乳腺癌气虚血瘀证小鼠模型的建立及益气活血法的干预研究

摘要

目的 建立气虚血瘀证乳腺癌荷瘤4-T1小鼠模型,并予益气活血中药干预,分析在气虚血瘀状态下中药干预对肿瘤微环境的影响.方法 雌性BALB/c小鼠48只,随机平均分为6组:空白对照组、单纯荷瘤组、气虚血瘀证荷瘤模型组(复合模型组)、复合模型低剂量组、复合模型中剂量组、复合模型高剂量组.单纯荷瘤组小鼠注射4T1单细胞悬液,复合模型组在单纯荷瘤组基础上注射利血平.从实验第15天起,复合模型低剂量组、中剂量组、高剂量组分别灌胃相应剂量中药(四君子汤合血府逐瘀汤),每日1次,连续28 d.分别于实验第7、14、21、28、35、42天对复合模型组及各中药干预组予症状量化评分表评估,第42天测量各组耳缘、脚趾、腹壁静脉微循环血流量,West-ern blot法测定肿瘤组织基质金属蛋白酶2(MMP2)、磷酸化细胞外信号调节激酶(p-ERK1/2)的表达.结果 复合模型组、复合模型低剂量组、复合模型中剂量组、复合模型高剂量组均出现气虚血瘀症状,低剂量组量化评分值自第28天低于复合模型组(P＜0.05),中剂量组自第21天分值低于复合模型组(P＜0.05).空白对照组静脉微循环血流量大于其余各组(P＜0.05),复合模型组小于单纯荷瘤组(P＜0.05),各中药干预组均比复合模型组提高(P＜0.01或P＜0.05).复合模型组原位瘤MMP-2表达高于单纯荷瘤组(P＜0.05),各中药干预组均低于复合模型组(P＜0.05).复合模型低剂量组、中剂量组原位瘤p-ERK1/2表达均低于复合模型组(P＜0.05或P＜0.01).结论 通过注射4T1单细胞悬液结合注射利血平方法建立的气虚血瘀证乳腺癌小鼠模型稳定可靠,符合中医证候特点.气虚血瘀证小鼠肿瘤组织内MMP-2、p-ERK1/2表达上调,益气活血中药干预后能纠正气虚血瘀状态同时下调MMP-2、p-ERK1/2的表达,抑制肿瘤生长.%Objective To establish a mice model of breast cancer 4-T1 with the pattern of qi deficiency and blood stasis and study the effects of qi-tonifying and blood-activating therapy on tumor microenvironment.Methods 48 female Balb/c mice were randomly divided into 6 groups:blank control group (group A),tumor-bearing model group (group B),tumor-bearing with qi-deficiency blood-stasis pattern composite model group (group C,composite model),composite model with low-dose herbal treatment group (group D),composite model with mid-dose herbal treatment group (group E),and composite model with high-dose herbal treatment group (group F).4-T1 monocyte suspension was injected to induce breast cancer model while reserpine injection was added to establish qi-deficiency blood-stasis composite pattern model.Herbal treatment (Si junzi Tang plus Xuefu Zhuyu Tang,i.e.four noble-ingredient decoction plus blood-activating stasis-removing decoction) of corresponding dose was offered to mice (intragastric administration) in group D,E and F from the 15th day daily for 28 days consecutively.Symptoms scores were evaluated on the 7th,14th,21st,28th,35th,and 42nd day.On the 42nd day blood flow of microcirculation in ear rim,toes and veins on the abdominal surface was detected.Matrix metalloproteinase-2 (MMP-2) and phospho-Erkl/2 (p-ERK1/2) in tumor issue were tested by using Western blot.Results Symptoms of qi deficiency and blood stasis were observed in group C,D,E and F.Symptom score of group D became lower than group C since day 28 (P ＜ 0.05).The local blood flow of mice in group A was higher than that of group C fom day 21 (P ＜ 0.O1).Blood flow in group C was lower than that in group B (P ＜0.05).Microcirculation all improved in group D,E and F compared with group B (P ＜0.01 or P ＜0.05).The expression of MMP-2 in mice in group C was higher than that in group B (P ＜ 0.05);lower in group D,E and F than that in group C (P ＜ 0.05).The expression of pERK1/2 in group C was slightly higher than that in group B (P ＞ 0.05);lower in group D and E than that in group C (P ＜ 0.01 or P ＜ 0.05).Conclusions The mice composite model of breast cancer with qi deficiency and blood stasis established in this study proves to be reliable and consistent with TCM pattern characteristic.MMP-2 and p-ERK1/2 were upregulated in mice tumor with the pattern of qi deficiency and blood stasis.Qi-tonifying and blood-activating herbal intervention could not only relive qi deficiency and blood stasis,but also downregulate the expression of MMP-2 and p-ERK1/2,and thus inhibit the growth of tumor.