Effect of Cisplatin Combined with Buzhong Yiqi Decoction on survivin Expression in Transplantation Tumor of Nude Mice with A549/DDP Cells
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摘要:目的
探讨顺铂联合补中益气汤对A549/DDP荷瘤裸鼠移植瘤survivin蛋白表达的影响。
方法30只BALB/c-nu裸小鼠随机分为5组,A:荷瘤对照组、B:顺铂组、C:顺铂联合低剂量补中益气汤组、D:顺铂联合中剂量补中益气汤组、E:顺铂联合高剂量补中益气汤组。25 d后处死全部裸小鼠。测量移植瘤体积、重量并计算药物抑瘤率;Real-time PCR法检测移植瘤组织survivin mRNA的表达;Western blot法检测移植瘤组织survivin蛋白的表达。
结果顺铂联合低、中、高剂量组均较荷瘤对照组、顺铂组移植瘤体积显著缩小,重量显著减轻,差异有统计学意义;顺铂抑瘤率随着补中益气汤的剂量增加而增大;顺铂组、顺铂联合低剂量组较荷瘤对照组移植瘤组织中survivin的基因和蛋白表达上调,而顺铂联合中、高剂量组较荷瘤对照组survivin的基因和蛋白表达下调,差异均有统计学意义;顺铂联合低、中、高剂量补中益气汤组较顺铂组移植瘤组织中survivin的基因和蛋白表达均下调,差异均有统计学意义。
结论顺铂联合补中益气汤可通过下调survivin的表达改善A549/DDP荷瘤裸鼠移植瘤对顺铂的耐药性。
Abstract:ObjectiveTo explore effect of cisplatin combined with Buzhong Yiqi decoction on survivin expression in transplantation tumor of nude mice with A549/DDP cells.
MethodsThirty BALB/c-nu mice were randomly divided into five groups: A group (tumor control group),B group (cisplatin group),C group (cisplatin with low dose of Buzhong Yiqi decoction),D group (cisplatin with medium dose of Buzhong Yiqi decoction),E group (cisplatin with high dose of Buzhong Yiqi decoction). DDP or DDP and Buzhong Yiqi decoction were treated from 9 to 33 day,lastly the mice were executed. The volume and weight of transplantation tumors were measured; tumor inhibitory rate was calculated. Real-time PCR was used to determine the expression of survivin mRNA; Western blot was adopted to detect the expression of survivin protein.
ResultsThe volume and weight of transplantation tumors in C,D and E groups were significantly smaller than those in A and B groups. Tumor inhibitory rates were increased with the increasing dose of Buzhong Yiqi decoction. Compared with A group,the survivin gene and protein levels of transplantation tumors in B and C groups were higher,however,the results were lower in D and E groups. The survivin gene and protein expressions of transplantation tumors in C,D and E groups were significantly down-regulated,compared with B group.
