摘要
目的:已发现人参中某些有效组分能增强和激活机体免疫系统,具有抗肿瘤、抗衰老、抗辐射等多种生物活性作用。选取人髓性白血病细胞株HL60为模型,探讨人参皂甙单体Rh2抑制其增殖和诱导凋亡的时效量效关系。方法:实验于2006-07/08在吉林医药学院临床检验实验室进行。①材料:人参皂甙单体Rh2购自吉林省宏久生物科技股份有限公司,批号050801,溶于pH7.4磷酸盐缓冲液中配成50g/L母液。人髓性白血病细胞株HL60购自中国科学院上海生物细胞研究所。②实验方法:取处于对数生长期的HL60细胞制备3×108L-1细胞悬液,接种后6h分别加入终浓度为5,10,20,40,80mg/L的人参皂甙单体Rh2100μL,于加药48h后采用MTT比色法检测人参皂甙单体Rh2对HL60细胞生长的抑制作用,计算抑制率和半数抑制浓度。选择半数抑制浓度的人参皂甙单体Rh2处理HL60细胞,分别于作用6,12,24,48,72h后采用MTT比色法测定抑制率,并与未加药对照组进行比较。半数抑制浓度的人参皂甙单体Rh2作用48h时倒置显微镜下观察HL60细胞形态,吉姆萨染色观察细胞凋亡情况。结果:①量效关系:人参皂甙单体Rh2浓度为5,10,20,40mg/L时,处理48h后HL60细胞生长抑制率呈升高趋势(F=9.45,P<0.01),具有明显的剂量依赖性;至80mg/L时抑制率与40mg/L基本相似。加药48h后半数抑制浓度为13.0mg/L。②时效关系:13.0mg/L人参皂甙单体Rh2处理HL60细胞6,12,24,48,72h后,抑制率逐渐升高(F=9.32,P<0.01),呈时间依赖性。③HL60细胞形态观察:与未加药对照组比较,经13.0mg/L人参皂甙单体Rh2处理后HL60细胞数量明显减少,细胞分散,体积缩小,细胞核呈碎片状。④凋亡细胞吉姆萨染色检测:经13.0mg/L人参皂甙单体Rh2处理后,HL60细胞出现核染色质浓缩、呈周边凝聚,核膜裂解形成凋亡小体、胞质出现空泡但胞膜完整的典型细胞凋亡形态学改变。结论:人参皂甙单体Rh2能有效抑制体外培养HL60细胞的增殖,并诱导其凋亡,干预效果呈时间和剂量依赖。
AIM： It is found that some active priciples of ginseng can enhance and activate body immune system, and have many bioactivities such as anti-tumor, anti-aging and anti-radiation. This study examined the effect of ginsenoside-Rh2 （GS-Rh2） on proliferation and apoptosis in human myeloid leukemia cell strain HL60, and analyzed the dose- and time-dependent manners of GS-Rh2. METHODS： Experiments were performed at the Department of Clinical Laboratory of Jilin Medical College from July to August in 2006. （1）Rh2 was purchased from Hongjiu Biotech Co., Ltd. （batch number 050801）, and prepared into 50 g/L stock solution by dissolving in pH 7.4 phosphate buffer saline. The HL60 cell strain was purchased from Shanghai Institute of Cell Biology of Chinese Academy of Sciences. （2）HL60 cells in logarithmic growth phase were inoculated into 3×10^8 L^-1 cell suspension. After the cells were cultured for 6 hours, 100 μL GS-Rh2 at different concentrations （5, 10, 20, 40, 80 mg/L） was added respectively. After the cells were administrated for 48 hours, cell inhibition ratio （IR） was evaluated by MTT colorimetric assay. The 50% inhibitory concentration （IC50） was worked out. HL60 cell was acted with this concentration for different time （6, 12, 24, 48 and 72 hours）, cell inhibition ratio （IR） at different time points was evaluated by MTT colorimetric assay, and compare it to the control. After the IC50 of GS-Rh2 acted for 48 hours, HL60 cells were observed with an inverted microscope. HL60 cell was stained by Giemsa, and the typical apoptosis cells were discovered. RESULTS：（1）Dose-effect relationship： When the concentration of GS-Rh2 was 5, 10, 20, 40 mg/L, the IR of GS-Rh2 to the growth of HL60 cells was increased gradually in obviously dose-dependent manner. The IR was similar between 80 mg/L and 40 mg/L. After the cells were administrated for 48 hours, the IC50 value was 13.0 mg/L. （2）Time-effect relationship： After the concentration of IC50 of GS-Rh2 （13.0 mg/L） acting on HL60 line at different time points （6, 12, 24, 48 and 72 hours）, cell IR was increased gradually （F=9.32, P 〈 0. 01） in obviously time-dependent manner. （3）Compared to the control, the density of HL60 cells dealt with the GS-Rh2 （13.0 mg/L） was low and the cells separated from each other with reduced volume. （4）After the cells dealt with GS-Rh2 of 13.0 mg/L, the typical apoptosis cells were discovered： showing disorganized, highly contracted and vacuolated, condensed cytoplasm, vanished karyotheca, ruptured cell nucleus and apoptotic body. CONCLUSION： GS-Rh2 can inhibit the growth and induce the apoptosis of HL60 cell line cultured in vitro in a time-and dose-dependent manner.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第12期2331-2334,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
