inhibits the growth of hypopharyngeal carcinoma xenografts in nude mice model

目的:通过构建下咽癌裸鼠移植瘤模型,研究二甲双胍对下咽癌体内生长的抑制作用及临床上治疗下咽癌的理论基础。方法:人下咽癌Fadu细胞悬液皮下接种法构建荷瘤鼠,接种2周后取皮下移植瘤块接种至裸鼠腋背部皮下,建立移植瘤动物模型。当移植瘤体积增大到约60mm3时,选取15只瘤块较均匀的裸鼠,随机均分为3组,分别为对照组(等体积的生理盐水)、低剂量二甲双胍治疗组[met40组,40mg/(kg·d)]和高剂量二甲双胍治疗组[(met200组,200mg/(kg·d)],连续28d每日腹腔注射给药1次,每天观察裸鼠的一般情况。治疗28d后,脱颈处死荷瘤鼠,取移植瘤,以免疫组织化学染色及Western Blot方法检测各组移植瘤内AMPK、p21、cyclin D1的表达情况。结果:(1)以Fadu细胞瘤块接种于裸鼠腋背部皮下,成功构建了下咽癌裸鼠移植瘤模型,在药物干预全程中,各组裸鼠一般情况良好,无特殊异常表现及药物不良反应发生;(2)药物干预28d后,2个二甲双胍治疗组的移植瘤平均体积均较对照组有显著抑制(P<0.05),met40组和met200组的抑瘤率分别为(23.30±7.17)%和(40.51±9.25)%;(3)移植瘤标本免疫组织化学染色结果显示,肿瘤内AMPK和p21蛋白的阳性表达平均吸收度值:met40组为0.262±0.046和0.305±0.091,met200组为0.308±0.087和0.477±0.114,均显著高于对照组(0.246±0.038和0.288±0.065),差异有统计学意义(P<0.05);cyclin D1蛋白阳性表达平均吸收度值:met40组为0.269±0.042,met200组为0.216±0.032,显著低于对照组(0.307±0.060),差异有统计学意义(P<0.05);(4)Western Blot方法检测AMPK和p21蛋白的灰度值结果显示:AMPK和p21蛋白met40组分别为1.124±0.085和0.594±0.063,met200组为1.783±0.128和0.970±0.079,均显著高于对照组(0.641±0.042和0.474±0.027),差异有统计学意义(P<0.05);cyclin D1蛋白met40组为0.781±0.055,met200组为0.711±0.046,显著低于对照组(1.608±0.101),差异有统计学意义(P<0.05)。结论:40mg/(kg·d)和200mg/(kg·d)浓度的二甲双胍均可有效抑制下咽癌裸鼠移植瘤模型的生长,抑制肿瘤细胞的增殖、促进其凋亡,且没有引起明显的不良反应,提示二甲双胍对于下咽癌患者的治疗具有较好的临床应用前景。

Objective：To investigated the effect of metformin on growth inhibition of hypopharyngeal carcinoma xenograft in nude mice and to study the theoretical basis of metformin for the treatment of hypopharyngeal carcinoma in clinical. Method： Fadu cells were inoculated subcutaneouly in nude mice on the right flank. After two weeks, the tumors were big enough to be implant into other nude mice. When the average tumor volume reached 60 mm^3, 15 mice were randomized into control （normal saline was administered daily with intraperitoneal injections） and treatment groups （metformin was given daily at 40 or 200 mg/kg body weight）. We observed the general condition of the mice. 28 clays treatment later,all the mice were sacrified and the tumors were used for immunohistochemical analyses and Western Blot to detect AMPK, p21,and cyelin D1. Result： ① To observe the course of metformin treatment, there were no severe adverse effects among the xenograft mice. ②After 28 days treatment, the average volumes of hypopharyngeal carcinoma xenografts of the metformin treated groups were significantly low compared with that of the control group. The inhibition ratio of metformin at the concentration of 40 mg/（kg · d） and 200 mg/（kg · d） to the growth of tumor was 23.30 %±7.17% and 40.51%±9.25%, respectively. ③The averageoptical density（OAD） of AMPK in tumors of the control, met40 and met200 group was 0. 246 ± 0. 038, 0.262±0.046,and0.308±0.087,respectively. The OAD of p21was 0. 288±0. 065 , 0. 305±0. 091,and 0. 477±0. 114, respectively. And the OAD of cyclin D1 was 1. 608±0. 101, 0. 781±0. 055, and 0. 711±0. 046 ,respectively. Both the OAD of AMPK and p21 in met40 and met200 group were observably higher than those of the control group （P〈0.05）. But the OAD of cyclin D1 in met40 and met200 group were remarkably lower than the control group（P〈0.05）.④The Western Blot drawed the consistant trend variation with immunohistochemical analyses. The gray value of AMPK in tumors of the control, met40 and met200 group was 0.641 ±0. 042 ,1.124 ± 0.085, and 1. 783±0. 128;The gray value of p21 was 0. 474±0. 027,0. 594±0. 063,and 0. 970±0. 079;The gray value of cyclin D1 was 1. 608±0. 101,0. 781±0. 055, and 0. 711 ± 0. 046. Conclusion： Metformin can suppress the growth of hypopharyngeal carcinoma xenograft in nude mice and inducing tumor cell apoptosis without any severe adverse effects.