ConclusionCisplatin combined with Buzhong Yiqi decoction could improve cisplatin resistance of transplantation tumor of nude mice with A549/DDP cells through down-regulating the gene and protein expression of survivin.
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0 引言
目前,临床上肺癌的治疗方法主要为手术、化疗、放疗、中医药等。肺癌诊断时多处于中晚期,往往丧失了手术的最佳时机,因此化疗是其常用的治疗手段。顺铂(cisplatin,DDP)是治疗肺癌的常用一线药物,其抗肿瘤的毒性和有效性在临床上得到了充分的肯定。但顺铂不良反应大且极易形成耐药,这严重影响其临床疗效,限制其临床应用[1-2]。有文献报道培土生金中药能减轻化疗产生的不良反应,有效改善肺癌耐药,提高化疗药物临床疗效[3-4]。本课题组前期实验研究也发现,补中益气汤(培土生金法的代表方药)含药血清能有效干预A549/DDP的耐药性[5-6] ,但培土生金理论在肺癌顺铂耐药形成过程中的分子机制尚未完全阐明。本实验通过观察顺铂联合补中益气汤对A549/DDP荷瘤裸鼠移植瘤生长的抑制作用和凋亡抑制蛋白survivin表达的影响,以明确化疗中培土生金法干预肺癌顺铂耐药的分子作用机制。
1 材料与方法
1.1 材料和细胞株
30只健康BALB/c-nu裸小鼠购自北京维通利华实验动物技术有限公司,6周龄,雄性,体质量(16~20)g,裸小鼠饲养于屏障级的实验动物中心,喂食高压灭菌饲料、纯净水,每日保证12 h光照。动物许可证号SCXK(京)2012-0001;A549/DDP细胞株购自中国医科院肿瘤研究中心细胞库。
1.2 药物及中药制备
顺铂购自大连美仑生物技术有限公司(批号MB1055)。根据新世纪版《方剂学》中规定用药量并结合临床常用剂量,补中益气汤的组方及剂量为黄芪18 g,甘草9 g,人参6 g,当归3 g,橘皮6 g,升麻6 g,柴胡6 g,白术9 g,全部药材均购自辽宁中医药大学附属医院药房,并通过辽宁中医药大学药理实验室的鉴定后,按成人推荐日用量常规煎煮,根据“人和动物之间按体表面积折算的等效剂量比值表”计算出小鼠等效剂量,并分别设高、中、低剂量5.82 g/kg、2.91 g/kg、1.46 g/kg,过滤、水浴蒸发至终浓度为每毫升含生药1 g,冷却后4℃冰箱保存备用。
1.3 试剂
RPMI 1640培养液和胎牛血清均购自美国HyClone公司(批号SH30058.02、SV30089.01);survivin、β-actin一抗、羊抗兔IgG-HRP、全蛋白提取试剂盒、BCA蛋白浓度测定试剂盒、ECL发光液均购自沈阳万类生物科技有限公司(批号WL01684、WL0002、WLA023、WLA019、WLA004、WLA003);Super M-MLV反转录酶、高纯度总RNA快速提取试剂盒均购自北京百泰克生物技术有限公司(批号PR6502、RP1201);引物由生工生物工程(上海)有限公司合成。
1.4 细胞培养
取处于对数生长期的A549/DDP细胞,弃去培养液,加入0.25%胰酶1 ml,消化2~5 min(肉眼观察瓶壁半透明的细胞层出现细针孔空隙,或倒置显微镜下细胞变钝圆,细胞间隙增大)即可终止消化。加入含10%胎牛血清RPMI 1640培养液5 ml,无菌吸管轻轻吹打,使之均匀悬浮。分装入两个培养瓶,传代继续培养。
1.5 成瘤模型的制备
30只BALB/c-nu裸小鼠适应性喂养2周,用无血清培养液将收集好的对数生长期A549/DDP细胞密度调整至5×107/ml,每只裸小鼠腋窝皮下接种0.2 ml肿瘤细胞。自接种之日起每天观察裸鼠状态及成瘤时间,造模成功24 h后,各组裸鼠分别做后续处理。
1.6 实验分组及用药
按照随机数字表法分为5组:A:荷瘤对照组、B:顺铂组、C:顺铂联合低剂量补中益气汤组、D:顺铂联合中剂量补中益气汤组、E:顺铂联合高剂量补中益气汤组,每组6只。A组蒸馏水灌胃,1毫升每只,2 次/天;B组0.01 g/kg顺铂腹腔注射,2 次/天;C、D、E组分别用高剂量(5.82 g/kg)、中剂量(2.91 g/kg)、低剂量(1.46 g/kg)补中益气汤灌胃,1毫升每只,2次/天,同时0.01 g/kg顺铂腹腔注射,2次/天。接种瘤细胞25 d后,无菌剥取移植瘤,测量皮下移植瘤的最大长径(a)和横径(b),计算肿瘤体积:肿瘤体积= a×b2/2,称重计算各组药物的抑瘤率:抑瘤率=(对照组瘤质量-药物组瘤质量)/对照组瘤质量×100%。
1.7 Real-time PCR法检测移植瘤组织中survivin mRNA的表达
按相应试剂盒说明提取样本总RNA,反转录得到对应的cDNA,最后进行PCR扩增。引物见表 1,β-actin为内参。本实验采用2-ΔΔCt分析方法。
