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乳宁Ⅱ号方对小鼠Ca761乳腺癌移植瘤细胞周期及p53和ras表达的影响

时间:2017-10-05 04:00:07来源:www.xielunwen.net 作者:admin 点击: 62 次
作者:陈前军,王一安,司徒红林,陆德铭,任黎萍,赖熙雯,刘鹏熙,林毅 【关键词】乳腺癌 [摘要]目的:通过研究乳宁Ⅱ号方对小鼠Ca761乳腺癌移植瘤细胞周期及p53、ras表达的影响,探讨乳宁Ⅱ号方抑制乳腺癌生长的机制。方法:建立小鼠Ca761乳腺癌移植模型,小鼠造模后随机分成生理盐水对照组、环磷酰胺(cyclophosphamide,CTX)治疗组、乳宁Ⅱ号方治疗组及乳宁Ⅱ号

        作者:陈前军,王一安,司徒红林,陆德铭,任黎萍,赖熙雯,刘鹏熙,林毅

【关键词】  乳腺癌

    [摘要]  目的:通过研究乳宁Ⅱ号方对小鼠Ca761乳腺癌移植瘤细胞周期及p53、ras表达的影响,探讨乳宁Ⅱ号方抑制乳腺癌生长的机制。方法:建立小鼠Ca761乳腺癌移植模型,小鼠造模后随机分成生理盐水对照组、环磷酰胺(cyclophosphamide, CTX)治疗组、乳宁Ⅱ号方治疗组及乳宁Ⅱ号方加CTX治疗组,每组12只。用流式细胞术检测经不同方法处理的荷瘤小鼠移植瘤细胞的细胞周期,同时运用免疫组化法测定移植瘤组织中p53和ras癌基因蛋白的表达,并进行组间比较。结果:乳宁Ⅱ号方治疗组、CTX治疗组与乳宁Ⅱ号方加CTX治疗组S期肿瘤细胞与生理盐水对照组比较明显减少(P<0.05);乳宁Ⅱ号方治疗组G0G1期肿瘤细胞百分比低于CTX治疗组(P<0.05),而G2M期细胞百分比高于CTX治疗组(P<0.05);乳宁Ⅱ号方治疗组与乳宁Ⅱ号方加CTX治疗组p53的表达均显著低于生理盐水对照组(P<0.05),而CTX对p53表达的影响不显著(P>0.05);乳宁Ⅱ号方治疗组、CTX治疗组与乳宁Ⅱ号方加CTX治疗组ras的表达均低于生理盐水对照组(P<0.05)。结论:乳宁Ⅱ号方可通过调控小鼠Ca761乳腺癌移植瘤细胞的细胞周期来影响肿瘤的生长,这一作用与其调控肿瘤组织p53和ras基因表达有关。

    [关键词]  中国医药学; 乳腺癌; 细胞分化; 癌基因蛋白类

      Effects of Runing RecipeⅡ on expressions of p53 and ras oncogene proteins and cell cycle of the transplanted Ca761 breast cancer in mice

      ABSTRACT  Objective: To explore the mechanisms of Runing RecipeⅡ (a recipe composed of traditional Chinese herbs) in inhibiting the growth of breast cancer by observing its effects on the expressions of p53 and ras oncogene proteins and cell cycle of the transplanted Ca761 breast cancer in mice. Methods: We established the breast cancer model by transplanting Ca761 cells in mice. The mice were randomly divided into 4 groups: normal saline control group, CTXtreated group, Runing RecipeⅡtreated group, and Runing RecipeⅡand CTXtreated group, with 12 mice in each group. We detected the cell cycle of the cancer cells in the mice’s transplanted tumor with flow cytometry and measured the expressions of p53 and ras oncogene proteins in the transplanted tumor with immunohistochemical methods. Results: The percentages of tumor cells in Sphase of the Runing RecipeⅡtreated group, CTXtreated group and Runing RecipeⅡand CTXtreated group were significantly lower than that of the normal saline control group respectively (P<0.05). The percentage of tumor cells in G0G1 phase of the Runing RecipeⅡtreated group was lower than that of the CTXtreated group (P<0.05), while the percentage of tumor cells in G2M phase was higher than that of the CTXtreated group. The immunoreactive scores (IRSs) of p53 in the Runing RecipeⅡtreated group and Runing RecipeⅡand CTXtreated group were significantly lower than that in the normal saline control group respectively (P<0.05). The effect of CTX on the expression of p53 was not significant. The IRSs of ras oncogene protein in the RecipeⅡtreated group, CTXtreated group and Runing RecipeⅡand CTXtreated group were lower than that in the normal saline control group respectively (P<0.05). Conclusion: Runing RecipeⅡ can inhibit the growth of Ca761 breast cancer in mice by controlling the cell cycle of the transplanted tumor. This may be related to its effect on the gene expressions of p53 and ras in the tumor tissue.

