温通法对S180荷瘤小鼠生存期及细胞凋亡的影响

Effect of Wentong Method on Survival and Apoptosis of S180 Tumor-Burdened Mice

目的:观察温通法对荷S180肿瘤细胞昆明小鼠生存期的影响,从诱导肿瘤细胞凋亡方面揭示温通法抑瘤机制。方法:建立S180小鼠模型,随机分为空白组、中药高剂量组、中药中剂量组、中药低剂量组、环磷酰胺组,每组16只,8只观察生存期,其余8只取材。中药高剂量组、中剂量组、低剂量组小鼠于造模次日给予不同浓度的中药灌胃,每只0.4 mL,每天1次;空白组于造模次日给予生理盐水灌胃,每只0.4 mL,每天1次;环磷酰胺组造模72 h后给予环磷酰胺0.3 mL腹腔注射,隔日1次;各组于造模第12天停止用药。每组处死8只小鼠,瘤体制作细胞悬液,采用Annexin-Ⅴ-FITC/PI双染,流式细胞仪检测细胞凋亡率;分光光度法检测Caspase-3活性。另外每组8个小鼠,记录各小鼠生存时间并做生存分析。结果:生存期实验中,空白组中位生存期为16 d,环磷酰胺组中位生存期为32 d,中药低剂量组中位生存期为24 d,中药中剂量组中位生存期为30 d,中药高剂量组中位生存期为35 d。中药3个剂量组随着剂量的增加中位生存期逐渐增高。经Log-rank假设检验,与空白组比较,中药中剂量组、高剂量组及环磷酰胺组中位生存期均较长,差异有统计学意义(P<0.05或P<0.01)。凋亡率检测实验中,中药高、中、低剂量组细胞凋亡率分别为(58.07±4.77)%、(54.89±3.29)%、(49.01±4.20)%,环磷酰胺组细胞凋亡率为(38.11±3.07)%,与空白组比较,差异均有统计学意义(P<0.05);中药3个剂量组随着剂量的增大细胞凋亡率有所增高;各组间两两比较,中药高剂量组与中药低剂量组比较,均有显著性差异(P<0.05);中药各剂量组细胞凋亡率均高于环磷酰胺组,差异有统计学意义(P<0.01)。与空白组比较,中药各剂量组及环磷酰胺组瘤细胞Caspase-3活性均较高,差异有统计学意义(P<0.01);中药3个剂量组间随着剂量的增大瘤细胞Caspase-3活性逐渐提高;中药各剂量组瘤细胞Caspase-3活性均高于环磷酰胺组,差异有统计学意义(P<0.01)。结论:温通法可明显延长S180荷瘤鼠生存期,诱导S180荷瘤鼠细胞凋亡,其作用机制可能是通过线粒体途径现实的。

Objective： To observe the effect of Wentong Method on survival of S180 tumor-burdened mice,reveal Wen Tong method on tumor suppressor mechanism from inducing apoptosis of tumor cells. Method： Establishing the mice model of S180,which were randomly divided into the black group,high dose group of Chinese herbs,middle dose group of it,low dose group of it,cyclophosphamide（ CTX） group,with 16 rats in each group： 8 rats were used to observe the survival time,the remaining 8 rats were used to obtain experimental material. We drench the mice with Chinese herbs in high,middle,low dose group in the next day with different concentrations,0. 4 mL/only,1 times a day; the blank group were given gavage with normal saline in the next day,0. 4 mL/only,1 times a day; CTX group were given CTX intra-peritoneal injection of 30 mL after 72 h,every other day for 1 times each; the medication is stopped on the twelfth day of modeling. Each group of 8 mice were killed,the tumor cell were made into suspension,using Annexin-V-FITC/PI double staining,cell apoptotic rate was detected by flow cytometry; Caspase-3 activity was detected with spectrophotometry. In addition,record survival time and make analysis of 8 mice in each group. Results： The median survival time was16 days in the blank group and 32 days in the cyclophosphamide group. The median survival time was 24 days in the low dose group and 30 days in the middle dose group,and the survival time was 35 days in high dose group of Chinese medicine in the median. The median survival time of the three dose groups increased with the increase of the dose. Compared with the blank group,the median survival time of the middle dose group,the high dose group and the cyclophosphamide group was longer than that of the blank group（ P〈0. 05 or P〈0. 01）,（ 58. 07 ± 4. 77） %,（ 54. 89 ± 3. 29） %,（ 49. 01 ± 4. 20） %,and the apoptotic rate of cyclophosphamide group was significantly higher than that of the control group（ P〈0. 05）（ 38. 11 ± 3. 07） %,compared with the blank group,the difference was statistically significant（ P〈0. 05）; Three doses of traditional Chinese medicine group with the dose increased the rate of apoptosis increased; between the two groups（ P〈0. 05）. The apoptotic rate of each dose group was higher than that of cyclophosphamide group（ P〈0. 01）. The difference was statistically significant（ P〈0. 01）. Compared with that of the blank group,the activity of Caspase-3 in the tumor cells of each group and the group of cyclophosphamide was higher（ P〈0. 01）.The activity of Caspase-3 was significantly higher than that of cyclophosphamide group（ P〈0. 01）. Conclusion： Wentong method can prolong the survival time of S180 tumor-burdened mice and induce the apoptosis of S180 tumor-burdened mice. The mechanism may act through the mitochondrial pathway.