表 1 Real-time PCR引物序列表Table 1 Oligonucleotide primer sets for real-time PCR1.8 Western blot法检测移植瘤组织中survivin蛋白的表达
按全蛋白提取试剂盒的说明提取瘤组织总蛋白,BSA法制备蛋白标准曲线,检测各蛋白样本的浓度。制备聚丙烯酰胺凝胶,等量的组织蛋白提取物上样,SDS-PAGE电泳分离,并转印到PVDF膜上。室温下,PVDF膜在封闭缓冲液中封闭1 h,孵育survivin一抗缓冲液(1:500),4℃过夜。次日,37℃孵育HRP-标记的二抗缓冲液(1:5 000)45 min。在PVDF膜上滴加ECL发光液,将胶片进行扫描,用凝胶图像分析系统(Gel-Pro-Analyzer软件)分析目标条带的吸光度值(OD)。实验结果用蛋白相对表达量表示,相对表达量=(待测组ODsurvivin/待测组ODβ-actin)/(对照组ODsurvivin/对照组ODβ-actin)。
1.9 统计学方法
使用SPSS17.0软件对数据进行统计学分析。计量资料用(x±s)表示,各组数据符合正态分布,方差齐时,采用单因素方差分析,两两比较采用SNK法;不符合参数检验者,采用多组计量资料秩和检验。P<0.05为差异有统计学意义。
2 结果
2.1 顺铂联合补中益气汤对各组裸小鼠的一般状态和体质量的影响
各组BALB-c小鼠荷瘤后活动良好,未出现明显的拒食、躁动、便溏等症状。荷瘤8 d后,各组BALB/c小鼠均有肿瘤结节生长,瘤块直径达到2~3 mm,经病理学诊断符合肺腺癌组织学特征。给药前和给药后各组裸小鼠体质量差异均无统计学意义(P=0.662,P=0.662,P=0.662,P=0.385; P=0.125,P=0.128,P=0.126,P=0.127),见表 2。
表 2 顺铂联合补中益气汤对各组裸小鼠体重的影响 (x±s,n=6)Table 2 Effect of cisplatin combined with Buzhong Yiqi decoction on body weight of nude mice in each group (x±s,n=6)2.2 顺铂联合补中益气汤对各组裸小鼠移植瘤的体积、重量及药物抑瘤率的影响
各组造模成功后移植瘤体积差异无统计学意义。给药25 d后,顺铂组较荷瘤对照组移植瘤体积轻度减小,重量显著减轻,但差异均无统计学意义(P=0.370,P=0.814);顺铂联合低、中、高剂量组均较荷瘤对照组、顺铂组移植瘤体积显著缩小,重量显著减轻,差异有统计学意义(P=0.033,P=0.045; P=0.011,P=0.018; P=0.000,P=0.000),且随着补中益气汤药物剂量的增加,移植瘤体积逐渐缩小,重量逐渐减轻,存在明显的剂量依赖性,见图 1。抑瘤率由高至低的药物依次为顺铂联合高剂量补中益气汤、顺铂联合中剂量补中益气汤、顺铂联合低剂量补中益气汤,见表 3。
表 3 顺铂联合补中益气汤对各组移植瘤体积、重量及抑瘤率的影响 (x±s,n=6)Table 3 Effect of cisplatin combined with Buzhong Yiqi decoction on volume,weight and inhibitory rate of transplantation tumor of nude mice in each group (x±s,n=6)2.3 顺铂联合补中益气汤对各组移植瘤组织中survivin mRNA表达的影响
Real-time PCR法检测survivin的DNA扩增产物,显示熔解曲线无杂峰,平均熔解温度一致,具有特异性。与荷瘤对照组相比,顺铂组、顺铂联合低剂量组移植瘤survivin mRNA表达升高,而顺铂联合中、高剂量组survivin mRNA表达降低,差异均有统计学意义(P=0.010,P=0.004,P=0.004,P=0.002);与顺铂组相比,顺铂联合低、中、高剂量补中益气汤组移植瘤survivin mRNA表达降低,差异均有统计学意义(P=0.005,P=0.004,P=0.002),且随着补中益气汤药物剂量的增加,survivin mRNA表达量逐渐降低,存在明显的剂量依赖性,见图 2、表 4。
表 4 顺铂联合补中益气汤对各组移植瘤组织中survivin mRNA和蛋白相对表达量的影响 (x±s,n=6)Table 4 Effect of cisplatin combined with Buzhong Yiqi decoction on survivin mRNA and protein relative expression of transplantation tumor in each group (x±s,n=6)2.4 顺铂联合补中益气汤对各组移植瘤组织中survivin蛋白表达的影响
