摘要目的观察自拟治癌方剂镇癌灵诱导胃癌细胞凋亡和抑制胃癌细胞体外生长的效应，为临床应用与新药开发奠定实验基础.

方法镇癌灵是由马钱子、三棱、红根地覃、白花蛇舌草等10余种中药、草药、植物组成的药方.将镇癌灵按成人每天正常使用剂量500 g(即10 g/ kg体重)，常规煎熬浓缩成500 mL汤液，4℃冰箱冷却，0.22 μm滤膜过滤除菌，以消毒的玻璃离心管分装成每支5 mL，-20℃冰箱储存备用.根据抗癌药物敏感性实验的方法，计算出体外药物敏感性实验所用药物的一般浓度为5 g/ L，即在含10%小牛血清的RPMI1640完全培养基中加入5 mL/ L的过滤汤剂为中剂量，0.5 mL/ L和50 mL/ L分别为低剂量和高剂量，常规培养SGC7901胃癌细胞，3 d换液一次. 根据直接记数法和MTT显色法检测细胞生长曲线与细胞增殖实验的同时，每隔36 h收集细胞进行细胞涂片染色及提取细胞基因组DNA电泳.结果生长曲线表明，经高、中、低剂量镇癌灵作用的胃癌细胞实验组的对数生长期分别为3 d～6.5 d，3 d～7 d，3 d～7.5 d，细胞倍增时间各为3 d，2.5 d，2.5 d；而非空白对照组细胞对数生长期为2 d～8 d，倍增时间为2 d. mTT显色法结果提示，高、中、低不同剂量实验组的细胞增殖速度或增殖指数以及细胞存活率均明显低于非实验组，差异均有非常显著性意义(P＜0.001).实验组细胞刷片染色显示有明显的细胞核固缩、核周边染色质加深、核断裂、凋亡小体现象，细胞DNA电泳可见梯状条带的凋亡特征，而空白对照组无此现象.结论自拟方剂镇癌灵含有解毒、消肿、镇痛、化痰、活血、益气、生津、降火等多种功效；从本实验结果表明，具有明显抑制体外培养胃癌细胞生长增殖的作用，其作用机制可能与镇癌灵能诱导胃癌细胞发生显著凋亡的作用有关.

中国图书馆分类号R 735.2

Experimental study of the inhibition affect of Zhen＇ailing on the growth of gastric cancer in vitro

XIAO Bing¹, XIAO Li-Chun², LAI zhuo-Sheng¹, ZHANG Ya-Li¹, ZHANG Zhen-Shu¹ and zHANG Wan-Dai¹¹Institute for Digestive Disease, Nanfang Hospital, The First military Medical University, Guangzhou 510515, Guangdong Province, China²Department of Traditional Chinese Medicine, The Second People＇s hospital, Fuzhou 350007, Fujian Province, China

Subject headingsZhen＇ailing/pharmacology; apoptosis; stomach neoplasm

AbstractAIMTo investigate apoptosis and growth inhibition effect of gastric carcinoma cell in vitro induced by the formulae Zhen＇ailing made by ourself, and establish the experimental basis for clinical application and development of new drugs.METHODSZhen＇ailing was composed of more than ten types of Chinese herbal medicine and plants including Vomiting nut (马钱子), Burreed tuber (三棱), Sage (红根地覃), oldenlandia (白花蛇舌草), et al. Zhen＇ailing was decocted and concentrated in 500 mL decoction according to the normal dosage of 500 g (10 g/ kg body weight) used for adults, cooled at 4℃; bacteria were filtered by 0.22μm filtration membrane, then it was put into 5 mL glass tube, stored at -20℃ refrigerator. The concentration of this formulae was divided into high, moderate and low dose, added by this decoction of 50 mL/ L, 5 mL/ L, 0.5 mL/ L respectively. In the anti-tumor drug＇s sensitive test in vitro SGC7901 stomach carcinoma cell was cultured in this complete medium. The growth curve and proliferation of cells were detected by MTT test and direct counting method, simultaneously cells were collected, smeared and stained, the genomic DNA was extracted for gel electrophoresis once every 36 hours.RESULTSThe growth curve indicated that the logarithmic growth phase of the experimental group of stomach carcinoma cells were 3 d-6.5 d, 3 d-7 d, 3 d-7.5 d, respectively by high, moderate and low dose of Zhen＇ailing, but the control group was 2 d-8 d and the doubling time was 2 d. The results of MTT test showed the cell proliferation rate or proliferation index and survival rate of experimental group by high, moderate and low dose of Zhen＇ailing were lower than control group. There was significant difference between the two groups (P＜0.01). the staining of brush cells of experimental group showed nuclear shrinkage, margination, nuclear fragmentation and formation of apoptotic bodies, and the characteristic ladder band on gel electrophoresis. NO changes were observed in control group.CONCLUSIONZhen＇ailing has multiple functions including promoting blood circulation, promoting salivation, removing heat, supplementing qi. The results of this experiment showed that the proliferation of stomach carcinoma cells cultured in vitro was inhibited by this formulae Zhen＇ailing and the mechanism was probably associated with the induction of apoptosis of stomach carcinoma cells.

