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目的:探讨从天然冬虫夏草中分离出的虫草拟青霉Cs-4菌株,经人工发酵所得发酵虫草菌粉对SMMC-7721人肝癌细胞的生长抑制作用。方法:20只体重(200±20)g雄性SD大鼠随机分为4组(每组5只):空白组及Cs-4低、中、高剂量组,空白组ig予蒸馏水,Cs-4低、中、高剂量组(0.77,1.54,3.08 g·kg-1,ig);给药3 d后,制备含药血清。肝癌细胞悬液调整至4×104/m L,96孔板每孔加入100μL,培养24 h后加入大鼠含药血清及顺铂注射液;干预48 h MTT法测各组细胞增殖抑制率。将SMMC-7721细胞悬液调整至2.8×104/m L,接种于6孔板培养24 h,加入大鼠含药血清及顺铂,干预培养48 h后Annexin V-FITC/PI染色,流式细胞仪检测各组细胞凋亡率;ELISA检测各组细胞上清液中血管内皮生长因子(VEGF),金属基质蛋白酶-2(MMP-2)表达量。结果:SMMC-7721细胞增殖被Cs-4含药血清明显抑制(与空白组比较P<0.05),低、中、高剂量组抑制率分别为12.2%,23.2%,38.1%,呈剂量依赖性(Cs-4不同剂量组间比较P<0.05)。Cs-4含药血清低、中、高剂量组细胞凋亡率分别为13.9%,23.5%,24.9%,均高于空白组(P<0.05)。Cs-4含药血清低、中、高剂量组细胞上清液中VEGF分别为443.5,436.9,411.0 ng·L-1,低于空白组(P<0.05);Cs-4低、中、高剂量组细胞上清液中MMP-2浓度与空白组比无显著差异。结论:Cs-4含药血清能抑制SMMC-7721细胞增殖,提高体外培养的SMMC-7721细胞凋亡率,抑制SMMC-7721细胞VEGF表达,提示Cs-4有抗肝癌作用,对SMMC-7721细胞MMP-2表达无显著影响。
OBJECTIVE: To investigate the inhibitory effect of Cordyceps militaris Cs-4 isolated from natural Cordyceps sinensis on the growth of SMMC-7721 human hepatocellular carcinoma cells. Methods: Twenty male Sprague-Dawley rats weighing 200 ± 20 g were randomly divided into 4 groups (n = 5 each): blank group and Cs-4 low, medium and high dose groups, blank group ig distilled water, Cs-4 Low, medium and high dose group (0.77,1.54,3.08 g · kg-1, ig); after 3 d administration, the drug-containing serum was prepared. Liver cancer cell suspension was adjusted to 4 × 104 / m L, 96 well plates were added to each well 100μL, cultured for 24 h after the drug-containing serum and cisplatin injection; intervention 48h MTT assay inhibition rate of each group of cell proliferation. The suspension of SMMC-7721 cells was adjusted to 2.8 × 10 4 / ml and cultured in 6-well plates for 24 h. The serum-containing SMMC-7721 cells and cisplatin were added and the cells were stained with Annexin V-FITC / PI for 48 h. The apoptosis rate of each group was detected by cytometry. The expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) in the supernatant of each group was detected by ELISA. Results: The proliferation of SMMC-7721 cells was significantly inhibited by Cs-4-containing serum (P <0.05 compared with the blank group), and the inhibition rates of SMMC-7721 cells were 12.2%, 23.2% and 38.1% (Cs-4 different dose groups compared P <0.05). The apoptosis rates of Cs-4-containing serum in low, medium and high dose groups were 13.9%, 23.5% and 24.9%, respectively, which were higher than those in blank group (P <0.05). The levels of VEGF in Cs-4-containing serum were 443.5, 436.9 and 411.0 ng · L-1, respectively, which were lower than those in the blank group (P <0.05), while the levels of Cs-4 in the medium, There was no significant difference in the concentration of MMP-2 between the dose group and the blank group. CONCLUSIONS: Cs-4-containing serum can inhibit the proliferation of SMMC-7721 cells, increase the apoptosis rate of SMMC-7721 cells and inhibit the expression of VEGF in SMMC-7721 cells, suggesting that Cs-4 can inhibit the proliferation of SMMC-7721 cells MMP-2 expression had no significant effect.