Western blot法检测survivin的蛋白表达量。与荷瘤对照组相比,顺铂组、顺铂联合低剂量组移植瘤survivin蛋白表达上调,而顺铂联合中、高剂量组survivin蛋白表达下调,差异均有统计学意义(P=0.008,P=0.004,P=0.004,P=0.002);与顺铂组相比,顺铂联合低、中、高剂量补中益气汤组移植瘤survivin蛋白表达下调,差异均有统计学意义(P=0.005,P=0.003,P=0.001),且随着补中益气汤药物剂量的增加,survivin蛋白表达量逐渐降低,存在明显的剂量依赖性,见图 3、表 4。
3 讨论
在中医学中,肺癌属“肺积”、“肺壅”、“息贲”、“息积”等范畴,正气不足或相对不足是发病的内在根据,邪气是发病的重要条件。正如清·沈金鳌《杂病源流犀烛》所云“邪积胸中,阻塞气道,气不得通,为痰……为血。皆邪正相搏,邪既胜,正不得制之,遂结成形而有块”。说明正气虚损以后,邪气乘虚袭肺,郁结胸中,肺气愤郁,宣降失司,积聚成痰,痰气瘀毒互结,日久形成肿块而成肺癌。在五行学说中,肺属金,脾胃属土,土能生金,故“脾有生肺之能,土旺而金生”之说。脾为肺之母,肺主气而脾益气,肺所主之气来源于脾,其司呼吸、主宣发肃降的功能皆赖脾胃运化水谷精微以灌溉滋养。脾土的强弱决定肺气的盛衰,肺气不足多与脾气虚弱有关。临床上,肺癌患者化疗后常出现脾胃虚弱、气血不足的表现,如虚羸少气、畏寒肢冷、食少纳差、食入难化,脘腹痞闷等,故改善肺癌耐药当以健脾益气为主[7]。
补中益气汤乃金元时期名医李东垣的传世名方,主要由黄芪、甘草、人参、当归、橘皮、升麻、柴胡、白术八味药组成,具有“补中益气,升阳举陷”之功效,可补脾虚生肺气。现代医学亦证实补中益气汤可治疗中晚期非小细胞肺癌,在调动脾胃运化功能,使气血生化有源的同时,还可增强患者的免疫力、减轻放化疗所致的不良反应[8]。本实验结果显示顺铂联合补中益气汤处理后移植瘤体积显著缩小,移植瘤重量显著减少,且随着补中益气汤药物剂量的逐渐增加,移植瘤体积逐渐缩小,移植瘤重量逐渐减少,存在明显的剂量依赖性,说明补中益气汤可改善A549/DDP细胞接种所致移植瘤对顺铂的抵抗性。
survivin基因是已发现的凋亡蛋白家族中最小的成员,但其凋亡抑制作用最强。survivin基因已经成为在肿瘤治疗中抗肿瘤药物的新靶点,其抑制细胞凋亡及有丝分裂,主要为负调控作用[9],且其过表达与耐药有关[10]。课题组前期体外实验发现顺铂持续处理A549/DDP细胞后survivin的表达会显著上调[11],补中益气汤可下调A549/DDP细胞中该基因的表达[12]。本实验旨通过在体实验进一步证实补中益气汤在肺腺癌化疗中改善顺铂耐药中所发挥的作用,结果显示A549/DDP荷瘤小鼠经持续灌饲顺铂后,移植瘤组织中survivin基因和蛋白水平的表达均有所增加,说明对于耐药个体而言,顺铂非但没有治疗作用,反而增强瘤组织对药物的抵抗性。然而,A549/DDP荷瘤小鼠持续灌饲顺铂联合补中益气汤后,移植瘤组织中survivin基因和蛋白水平的表达有所减少,尤其是顺铂联合中、高剂量补中益气汤组,survivin的表达较顺铂组和荷瘤对照组均显著减少,说明中、高剂量的补中益气汤可改善耐药个体对顺铂产生的耐药性,而低剂量的补中益气汤的效果欠佳。本研究进一步证实顺铂联合补中益气汤可下调A549/DDP荷瘤裸鼠移植瘤组织中survivin的表达,为临床寻求中医药改善肺癌顺铂耐药的有效靶点提供可参考的实验依据。
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表 1 Real-time PCR引物序列表
Table 1 Oligonucleotide primer sets for real-time PCR
表 2 顺铂联合补中益气汤对各组裸小鼠体重的影响 (x±s,n=6)
Table 2 Effect of cisplatin combined with Buzhong Yiqi decoction on body weight of nude mice in each group (x±s,n=6)
表 3 顺铂联合补中益气汤对各组移植瘤体积、重量及抑瘤率的影响 (x±s,n=6)
Table 3 Effect of cisplatin combined with Buzhong Yiqi decoction on volume,weight and inhibitory rate of transplantation tumor of nude mice in each group (x±s,n=6)
表 4 顺铂联合补中益气汤对各组移植瘤组织中survivin mRNA和蛋白相对表达量的影响 (x±s,n=6)
Table 4 Effect of cisplatin combined with Buzhong Yiqi decoction on survivin mRNA and protein relative expression of transplantation tumor in each group (x±s,n=6)
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