    KEY WORDS  Chinese medicine and pharmacology; breast cancer; cell differentiation; oncoproteins

     肿瘤细胞细胞周期失去有效控制是肿瘤增殖的主要机制之一。p53与ras基因的变异是细胞周期失调控的重要原因,p53诱导p21表达共同参与细胞周期的调控[1,2]。乳宁Ⅱ号方是陆德铭教授根据多年防治乳腺癌的经验,以益气养阴,调冲任,佐以解毒为大法研制而成。既往研究表明该药对小鼠乳腺癌细胞的生长具有抑制作用[3,4],本研究拟从该药对肿瘤细胞分化及p53和ras蛋白表达的影响探讨其抑制乳腺癌生长的机制。

    1  材料与方法

    1.1  实验动物  615系小鼠48只,雌性,体质量20 g左右,6周龄,购自协和医科大学实验动物学部实验动物繁育场。

    1.2  药物与试剂  乳宁Ⅱ号方由生黄芪、枸杞子、仙灵脾、莪术等中药组成,由上海中医药大学龙华医院制剂室制成含生药3.2 g/ml的水煎浓缩液。环磷酰胺(cyclophosphamide, CTX)由上海华联制药有限公司生产,沪卫准字(1995)第012034号,200 mg/瓶,用生理盐水稀释成4 mg/ml。免疫组化试剂p53(工作浓度为1∶50)与ras(工作浓度为1∶100)单抗购自丹麦Dako公司。

    1.3  瘤株与造模  615系小鼠乳腺癌细胞株Ca761由大连医科大学病理生理学教研室钱振超教授惠赠。将2×106个Ca761细胞移植于615系小鼠右腋皮下,建立小鼠乳腺癌移植模型[3]。接种次日开始用药,用量按人与动物体表面积折算[5]。

    1.4  实验动物分组  造模后的小鼠随机分成4组,每组12只。生理盐水对照组以生理盐水0.2 ml/d灌胃;CTX治疗组以CTX 40 mg・kg1・d1腹腔注射;乳宁Ⅱ号方治疗组以乳宁Ⅱ号方30 g・kg1・d1灌胃;乳宁Ⅱ号方加CTX治疗组以乳宁Ⅱ号方30 g・kg1・d1灌胃和CTX 40 mg・kg1・d1腹腔注射。各组处理时间均为21 d。

    1.5  观察指标

    1.5.1  细胞周期  每组取8个肿瘤组织标本,运用流式细胞仪检测肿瘤组织细胞周期。

    1.5.2  p53及ras蛋白表达  每组取10个肿瘤组织标本,用免疫组织化学ABC法。p53检测以细胞核出现棕色颗粒者为阳性细胞,ras检测以细胞浆出现棕色颗粒者为阳性细胞。结果判断采用免疫反应积分(immunoreactive score, IRS)标准[6]。该计分法避免了单一阳性细胞数或阳性强度计分的缺点,能较完整地反映免疫组化反应结果。具体计算方法如下。着色强度(staining intensity, SI)计分:着色阴性为0分;着色弱、中、强依次为1、2、3分。阳性细胞百分数(percentage of positive cells, PP)计分:无阳性细胞为0分;阳性细胞数≤10%为1分,11%~50%为2分,51%~80%为3分;>80%为4分。总积分:IRS=SI×PP。

    1.6  统计学方法  数据用x±s表示,组间数据比较采用单因素方差分析。

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