0引言

镇癌灵是根据闽西民间用于治疗消化道肿瘤的验方加减制作而成的汤剂.为了证实这一药物的抗癌效应及探讨其作用机制，我们观察了这一汤剂对体外培养的胃癌细胞生物学行为的影响.

1材料和方法

1.1镇癌灵汤剂由马钱子、三棱、红根地覃、白花蛇舌草、虎杖、鳖甲、牡蛎、半枝莲等10种中药、草药、植物组成的药方，患者每天正常使用剂量共500 g(即10 g/ kg体重)，常规煎熬浓缩成500 mL汤液，4℃冰箱冷却，0.22μm滤膜过滤除菌，以消毒的玻璃离心管分装成每支5 mL，-20℃冰箱储存备用.

1.2细胞与试剂SGC7901胃癌细胞由本所保存细胞，RPMI1640、胎牛血清、MTT、琼脂糖、蛋白酶k、限制性内切酶EcoRⅠ、DMSO、平衡酚、RNase a、Tris碱等分别购于Sigma公司和Promega公司.

1.3细胞培养与药物应用根据抗癌药物敏感性实验的方法^［1］，计算出体外药物敏感性实验所用药物的一般浓度为5 g/ L，即在含10%小牛血清的RPMI1640完全培养基中加入5 mL/ L的过滤汤剂为中剂量，0.5 mL/ L和50 mL/ L分别为低剂量和高剂量，常规培养SGC7901胃癌细胞，3 d换液一次.

1.4细胞生长曲线测定取生长状态良好的细胞，采用一般传代方法进行消化，制成细胞悬液.经记数后，精确地按1×10⁴ cells分别接种于24孔培养板内，共接种8个24孔板，分成非诱导组、低剂量组、中剂量组、高剂量组，每孔培养液为1 mL. 每天各组细胞取4～5孔细胞进行计数，并计算均值. 共记数10 d，以培养时间为横轴，细胞数(对数)为纵轴，描绘曲线.

1.5MTT法细胞增殖实验0.25%胰蛋白酶消化SGC7901细胞，用含10%胎牛血清的RPMI1640制成细胞悬液，以每孔2×10³ cells接种于96孔板中，每孔培养液200 μL. 各组细胞均分别按48 h，72 h，96 h，120 h培养后，每孔加入MTT溶液(5 g/ L)20 μL，37℃，继续孵育4 h，终止培养，小心吸弃孔内培养上清液. 每孔加入150 μL DMSO，振荡10min，使结晶物充分溶解.选择490 nm波长，在酶联免疫检测仪上测定各孔光吸收值.以时间为横轴，光吸收值为纵轴绘制细胞增殖曲线.并计算细胞存活率(实验组吸光值/ 对照组吸光值×100%).

1.6细胞扒片与HE染色^［2］将高压消毒的玻片置于大小为120 mm的培养皿内，加入细胞悬液，基本培养条件与分组同上.同时，每组细胞均加入1.25 μmol/ L的VM26，培养24 h，然后撤去VM26，继续以镇癌灵和完全培养基培养细胞36 h，取出细胞扒片，2.5%戊二醛固定细胞，常规染色、洗片、复染、脱水、透明、封片，显微镜下观察细胞形态.

1.7细胞基因组DNA提取与电泳^［3］各组细胞在培养瓶内生长过程中，由于诱导剂的作用，部分细胞可逐渐发生死亡，不能贴壁，浮于培养液中.每天将培养上清离心后收集细胞，当收集至约有1×10⁶细胞时，加入细胞变性液(含100 mg/ L蛋白酶k，100 mmol/ L Tris-Cl，15 mmol/ L NaCl, 10 mmol/ L EDTA, 0.4% sDS)400 μL，混匀，37℃保温18 h，平衡酚与氯仿抽提二次，取上清，加入1/10体积3 mol/ L乙酸钠(pH 5.2)和2.5倍体积无水乙醇，离心沉淀.用含20 g/ L RNase A的TE缓冲液100 μL溶解DNA. 1%普通琼脂糖常规上样电泳(100V，1 h). 暗室紫外线透射仪观察电泳结果.

2结果

2.1细胞生长曲线各组细胞生长曲线结果见图1，非诱导株的细胞最大增殖数约为1.1×10⁵，对数生长期在2 d～8 d之间，细胞倍增时间为2 d.高、中、低剂量诱导株最大增殖数各为3.8×10⁴，5.3×10⁴，6.6×10<