叶酸在体外对人肝癌细胞生长的抑制作用

Inhibitory effects of folic acid on the in vitro growth of human hepatic cancer cell line

目的:体外采用叶酸对人肝癌细胞株SMMC-7721进行处理,分析叶酸抑制人肝癌细胞生长的作用。方法:实验于2003-09/2005-08在重庆医科大学完成。①人肝癌细胞SMMC-7721细胞株由四川大学华西校区分子生物学教研室提供;人胚肌腱细胞由四川大学华西校区分子生物学教研室提供。②细胞生长实验:常规培养细胞,待处于对数生长期时接种于24孔培养板内,每孔调整细胞数为1×104个,共分7组:加入叶酸,使各组叶酸的终浓度分别为15,75,375,750,1875nmol/L;加入顺铂,使顺铂组的终浓度为33nmol/L;以未加受试物、只单纯加入培养基为阴性对照组。通过绘制生长曲线计算各组细胞的增殖抑制率。③四唑盐比色实验:取对数生长期生长状态良好的SMMC-7721和人胚腱细胞,分别接种于96孔培养板,共分7组:加入叶酸,使各组叶酸的终浓度分别为15,75,375,750,1875nmol/L;加入顺铂,使顺铂组的终浓度为33nmol/L;以未加受试物、只单纯加入培养基为阴性对照组。继续培养24h后加入四唑盐(5g/L,20μL/孔)孵育。通过酶联免疫检测仪计算平均吸光度值和抑制率。④流式细胞仪检测细胞周期和凋亡率。结果:①叶酸对人肝癌细胞株体外增殖的影响:叶酸浓度为75,375,750,1875nmol/L时可显著抑制SMMC-7721细胞的增殖(P<0.05);随时间的延长和剂量的增加,SMMC-7721细胞的抑制率呈上升趋势,其生长曲线较阴性对照下移。②叶酸对肝癌细胞、人胚肌腱细胞活性及功能状态的影响:叶酸对人胚肌腱细胞的活性和功能状态无影响,但对人肝癌细胞株有抑制作用。③叶酸对SMMC-7721细胞周期各时相分布及凋亡率的影响:与S期及G2/M期比较,G0/G1期细胞比例增大(P<0.01或P<0.05),使其受阻于G0/G1期,细胞生长停滞,细胞凋亡率随浓度的增加而明显上升。结论:叶酸对人胚肌腱细胞无抑制作用,表明叶酸对正常人体细胞无不良影响;但叶酸对癌细胞存在明显的抑制作用。

AIM： To study the antitumor effects of folic acid after the human hepatic cancer cell line SMMC-7721 management by folic acid in vitro. METHODS： The experiment was done in Chongqing University of Medical Sciences from September 2003 to August 2005. ①Both hepatic cancer cell line SMMC-7721 and human embryonic tendon cells （HETCs） were supplied by Molecular Biology Staff Room, West China Campus of Sichuan University.②Cell growth curve analysis： The formally cultured at log phase cells were inoculated in 24-hole culture plate, with 1×10^4 cells in each hole, and were divided into 7 groups： The addition of folic acid concentrated its own final density as 15, 75, 375, 750 and 1 875 nmol/L, respectively; The addition of cis-dichlorediamine platinum （CDDP） made its final concentration as 33 nmol/L; And negative control group was added cultured medium only. By growth curves, the inhibition rate of cells were calculated. ③Methylthiazol tetrazolium （MTT） colorimetric assay： The cell line SMMC-7721 and HETCs of good growth at log phase were inoculated into 96-hole culture plate, and the assignment was the same as above. But 24 hours later, the cells were incubated in medium adding MTY （5 g/L, 20 p,L per hole）. The average optical density （OD） and inhibition rate were counted by Universal Microplate Spectrophotometer. ④Cell cycle and cell apoptosis rate were assayed by flow cytometry. RESULTS： ①The influence of folic acid on the growth of SMMC-7721 in vitro： Folic acid at concentrations of 75, 375, 750 and 1 875 nmol/L could inhibit the growth of SMMC-7721 significantly （P〈0.05）; The inhibition rate was increased in the dose-effect and time-effect manners, whereas the growth curve tended to decrease compared with the negative controls.②The influence of folic acid on the activity and function of SMMC-7721 and HETCs： Folic acid had no effects on the activity and function of HETCs, but inhibited those of SMMC-7721. ③The influence of folic acid on cell cycle and apoptosis rate of SMMS-7721： The percentage of G0/G1 cells was rising obviously in a dose-effect manner, compared with S and G2/M cells （P 〈 0.01 or P 〈 0.05）. Cell cycle analysis showed that folic acid could make the SMMC-7721 cells stop growing in G0/G1 phase. CONCLUSION： The noninhibitory effects of folic acid on HETCs indicate that folic acid has slight influence on normal human cells, but it can inhibit the cancer cells significantly.